Darya Zibrova

Rolf Grempler

Rüdiger Streicher

Dr Stefan G Kauschke
email: stefan.kauschke@boehringer-ingelheim.com

Keywords: glycogen phosphorylase, G L, glycogen synthesis, glycogen metabolism, glycogen phosphorylase inhibitor, type 2 diabetes

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Inhibition of the interaction between protein phosphatase 1 glycogen-targeting subunit and glycogen phosphorylase increases glycogen synthesis in primary rat hepatocytes

Darya Zibrova, Rolf Grempler, Ruediger Streicher, Stefan G. Kauschke

To cite this version:

Darya Zibrova, Rolf Grempler, Ruediger Streicher, Stefan G. Kauschke. Inhibition of the interaction between protein phosphatase 1 glycogen-targeting subunit and glycogen phosphorylase increases glycogen synthesis in primary rat hepatocytes. Biochemical Journal, 2008, 412 (2), pp.359-366. [START_REF] Doherty | Loss of the hepatic glycogen-binding subunit (GL) of protein phosphatase 1 underlies deficient glycogen synthesis in insulin-dependent diabetic rats and in adrenalectomized starved rats[END_REF].1042/BJ20071483. hal-00478906

INTRODUCTION

Type 2 diabetes is a polygenic disease characterized by peripheral insulin resistance, elevated hepatic glucose production and defects in pancreatic insulin secretion [START_REF] Defronzo | Pharmacologic therapy for type 2 diabetes mellitus[END_REF]. The relative importance of each of these abnormalities for the aetiology and progression of type 2 diabetes is not fully established to date. However, it is generally accepted that excessive glucose production by the liver contributes significantly to the diabetic hyperglycaemia [START_REF] Consoli | Role of liver in pathophysiology of NIDDM[END_REF][START_REF] Defronzo | Pathogenesis of NIDDM. A balanced overview[END_REF][START_REF] Edelman | Type II diabetes mellitus[END_REF]. In the diabetic liver, glucose production is increased, in part, due to the elevated glycogenolysis as a consequence of the enhanced activity of GP. Therefore, the blockade of glycogenolysis via the inhibition of GP has been proposed as one of the therapeutic strategies to improve the glycaemic control in type 2 diabetes.

A number of GPis with different GP binding sites and hence distinct profiles with respect to potency, kinetics, synergism with glucose, and ability to promote the dephosphorylation of GP have been described [START_REF] Treadway | Glycogen phosphorylase inhibitors for treatment of type 2 diabetes mellitus[END_REF]. However, in spite of the variety of GPis discovered, certain critical issues remain associated with a direct inhibition of the GP enzyme as an anti-diabetic strategy. Among these are the potential risks of hypoglycaemia and potentially negative impact on muscle function. Foreseeing the latter one is supported, first, by a study showing that the pharmacological inhibition of GP in a perfused muscle system impairs the aerobic muscle function during prolonged contraction [START_REF] Baker | The experimental type 2 diabetes therapy glycogen phosphorylase inhibition can impair aerobic muscle function during prolonged contraction[END_REF]. And second, the exercise capacity is impaired in patients with McArdle's disease, subtype V of glycogen-storage diseases caused by GP deficiency in muscle [START_REF] Coakley | Relationship between ammonia, heart rate, and exertion in McArdle's disease[END_REF][START_REF] Sahlin | Impaired oxidative metabolism increases adenine nucleotide breakdown in McArdle's disease[END_REF]. Therefore, alternative strategies for the correction of glucose disposal would be of interest.

In recent years, the interaction between GP and the liver glycogen targeting subunit (G L ) of protein phosphatase 1 (PP1) has emerged as a potential novel target for the treatment of type 2 diabetes, as the G L -GP interaction plays an important role in the allosteric regulation of glycogen synthesis [START_REF] Cohen | The twentieth century struggle to decipher insulin signalling[END_REF]. Glycogen synthesis itself is regulated via changes in the phosphorylation state of glycogen synthase (GS). The key regulator of the phosphorylation state of GS is PP1 which dephosphorylates the enzyme and brings it to the active state (GSa).

In the liver, targeting of PP1 to glycogen particles by G L increases PP1 activity towards GS [START_REF] Doherty | Loss of the hepatic glycogen-binding subunit (GL) of protein phosphatase 1 underlies deficient glycogen synthesis in insulin-dependent diabetic rats and in adrenalectomized starved rats[END_REF][START_REF] Moorhead | Purification of the hepatic glycogen-associated form of protein phosphatase-1 by microcystin-Sepharose affinity chromatography[END_REF]. However, apart from targeting PP1 to glycogen, G L also interacts by its C-terminus with the active form of GP (GPa). This interaction suppresses the activity of PP1 towards GS and thereby inhibits glycogen synthesis [START_REF] Alemany | Phosphorylase a is an allosteric inhibitor of the glycogen and microsomal forms of rat hepatic protein phosphatase-1[END_REF]. This allosteric regulation of PP1 by GPa allows glycogen synthesis to be switched off when the glycogenolytic pathway is active. However, in the diabetic liver, increased conversion of GP to its active (a) form sustains the allosteric inhibition of glycogen synthesis, furthermore depleting glycogen pools and contributing to the elevated glucose production. Under these circumstances, an inhibition of the G L -GP interaction would be of particular relevance as an anti-diabetic strategy as it would help to counteract the abnormally increased hepatic glucose output. However, no compounds working via this novel mechanism have been described so far.

In the present study, following the identification and initial characterization of a G L -GP inhibitor (G L -GPi), the effects of this tool compound on glycogen metabolism were analyzed in comparison with two direct GP inhibitors. Using primary rat hepatocytes, we demonstrate here that the pharmacological prevention of the G L -GP interaction increases both glycogen synthesis rate and glycogen amounts at elevated glucose levels similarly to direct GPis, however without inhibiting the activity of GP. S t a g e 2 ( a ) P O S T -P R I N T

EXPERIMENTAL Isolation and culture of primary rat hepatocytes

Primary hepatocytes were isolated by collagenase perfusion from the livers of male Wistar [Crl:Wi(Han)] rats weighting 200-300 g (Charles River, Sulzfeld, Germany). The cells were suspended in William's medium E (Invitrogen, Karlsruhe, Germany) supplemented with 5% (v/v) foetal calf serum, 17 nM insulin (Invitrogen), 100 nM dexamethasone and 2 mM glutamine (Sigma, Taufkirchen, Germany), 0.1 mg/ml gentamicin (Cambrex BioScience, Walkersville, MD) and seeded at 1.1×10 4 or 1.1×10 5 cells per well in 96-or 6-well collagen Icoated plates (Becton Dickenson Labware, Bedford, MA), respectively. After an attachment phase of 3 h, the medium was replaced by serum-free Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 5 mM glucose and other additives as described above (unless otherwise indicated). The cells were incubated overnight (16-18 h) prior to compound treatments.

Compounds

G L -GPi was identified in a scintillation proximity assay (Amersham Biosciences, Inc) using

Compound treatments

Following the exchange of the overnight culture medium to serum-free DMEM containing 2.5 mM or 10 mM glucose and additives as described above, the cells were incubated with compounds for 2 or 6 h. Cells treated with 0.25% (v/v) DMSO served as solvent controls. To stop ongoing biological processes, the cells were washed with ice-cold PBS and immediately stored at -80°C until further usage.

GP activity measurement

Enzymatic activity of GP was measured in the glycogenolytic direction using a phosphoglucomutase/glucose-6-phosphate dehydrogenase-coupled assay. For this, hepatocytes were cultured in 6-well plates and scraped in lysis buffer containing 50 mM HEPES, pH 7. rotator. The GS/GST-glycogenin complexes caught by glutathione-sepharose were recovered by centrifugation at 500 g for 5 min at 4°C and washed sequentially with washing buffer 1 (lysis buffer supplemented with 154 mM NaCl) and washing buffer 2 (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 1 mM EGTA) twice each. GS bound to GST-glycogenin was eluted with elution buffer (washing buffer 2 supplemented with 20 mM reduced glutathione) for 10 min at room temperature. The protein concentration in the eluate was determined according to the Bradford method.

Dephosphorylation of the recombinant mouse liver GS Mouse liver GS was suspended in the reaction buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM DTT, 0.1 mM EGTA, 0.025% Tween 20) to the final concentration of 1.25 µg/ml. The dephosphorylation of GS with catalytic subunit of the α-isoform of rabbit recombinant PP1 (25 U/ml final concentration) (Sigma) was performed for 30 min at 30°C in the absence or presence of 17 mM Na 3 VO 4 . The reaction was terminated by adding protein loading buffer (Roth, Karlsruhe, Germany) and the samples were analyzed by immunoblotting.

GS activity assay

To measure GS activity, hepatocytes were cultured in 6-well plates and lysed in 20 mM Tris HCl, pH 7.4, 50 mM NaF, 5 mM Na 3 VO 4 , 1 mM EDTA, 1 mM EGTA, 0.1% (v/v) βmercaptoethanol, 1% (v/v) Triton X-100 and protease inhibitor mix. After a centrifugation step (13 000 g, 10 min, 4°C), aliquots of the supernatants (15 µl/reaction at 1.5 mg/ml protein) were suspended in the assay mix (30 µl/reaction) composed of 50 mM Tris HCl, pH 7.4, 25 mM NaF, 20 mM EDTA, 1 mg/ml glycogen, 0.4 mM UDP-glucose, in the presence of either 0.1 mM or 10 mM glucose-6-phosphate. 0.075 µCi of [ 14 C]UDP-glucose was added to each well to obtain a final specific activity of 20.3 mCi/mmol. After a 30 min incubation at 37°C, glycogen precipitation (2h, 4°C) and liquid scintillation counting were performed. The ratio of activities at 0.1 mM (active GS) and 10 mM glucose-6-phosphate (total GS) was used to determine the degree of GS activation.

To measure the activity of mouse liver GS, the recombinant protein was suspended in the reaction buffer (composition as described above) to the final concentration of 0.03 mg/ml and pre-incubated with the G L -GPi (0.3-100 µM) or the catalytic subunit of the α-isoform of rabbit recombinant PP1 (147 U/ml final concentration) for 30 min at 30°C. Aliquots (15 µl) from each reaction were assayed for GS enzymatic activity as described above for the cellular lysates.

Preparation of glycogen-enriched fractions

Cell lysates obtained as described for the determination of GP activity, were sequentially centrifuged for 10 min at 1 000 g and 15 min at 10 000 g, 4°C. The resulting supernatants were further centrifuged at 100 000 g (40 min, 4°C). The pellets obtained in the last step were referred to as the glycogen-enriched fraction. They were resuspended in detergent-free lysis buffer, the protein concentrations in the samples were determined according to the Bradford method.

Immunoblotting 10 µg protein/well of cell lysates or glycogen-enriched fractions was electrophoretically separated by SDS-PAGE and transferred onto nitrocellulose membranes according to standard procedures. The membranes were blocked in Tris-buffered saline containing 0.25% (v/v) Tween 20 (TBST) and 5% (w/v) non-fat dry milk and further incubated with antibodies diluted in TBST containing 0.5% (w/v) milk. The primary antibodies were polyclonal anti-GP (Acris Antibodies, Hiddenhausen, Germany), anti-phospho(Ser 14 )-GP, anti-GS, antiphospho(Ser 641 )-GS (Cell Signaling Technology, Beverley, MA), all used at 1:1000 dilution, and monoclonal anti-γ-tubulin (1:5000) (Sigma). Following the incubation with the respective HRP-labelled secondary antibodies, the blots were developed with a Lumi-LightPLUS chemiluminescent substrate (Roche Diagnostics) and signals were detected using a Fuji LAS 3000 Imaging station (Fuji Photo Film Co., Tokyo, Japan).

Glycogen synthesis assay

To determine the rate of glycogen synthesis, primary rat hepatocytes were cultured in 96-well plates for overnight in serum-free William's medium E supplemented as described above.

After washing with PBS (37°C), the medium was changed to Krebs-Ringer bicarbonate HEPES buffer containing 10 mM glucose and 0.1% (w/v) BSA. Compounds were applied using a concentration range from 0.01 to 10 µM and 0.5 µCi of [ 14 C]glucose was added to each well to obtain a final specific activity of 11.2 mCi/mmol. After 2 h incubation at 37°C, the cells were lysed with 1 M NaOH (100 µl/well), whereupon the lysates were transferred to the glass fibre filter plates (Millipore) pre-loaded with 100% ethanol (100 µl/well) in order to precipitate the glycogen for 2 h at 4°C. The incorporation of [ 14 C]glucose into glycogen was measured by liquid scintillation counting.

Glycogen hydrolysis and glucose determination

For the determination of glycogen levels, aliquots (10 µg) of the glycogen-enriched fractions were used for glycogen digestion by amyloglucosidase (300 µl/sample at an activity of 4.5 U/ml in 0.2 M NaAc, pH 4.8). Following an incubation for 2 h at 37°C, the samples were neutralized with 0.25 M NaOH and the glucose concentrations were determined fluorometrically using the Amplex Red reagent (Molecular Probes, Eugene, OR).

Statistical analysis

Results are presented as means ± standard error of the mean (SEM). For group comparisons the unpaired Student's t-test was used. Values of p<0.05 were considered to differ significantly. For all read-outs, a representative or pooled data of minimally three experiments are shown.

RESULTS

Identification and characterization of an inhibitor of the G L -GP interaction

An inhibitor of the G L -GP interaction (G L -GPi) was identified in a miniaturized scintillation proximity assay. This inhibitor suppressed the interaction between a biotin-labelled G L peptide and recombinant human GPa with an IC 50 of 0.15 µM (Table 1). G L -GPi had no effect on the enzymatic activity of GP at all concentrations tested in vitro (IC 50 >30 µM). Two direct GPis, CP-320,626 [START_REF] Oikonomakos | A new allosteric site in glycogen phosphorylase b as a target for drug interactions[END_REF] and compound A (Bsp.1 in [Aventis Pharma Deutschland GmbH: DE 10335092 B3 (February 3, 2005)]), were used as reference compounds in all experiments. These two compounds inhibited GP activity with IC 50 's of 1.4 µM (CP-320,626) and 2 µM (compound A). With respect to the G L -GP interaction, CP-320,626 also suppressed the interaction with an IC 50 of 1 µM, whereas compound A did not affect the G L -GP interaction. G L -GPi was tested in primary rat hepatocytes using a crystal violet staining [START_REF] Grempler | Inhibition of SH2-domain containing inositol phosphatase 2 (SHIP2) in insulin producing INS1E cells improves insulin signal transduction and induces proliferation[END_REF] and showed no cytotoxic effect at concentrations up to 100 µM (data not shown).

Thus G L -GPi, an inhibitor of the G L -GP interaction that showed no inhibition of the enzymatic activity of GP in vitro was identified.

G L -GPi increases glycogen synthesis similarly to direct GPis

Since the prevention of the G L -GP interaction was predicted to increase glycogen synthesis, the ability of G L -GPi to increase the rate of glycogen synthesis was tested in primary rat hepatocytes used for this and further experiments as model system. A range of compound concentrations (0.01-10 µM) was used and the rate of [ 14 C]glucose incorporation into glycogen was analyzed. G L -GPi increased the rate of glycogen synthesis in a dose-dependent fashion similarly to both direct GPis (Figure 1) with compound A being the most potent compound (EC 50 0.3 µM), while the potencies of G L -GPi (EC 50 1.3 µM) and CP-320,626 (EC 50 1.8 µM) were comparable. To work under stable experimental conditions, compound concentrations leading to a plateau effect in the glycogen synthesis assay were used for all subsequent experiments. They were 10 µM for G L -GPi and CP-320,626 and 3 µM for compound A.

It has been shown that the prevention of the G L -GP interaction increases glycogen synthesis rate.

G L -GPi elevates glycogen storage but does not prevent glycogen mobilization in contrast to direct GPis

To investigate whether the newly identified inhibitor of the G L -GP interaction increases glycogen storage, the effect of G L -GPi on glycogen levels was measured and compared to the effects of direct GPis. At elevated glucose concentrations (10 mM), the glycogen amounts were increased to a similar extent (2.6-fold) by the G L -GPi and direct GPis after 2 h of treatment (Figure 2A). However at prolonged treatment (6 h), glycogen levels continued to increase with direct GPis but plateaued with G L -GPi. To further discriminate the effects of a direct inhibition of GP and the G L -GP inhibition, similar treatments were performed at low glucose concentration (2.5 mM) to mimic the state of hypoglycaemia. Under these conditions, basal glycogen levels declined without compound treatment (Figure 2B), indicating glycogen store mobilization over time. However, with direct GPis, glycogen amounts not only remained at the basal level after 2 h of treatment, but further increased after prolonged treatment (2.5fold higher than starting point (0 h)). In contrast, in hepatocytes treated with G L -GPi no increase in glycogen levels but a delayed mobilization of glycogen pools was observed at 2.5 mM glucose. Thus, the G L -GP inhibitor increased glycogen storage at elevated glucose levels but did not prevent its mobilization at low glucose levels. The molecular mechanisms underlying the differential effects of G L -GPi on glycogen levels in comparison to GPis were investigated. First, the effects on the phosphorylation state and the activity of GS were analyzed. Both G L -GPi as well as two direct GPis significantly reduced the phosphorylation status of GS at both 10 and 2.5 mM glucose concentrations after 2 h of treatment (Figure 3A,B). At elevated glucose levels this effect decreased over time (small but significant decrease in GS phosphorylation after 6 h of treatment), while at low glucose levels the effect on GS phosphorylation was rather short lasting (no significant effect after 6 h of treatment). In order to test whether the dephosphorylation of GS translated into an activation of the enzyme, the enzymatic activity of GS was measured. As expected, GS activity was significantly increased by G L -GPi similarly to both direct GPis (Figure 3C). At the elevated glucose concentrations, this effect was more pronounced at 2 h (200-250% activity) compared to 6 h (150-170% activity) of compound treatment, indicating that the activity measured reflects the phosphorylation status of GS. A similar pattern in affecting the GS activity was observed at 2.5 mM glucose.

Overall, both direct GP inhibition and the prevention of the G L -GP interaction led to the dephosphorylation and consequent activation of GS.

G L -GPi has no effect on the activity of recombinant liver GS

To ensure that the activation of GS in hepatocytes is not attributed to a direct effect of G L -GPi on the enzyme, the effect of G L -GPi on the activity of recombinant mouse liver GS was measured. Treatment of GS with the catalytic subunit of recombinant PP1 caused a complete dephosphorylation of GS (Figure 4A). This effect was reduced by 17 mM sodium orthovanadate, a nonspecific protein phosphate inhibitor. Furthermore, treatment with PP1 increased the enzymatic activity of GS at both 0.1 mM glucose 6-phosphate (over 20-fold) (Figure 4B) and 10 mM glucose 6-phosphate (2-fold increase, Figure 4C). As expected, 10 mM glucose 6-phosphate per se had a strong stimulatory effect on GS activity. In contrast, G L -GPi had no effect on GS activity at the concentrations up to 100 µM at both 0.1 and 10 mM glucose 6-phosphate. These data show that the activation of GS in hepatocytes is not caused by a direct effect of G L -GPi on GS.

Inhibition of the G L -GP interaction does not inactivate and dephosphorylate cellular GP

To confirm that the prevention of the G L -GP interaction does not inhibit the cellular activity of GP, the effect of G L -GPi on the endogenous activity of GP in primary rat hepatocytes was analyzed. The activity of the enzyme measured in the glycogenolytic direction was significantly reduced (by 58-86%) by both direct GPis independent of the glucose concentrations used at both 2 and 6 h (Figure 5A). In contrast, G L -GPi did not cause any reduction in the enzymatic activity of GP at any of the glucose concentrations or time points tested. Rather a slight, non-significant increase in GP activity at 6 h of treatment was visible.

The reduction in GP activity with the direct GPis was associated with a decreased level of GP phosphorylation. A nearly complete abolishment of the GP phosphorylation at serine 14 was observed with the direct GPis at both glucose concentrations after 2 h treatment, slowly recovering thereafter (Figure 5B,C). In contrast, the phosphorylation status of GP was not influenced following a 2 h treatment with the G L -GP inhibitor at both glucose concentrations, correlating with the unchanged GP activity. At the 6 h time point, it was slightly but nonsignificantly increased, reflecting a possible trend towards an activation of GP. Overall, in contrast to direct GPis, the inhibition of the G L -GP interaction does not reduce neither the cellular activity of GP nor its phosphorylation over 6 h. Since changes in the subcellular compartmentalization of key enzymes is one of the mechanisms involved in the regulation of glycogen metabolism [START_REF] Aiston | Glucose 6-phosphate causes translocation of phosphorylase in hepatocytes and inactivates the enzyme synergistically with glucose[END_REF], the impact of G L -GPi and direct GPis on the amounts of GP found in the glycogen-enriched fraction was analyzed.

In the immunoblotted samples normalized to the protein content, significant differences in GP signals were observed with profound reduction in GP amounts following the treatment with direct GPis compared to the treatment with G L -GPi as the most prominent feature (Figure 6A). To assess whether the stoichiometry of GP-glycogen binding was altered upon compound action, the relative amounts of GP were normalized to the glycogen content of the same samples. The GP/glycogen ratios were significantly reduced (by 80-90%) by both GPis but only moderately (by 36%) by G L -GPi at 10 mM glucose compared to DMSO control at 0 h time point (Figure 6B). At 2.5 mM glucose, the GP/glycogen ratios remained unchanged following the treatment with G L -GPi and were again significantly reduced by direct GPis (by 70-85%). These data show that in contrast to G L -GPi, the treatment with direct GP inhibitors pronouncedly displaces GP from glycogen particles, thereby decreasing the ratio of GP per glycogen.

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DISCUSSION

It has been hypothesized that a drug blocking the G L -GP interaction would activate GS and increase glycogen synthesis, and could thereby potentially attenuate the state of diabetic hyperglycaemia [START_REF] Cohen | The twentieth century struggle to decipher insulin signalling[END_REF]. For the first time, we describe here the characterization of an inhibitor of the G L -GP interaction. This inhibitor suppressed the interaction between the G L peptide and GPa with an IC 50 of 150 nM. Importantly, it did not inhibit the enzymatic activity of GP. These data suggest that displacement of G L from GP does not cause significant conformational changes that would reduce the activity of GP. This is in agreement with our recent x-ray crystallographic study showing no large conformational differences between hlGPa crystallized alone or in a complex with a G L peptide [START_REF] Pautsch | Molecular recognition of the protein phosphatase 1 glycogen targeting subunit by glycogen phosphorylase[END_REF].

The direct GP inhibitor CP-320,626 also caused an inhibitory effect on the interaction between the G L and GPa with an IC 50 of 1 µM, which is in the same concentration range as the IC 50 for GPa inhibition (IC 50 1.4 µM). As has been shown recently, a related compound CP-316,819 is also capable of blocking the G L -GP interaction [START_REF] Kelsall | The hepatic PP1 glycogen-targeting subunit interaction with phosphorylase a can be blocked by C-terminal tyrosine deletion or an indole drug[END_REF]. Both compounds are members of the indole-2-carboxamide series of phosphorylase inhibitors [START_REF] Hoover | Indole-2-carboxamide inhibitors of human liver glycogen phosphorylase[END_REF]. The G L protein binds to the AMP binding site of GP [START_REF] Pautsch | Molecular recognition of the protein phosphatase 1 glycogen targeting subunit by glycogen phosphorylase[END_REF], which is distinct from the indole-binding site on GP. Therefore, the effect of these compounds on the G L -GP interaction cannot be explained by an overlap of these two sites. More likely, these compounds induce conformational changes in the GP protein, which prevents its binding to G L . The compound A, another potent GPi that belongs to the class of acyl ureas, did not affect the G L -GP interaction in the present study despite the fact that compounds of this class have been shown to bind to the AMP binding site of GP [START_REF] Klabunde | Acyl ureas as human liver glycogen phosphorylase inhibitors for the treatment of type 2 diabetes[END_REF]. This indicates differences in binding to this area of the GP protein by G L and compound A.

It was previously reported that in hepatocytes, the action of CP-320,626 was associated with the conversion of GP from phosphorylated a to dephosphorylated b form [20]. In the present study we confirmed this effect of CP-320,626 and showed that the action of a member of acyl urea class of GP inhibitors, compound A, was also associated with the dephosphorylation of GP. In contrast, G L -GPi did not lead to a GP dephosphorylation at 2 h, which correlated with the unchanged GP activity. At 6 h, a slight increase in GP phosphorylation was observed, which explains the trend towards an increased GP activity after a prolonged treatment using the G L -GPi. Based on this trend, we cannot rule out a possible feed-back regulation that might be switched on in order to counteract a sustained stimulation of glycogen synthesis. Further studies should be undertaken to clarify this phenomenon.

In hepatocytes, the inactivation of GP by CP-320,626 led to an increase in glycogen synthesis [START_REF] Aiston | Inactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesis[END_REF][START_REF] Aiston | Hepatic glycogen synthesis is highly sensitive to phosphorylase activity: evidence from metabolic control analysis[END_REF]. In the present study we provide the first pharmacological evidence demonstrating that prevention of the G L -GP interaction is sufficient to increase glycogen synthesis in hepatocytes without inhibiting the activity of GP. G L -GPi consistently increased GS activity similarly to both direct GPis, which correlated well with a reduced GS phosphorylation at most conditions. We did not see this correlation at 6 h of treatment at low glucose where no decrease in GS phosphorylation was measurable. A possible explanation for this discrepancy could be a lower sensitivity of the GS phosphoblot compared to radioactive method of GS activity determination. G L -GPi increased the rate of glycogen synthesis similarly to direct GPis. Since G L -GPi did not have any effect on the activity of recombinant GS, the increases in GS activity and glycogen synthesis rate caused by G L -GPi can be entirely attributed to the inhibition of the G L -GP interaction. Modulation of the subcellular compartmentalization of key enzymes is one of the mechanisms involved in the regulation of glycogen metabolism [START_REF] Aiston | Glucose 6-phosphate causes translocation of phosphorylase in hepatocytes and inactivates the enzyme synergistically with glucose[END_REF]. Therefore, we have investigated the impact of the compounds on the association of GP with the glycogen-enriched fractions. Direct GPis significantly reduced the amounts of GP present in glycogen-enriched fractions compared to the treatment with G L -GPi. In addition, direct GPis profoundly reduced the GP/glycogen ratios at both 10 mM and 2.5 mM glucose concentrations. In contrast, the G L -GP inhibitor only moderately reduced the GP/glycogen ratio at 10 mM glucose, possibly due to a saturation of the GP/glycogen binding at high glycogen levels, and did not change the GP/glycogen ratio at 2.5 mM glucose. These data demonstrate that the effects of G L -GPi and direct GPis on the stoichiometry of GP/glycogen binding are different with a pronounced and consistent displacement of GP from the glycogen particles by direct GPis. A possible explanation for the effect of direct GPis could be a weaker binding of GP to glycogen particles due to the loss of phosphorylation/activity. We show here for the first time that a pharmacological interference with the G L -GP interaction is sufficient to increase glycogen levels without inhibiting the activity of GP. G L -GPi increased glycogen levels with an efficacy similar to direct GPis following 2 h treatment at 10 mM glucose. Prolonged treatment up to 6 h with G L -GPi did not lead to a further increase in glycogen levels. This observation could be explained by a futile glycogenolysisglycogen synthesis cycle that occurs due to the co-existence of the activated forms of both glycogen phosphorylase and glycogen synthase [START_REF] David | Simultaneous synthesis and degradation of rat liver glycogen. An in vivo nuclear magnetic resonance spectroscopic study[END_REF][START_REF] Shulman | 13C NMR studies of glycogen turnover in the perfused rat liver[END_REF]. The higher efficacy towards elevating glycogen levels demonstrated by direct GPis at 6 h can be attributed to an increase in glycogen synthesis and a simultaneous prevention of glycogenolysis. A reduction of GP within the glycogen-enriched fraction upon treatment with direct GPis, as demonstrated here, could additionally potentiate the action of these compounds, for example by a delayed recruitment of GP to the glycogen particles as a site of action. At 2.5 mM glucose, G L -GPi did not lead to glycogen accumulation and rather allowed its mobilization over time, indicating that at low glucose levels the natural regulation of glycogen metabolism favouring glycogenolysis is functional in the presence of the G L -GPi. The ability of direct GPis to increase glycogen levels with high efficacy even at 2.5 mM glucose is explained by their glucose-independent effects on GP activity and phosphorylation. This glucose independence observed with CP-320,626 is in line with a previous report showing that this compound inhibited liver GP in vitro and in primary rat hepatocytes independent of the glucose concentration [START_REF] Andersen | The effect of glucose on the potency of two distinct glycogen phosphorylase inhibitors[END_REF].

In this study we show that G L -GPi does not prevent the mobilization of glycogen because the activity of GP is not impaired. Based on this observation it is tempting to speculate that the risk of hypoglycaemia upon an inhibition of the G L -GP interaction could be lower compared to an inhibition of the GP enzyme. In addition, associated with a direct inhibition of GP are potential side effects in muscle. It has been shown that a pharmacological inhibition of GP impairs the aerobic muscle function during prolonged contraction [START_REF] Baker | The experimental type 2 diabetes therapy glycogen phosphorylase inhibition can impair aerobic muscle function during prolonged contraction[END_REF]. Furthermore, genetic deficiency in muscle glycogen phosphorylase, known as glycogenosis type V or McArdle's disease [START_REF] Gordon | Glycogenosis type V or McArdle's disease[END_REF], significantly reduces the exercise capacity in these patients. Because G L protein is also expressed in human muscle [START_REF] Munro | Human skeletal muscle expresses a glycogen-targeting subunit of PP1 that is identical to the insulin-sensitive glycogen-targeting subunit G(L) of liver[END_REF], G L -GP inhibitors could also be functional in this tissue. But since the inhibition of the G L -GP interaction allows glycogen mobilization, the detrimental effects in muscle are not foreseen for G L -GP inhibitors. Therefore, a compound working via this novel mechanism could potentially offer a better risk-benefit profile, however at this stage it is too early to predict whether this will provide clinical advantages.

In summary, we have identified an inhibitor of the G L -GP interaction and provide first evidence that the pharmacological prevention of the G L -GP interaction increases both glycogen synthesis and glycogen accumulation in primary rat hepatocytes. In contrast to direct GP inhibitors, these effects were achieved without inhibiting GP activity. Taken together, our study suggests that the interaction between G L and GP plays an important role in the regulation of the glycogen metabolism and supports the hypothesis that the prevention of this interaction might be an attractive novel anti-diabetic strategy.

Figure 4 G L -GPi does not influence enzymatic activity of GS

Recombinant mouse liver GS was treated with PP1 (25 U/ml) in the presence or absence of 17 mM sodium orthovanadate for 30 minutes at 30°C followed by determination of phosphorylation state of the enzyme using anti-phospho(Ser641)-GS antibody and anti-GS antibody as an internal control (A). In separate experiments, recombinant GS protein was treated with G L -GPi (0.3-100 µM) or catalytic subunit of PP1 (147 U/ml) as a positive control for 30 minutes at 30°C followed by the determination of GS activity at 0.1 and 10 mM glucose 6-phosphate (B, C). The activity of the GS was determined from the activity of [ 14 C]UDP-glucose incorporated into glycogen. Significances of differences are: ** p<0.01 and *** p<0.001.

Figure 5 Inactivation (A) and dephosphorylation (B, C) of glycogen phosphorylase by direct GPis but not G L -GPi

Primary rat hepatocytes were treated for 2 or 6 h with 10 µM G L -GPi, 3µM compound A, 10 µM CP-320,626 or 0.25% DMSO at 10 mM or 2.5 mM glucose. Cell lysates were analyzed with respect to GP activity (A) and GP phosphorylation (B, C) using anti-phospho(Ser 14 )-GP antibody (upper panel); anti-GP antibody ((B), bottom panel) were used as internal control.

Values of GP activity (A) and densitometric analysis of phosphoblots (C) are presented as means±SEM for three experiments, * p<0.05 vs. respective vehicle controls.

  Biochemical Journal Immediate Publication. Published on 26 Feb 2008 as manuscript BJ20071483 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071483 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 26 Feb 2008 as manuscript BJ20071483 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071483 S t a g e 2 ( a ) P O S T -P R I N T Both G L -GPi and direct GPis promote the dephosphorylation and activation of GS

Biochemical

  Journal Immediate Publication. Published on 26 Feb 2008 as manuscript BJ20071483 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071483 S t a g e 2 ( a ) P O S T -P R I N T Direct GPis but not G L -GPi reduce the amounts of GP in the glycogen fraction

  Journal Immediate Publication. Published on 26 Feb 2008 as manuscript BJ20071483 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071483 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1

 1 FIGURE LEGENDSFigure1Both G L -GPi and direct GPis increase glycogen synthesis in a dose-dependent manner Primary rat hepatocytes were cultured in Krebs-Ringer-bicarbonate/HEPES buffer containing 10 mM glucose and treated with compounds used in a range of concentrations for 2 h in the presence of [ 14 C]glucose. The rate of glycogen synthesis was determined from the activity of [ 14 C]glucose incorporated into glycogen. A representative of minimally three experiments is shown.

Figure 2 G

 2 Figure 2 G L -GPi modulates the storage of glycogen in a glucose-dependent mannerAfter overnight incubation at 5 mM glucose, primary rat hepatocytes were treated for 2 or 6 h with 10 µM G L -GPi, 3 µM compound A, 10 µM CP-320,626 or 0.25% DMSO (solvent control) at 10 mM (A) or 2.5 mM (B) glucose, and glycogen amounts were determined. Glycogen levels at 0 h time point represent the glycogen content prior to the compound application. Glycogen amounts are expressed as nmol glucose equivalents per mg protein content in the glycogen-enriched fraction used for the assay. Values are presented as means±SEM of four independent experiments performed in duplicate. Significances of differences are: ** p<0.01 and *** p<0.001.

Figure 3

 3 Figure 3 Dephosphorylation and activation of glycogen synthase by G L -GPi and GPisPrimary rat hepatocytes were treated for 2 or 6 h with G L -GPi (10 µM), compound A (3 µM), CP-320,626 (10 µM) or 0.25% DMSO at 10 mM or 2.5 mM glucose. Cells were harvested and the lysates were used for the determination of GS phosphorylation (A, B) and GS activity (C). The phosphorylation status was determined using anti-phospho(Ser 641 )-GS antibody and anti-GS antibody as an internal control. (B) Densitometric analysis of three phosphoblots. (C) Glycogen synthase activity. Data are expressed as the activity ratio defined as the ratio of the activity measured at 0.1 mM glucose 6-phosphate relative to activity in the presence of 10 mM glucose 6-phosphate. Values are presented as means±SEM of three experiments, *p<0.05 vs. respective vehicle controls.

Figure 6 Direct

 6 Figure 6 Direct GPis but not G L -GPi reduce amounts of GP in glycogen fractionGlycogen-enriched fractions obtained by differential centrifugation were analyzed by immunoblotting (A) with anti-GP antibody after the incubation of hepatocytes for 2 or 6 h with 10 µM G L -GPi, 3 µM compound A, 10 µM CP-320,626 or 0.25% DMSO at 10 mM or 2.5 mM glucose. Immunoblot analysis with anti-γ-tubulin antibody (A, bottom panel) using the same samples served as an internal control. (B) Relative amounts of GP in the glycogenenriched fractions corrected to the glycogen amounts in the same samples (GP/glycogen ratio). Values are presented as means±SEM of four independent experiments, * p<0.05 vs. respective vehicle control at 0h time point (state prior to the application of compounds), # p<0.05 vs. respective treatments with G L -GPi.

2 Biochemical

 2 Figure 1

Figure 3 G

 3 Figure 3

Figure 4 -

 4 Figure 4

Table 1 . Inhibition of G L -GPa interaction by G L -GPi and recombinant human liver GPa by direct GPis Interaction

 1 between biotin-labelled G L peptide and recombinant human liver [ 33 P]radiolabelled GPa was assessed by a scintillation proximity assay (SPA). Human liver GPa activity was measured in the direction of glycogenolysis. The IC 50 values given are means of pooled data from 4-6 experiments.

	______________________________________________________
	Compound	G L -GPa interaction	Human liver GPa
		(SPA assay), IC 50 , µM	IC 50 , µM
	______________________________________________________
	G L -GPi	0.15	> 30
	CP-320,626	1	1.4
	Compound A	> 30	2

_____________________________________________________________________________________________________________ Biochemical Journal Immediate Publication. Published on 26 Feb 2008 as manuscript BJ20071483 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071483 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 26 Feb 2008 as manuscript BJ20071483 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071483 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

We gratefully acknowledge Petra Wieland and René Schiller for their assistance with the biochemical assays, Sabine Mayer-Bolte for her help with the isolation of primary rat hepatocytes and Eva Schlegel for her help in the purification of recombinant proteins. We are also thankful to Patricia T. W. Cohen (University of Dundee, UK) for providing us the phospho-specific GP and GS antibodies and Kei Sakamoto (University of Dundee, UK) for the constructs and protocols for the expression of recombinant GS.


Verna Lavender

Setareh Chong

Katherine Ralphs

Adrian J Wolstenholme

Barbara J Reaves
email: b.j.reaves@bath.ac.uk

Dr Craig Montell

Increasing expression of calcium permeable TRPC3 and TRPC7 channels enhances constitutive secretion

Keywords: Transient receptor potential, cation channel trafficking, secretory pathway, calcium 2

The hTRPC (human transient receptor potential canonical) family of nonselective cation channels are proposed to mediate calcium influx across the plasma membrane via PLC-coupled receptors. Heterologously expressed hTRPC3 and hTRPC7 have been localised at the cell surface, however, a large intracellular component has also been noted but not characterised. In this study we have investigated the intracellular pool in COS-7 cells and have shown co-localisation with markers for both the trans-Golgi network (TGN) and the cis-Golgi cisternae by immunofluorescence microscopy. Addition of Brefeldin A to cells expressing hTRPC3 or hTRPC7 resulted in the redistribution of the Golgi component to the endoplasmic reticulum indicating this pool is present in both the Golgi stack and the TGN. Expression of either TRPC3 or TRPC7, but not TRPC1 or the cell surface marker CD8, resulted in a 2-4 fold increase of secreted alkaline phosphatase in the extracellular media. Based on these data, we propose that an additional function of these members of the hTRPC family may be to enhance secretion either by affecting transport through the Golgi stack or increasing fusion at the plasma membrane.

INTRODUCTION

The transient receptor potential (TRP) protein superfamily consists of calcium permeable non-selective cation channels first described in a Drosophila phototransduction mutant associated with a defect in light induced calcium entry [START_REF] Hardie | The trp gene is essential for a lightactivated Ca2+ channel in Drosophila photoreceptors[END_REF].

The number of mammalian homologues of Drosophila TRP (dTRP) that have been identified over the past decade have expanded into a superfamily which has been further divided into seven subfamilies (reviewed in [START_REF] Montell | Drosophila TRP channels[END_REF]). Except for the lack of a voltage sensor in S4, the structure all of the TRP channels resembles that of voltagegated ion channels; it includes six predicted transmembrane segments with N-terminal and C-terminal cytoplasmic tails and a re-entrant pore loop between S5 and S6. In addition, analogously to Kv channels, TRPs most likely form tetrameric structures.

In contrast to other ion channel families, the TRP family members are predominantly grouped according to sequence homology and are diverse in terms of modes of activation, ion selectivity, tissue distribution and function both between and within each subfamily. TRPC or 'classical' (sometimes referred to as canonical) TRPs are most closely related to dTRP and are also subdivided into a further 4 groups based on sequence similarity: TRPC1, and TRPC2 (a pseudogene in humans) in their own categories, TRPC4 and TRPC5 forming the third grouping and TRPC3, TRPC6 and TRPC7 forming the fourth subfamily. All of the TRPC channels open in response to activation of PLC which leads to the production of IP 3 and diacylglycerol (DAG).

The issue of whether (or which) TRPC family members are activated by IP 3 and subsequent depletion of intracellular stores, or an increase in DAG remains controversial [START_REF] Putney | Recent breakthroughs in the molecular mechanism of capacitative calcium entry (with thoughts on how we got here)[END_REF]. Alternatively, or in combination with the mechanisms described above, activation of calcium influx through TRP channels also occurs by insertion of a vesicular pool into the plasma membrane [START_REF] Cayouette | Exocytotic insertion of TRPC6 channels into the plasma membrane upon Gq Protein-coupled receptor activation[END_REF][START_REF] Bezzerides | Rapid vesicular translocation and insertion of TRP channels[END_REF][START_REF] Singh | VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx[END_REF].

Not only are there differences in tissue distribution, function and activation within TRP families, it is also becoming clear that there are differences in the subcellular distribution of these channels as well. Localization studies in conjunction with electrophysiological experiments have demonstrated the presence of both heterologously expressed and endogenous TRP proteins at the plasma as predicted by a role in calcium influx [START_REF] Hofmann | Subunit composition of mammalian transient receptor potential channels in living cells[END_REF]. However, it is apparent that large intracellular pools of TRP proteins also exist. As would be predicted from the insertional activation model, a number of studies report localisation of TRP channels including the C. elegans TRP, TRP-3 [START_REF] Xu | A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization[END_REF], TRPC3 [START_REF] Singh | VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx[END_REF], TRPV5 amd TRPV6 [START_REF] Hoenderop | Homo-and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6[END_REF], TRPC5 [START_REF] Bezzerides | Rapid vesicular translocation and insertion of TRP channels[END_REF] and TRPV1 [START_REF] Morenilla-Palao | Regulated exocytosis contributes to protein kinase C potentiation of vanilloid receptor activity[END_REF] to vesicular pools which then fuse with the plasma membrane after stimulation.

More surprisingly, TRPs have recently been implicated in endosome/lysosome function after the gene responsible for the neurogenetic disorder mucolipidosis type IV was cloned and found to encode the group 2 TRP, TRPML (mucolipin-1).

TRPML and its homologue in C. elegans, CUP-5, have both been localised to late endosomes/lysosomes and have been proposed to have a role either in lysosome biogenesis [START_REF] Piper | CUPpling calcium to lysosomal biogenesis[END_REF] and/or regulation of lysosomal [START_REF] Soyombo | TRP-ML1 regulates lysosomal pH and lysosomal lipid hydrolytic activity[END_REF]. Another subcellular compartment in which several types of calcium channels reside is the ER. Both folding and assembly of channels occurs in this compartment and consequently it has been difficult to establish whether TRP channels actually function in this organelle or are merely being assembled and transported; a recent report suggests that the cold/menthol-sensitive TRPM8 channel is not only resident in the ER but has a role as an ER calcium-release channel [START_REF] Thebault | Novel role of cold/menthol-sensitive TRPM8 in the activation of store-operated channels in LNCaP human prostate cancer epithelial cells[END_REF]. Three distinct locations, the ER, plasma membrane and Golgi complex, have been reported for another group 2 TRP, TRPP2 (polycystin-2), mutations in which cause polycystic kidney disease (reviewed in [START_REF] Kottgen | Subcellular localization and trafficking of polycystins[END_REF]).

Thus, in addition to their function as calcium influx channels at the plasma membrane, an increasing number of TRP channels have been demonstrated to act intracellularly in a variety of different organelles.

In this study, we present a detailed analysis of the subcellular localisation of the intracellular pools of hTRPC1, 3 and 7 and propose a novel role for both hTRPC3 and hTRPC7 in enhancing constitutive secretion. suspension was centrifuged at 3,000 x g for 15 minutes, 4 o C and the supernatant then centrifuged at 50,000 x g for 30 minutes. The pellet was resuspended in Buffer A and protein determinations conducted using the BioRad protein assay. 20 g of the crude membrane pellet was resolved on an 8% SDS Tris-glycine gel and the gel transferred to nitrocellulose. Monoclonal anti-myc 4A6 (Upstate, 1:1000) was used to detect hTRPC3 in COS-hTRPC3-1 cells and polyclonal anti-TRPC7 for detection in GH4C 1 cells (1:1000), followed by anti-mouse IgG-HRP or anti-rabbit IgG-HRP (Amersham Biosciences, LittleChalfont, Buckinghamsire, UK) respectively. Detection was carried out with ECL Western Blotting Detection reagent (Amersham, U.K.).

Densitometry was conducted using the Epi Chemi II Darkroom and captured luminescence imaged using LABWORKS software (UVP Lab Products, Cambridge, U.K.).

SEAP Assays

COS-7 cells and clonal COS-7 cells stably expressing N-terminal myc-tagged human TRPC3, were transiently transfected with pSEAP2-Control mammalian expression vector (BD Biosciences Clontech, Palo Alto, CA) using GeneJuice Transfection Reagent (Novagen, La Jolla, CA)for 24 hours followed by induction with 50 M ZnCl 2 for a further 24 hours. Samples of media were harvested at 48 and 72 hours post-transfection, and the level of the heat-stable secreted placental alkaline phosphatase in 15 l aliquots was determined using BD Great EscAPe SEAP Chemiluminescent assay (BD Biosciences Clontech, Palo Alto, CA). Assays were conducted in transparent 96-well flat-bottomed microtitre plates, and luminescence was detected for 10 s integrals using a FARcyte plate luminometer (Amersham Pharmacia Biotech, Little Chalfont, Bucks). COS-7 cells were also transiently cotransfected with pSEAP2-Control mammalian expression vector and secondary recombinant mammalian expression plasmids. Extracellular media was harvested and assayed as described above. Immunofluorescence microscopy was performed on the pSEAP2-Control transfected cells to ensure the transfection efficiency was constant between the different sample groups.

Immunocytochemistry

Indirect immunofluorescence microscopy was performed as described previously [START_REF] Reaves | Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGNspecific integral membrane protein, TGN38[END_REF]. Briefly, cells were washed with PBS and fixed for 20 min at room temperature using 2% (w/v) paraformaldehyde and permeablised for 5 min with methanol at -20°C or 0.1% (v/v) Triton X-100 at room temperature, blocked with 10% (v/v) FCS in PBS for 20 min followed by addition of primary antibody at the dilutions indicated above. After washing, secondary antibody was added and the cells incubated 20 min followed by washing and mounting with Mowiol® (Calbiochem).

When required, cultured cells were incubated with 5 ug/ml Brefeldin A and incubated for 2hrs at 37°C as previously described [START_REF] Reaves | Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGNspecific integral membrane protein, TGN38[END_REF].

Imaging

Statistical analysis

Results are presented as means±S.E.M. with the number of experiments indicated in the legend.

RESULTS

Localisation of transiently expressed hTRPC1, hTRPC3and hTRPC7 in COS-7 cells

A number of different approaches including immunocytochemistry have been used to analyse the expression and distribution of TRPC proteins in various tissues as well as heterologously expressing cell lines. Although expression at the plasma membrane, in vesicles and in the ER have been documented for many of the TRP family members, several reports allude to a large intracellular pool of hTRPC3 that also appears to colocalise with the Golgi complex [19][START_REF] Mckay | Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3[END_REF][START_REF] Vandebrouck | Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers[END_REF]; this observation, however, has not been characterised further. In order to determine the exact localisation of the intracellular pool of hTRPC3, we first investigated the localisation pattern of Nterminally tagged constructs of hTRPC1, hTRPC3 and hTRPC7 in COS-7 cells. We used this cell line since the flattened morphology and large size enables clear identification of intracellular compartments and structures.

We transiently transfected COS-7 cells with a full-length construct of hTRPC3 containing an N-terminal myc epitope (Myc-hTRPC3) [START_REF] Xu | Coassembly of TRP and TRPL produces a distinct store-operated conductance[END_REF]. As shown in Figure 1A (middle image), over-expressed Myc-hTRPC3 is concentrated in a perinuclear location as well as in punctate structures throughout the periphery and at the plasma membrane. A similar distribution was seen for FLAG-hTRPC7 (Figure 1A, last image), however a more distinctive plasma membrane localisation was also observed.

In contrast to the results obtained with hTRPC3 and hTRPC7, our results indicate that FLAG-hTRPC1 [START_REF] Xu | Coassembly of TRP and TRPL produces a distinct store-operated conductance[END_REF][START_REF] Wes | TRPC1, a human homolog of a Drosophila store-operated channel[END_REF] localises predominantly to the endoplasmic reticulum (ER) in COS-7 cells (Figure 1A, first image).

In order to determine whether the large intracellular pool of hTRPC3 is merely due to overexpression and consequent saturation within the exocytic pathway resulting in the Golgi-like distribution, COS-7 cells were stably transfected with Myc-hTRPC3 under the regulation of the inducible metallothionein promoter contained in the expression vector pMEP4 (COS-hTRPC3-1). Even under uninduced conditions some expression was detected by both immunofluorescence and immunoblotting (Figure 1B andC). However, at these low levels of expression the distribution of hTRPC3 in these cells is similar to that after transient transfection with little or no detectable signal apparent at the plasma membrane and the majority of signal appearing intracellularly (Figure 1B). With increasing expression levels, the concentration in the perinuclear area and in punctate structures increases without a concomitant increase at the cell surface. These data suggest that the perinuclear localisation is not simply due to overexpression resulting in an increased concentration in the secretory pathway.

In order to confirm the distribution data described above we also conducted cotransfection experiments with hTRPC1 and hTRPC3. Based on previous immunoprecipitation data [START_REF] Goel | Selective association of TRPC channel subunits in rat brain synaptosomes[END_REF], hTRPC1 and hTRPC3 would not be expected to interact and indeed the localisation of hTRPC1 did not appear to alter upon cotransfection with hTRPC3 [START_REF] Hofmann | Subunit composition of mammalian transient receptor potential channels in living cells[END_REF]; the intracellular distribution of hTRPC3, however, was not investigated in these experiments. In contrast, recent reports based on immunoprecipitation data suggest TRPC1 and TRPC3 will heteromultimerize [24].

Results presented in Figure 2A demonstrate that COS-7 cells co-transfected with hTRPC1 and hTRPC3 exhibited expression patterns for both proteins that resembled the pattern in singly-transfected cells. Minimal colocalization was apparent and the intracellular pool of hTRPC3 remained unaffected.

The intracellular pool of hTRPC3and hTRPC7 localise to both the trans-Golgi network (TGN) and the the Golgi stack

One model for TRP activation includes an insertion into the plasma membrane from an intracellular pool. There is a growing body of evidence to support this model (reviewed in [START_REF] Cayouette | Intracellular trafficking of TRP channels[END_REF]; however, although there is evidence for rapid insertion of TRPCs from a sub-plasmalemmal pool using TIRF microscopy [START_REF] Bezzerides | Rapid vesicular translocation and insertion of TRP channels[END_REF], the possibility of contributions by other intracellular pools to this process has not been investigated.

Recycling from the TGN through the plasma membrane has been well characterised for a number of proteins including TGN38 [START_REF] Reaves | Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGNspecific integral membrane protein, TGN38[END_REF], and furin [27]. Precedence for transport of receptors from the TGN to the cell surface upon stimulation has also been demonstrated for two members of the tumour necrosis factor receptor family, TNFR1

and Fas [START_REF] Storey | The p55 tumour necrosis factor receptor TNFR1 contains a trans-Golgi network localization signal in the C-terminal region of its cytoplasmic tail[END_REF]. Although the majority of the intracellular pool of hTRPC3 and hTRPC7 colocalised with TGN46, the co-localization was not complete. The possibility that some of the hTRPC3 concentrates in the Golgi stack was approached initially by conducting double labelling with hTRPC3 and a marker for the cis Golgi, Gpp130 [START_REF] Linstedt | Sequence and overexpression of GPP130/GIMPc: evidence for saturable pHsensitive targeting of a type II early Golgi membrane protein[END_REF]. As for TGN46, there was also extensive co-localization of hTRPC3 (Figure 3, top panel) with Gpp130. This approach, however, does not provide definitive means for the dissection of the TGN from the Golgi stack. Brefeldin A (BFA) is a fungal metabolite which inhibits ADF-ribosylation factors and consequently coat formation; the consequence of this inhibition is the disassembly and redistribution of the Golgi complex. In contrast to the TGN which collapses to a small dot around the microtubule organizing centre (MTOC), BFA causes the Golgi stack to be redistributed into the ER in COS-7 cells [START_REF] Reaves | Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGNspecific integral membrane protein, TGN38[END_REF]. Surprisingly, after BFA treatment, hTRPC3 and hTRPC7 clearly redistribute with Gpp130 into the ER (Figure 3 panel 2 & 4) whereas TGN46 appeared in the classic 'BFA dot' (Figure 3, panel 3). This indicates that hTRPC3 localises not only to the TGN but also to the Golgi stack.

Endogenous TRPC7 localises to the Golgi complex in a rat pituitary cell line, GH 4 C 1

A key question concerning possible effects of overexpression in transfected cells on subcellular distribution prompted us to examine various cell lines to see if we could detect endogenous protein using immunofluorescence microscopy.

Unfortunately, none of the cell lines tested gave an unequivocal signal with either a commercially available or our own polyclonal anti-hTRPC3 antibody. However, as TRPC7 appears to have a similar distribution to TRPC3 and TRPC7 mRNA levels have been reported be highest in pituitary [START_REF] Riccio | mRNA distribution analysis of human TRPC family in CNS and peripheral tissues[END_REF], we used a polyclonal anti-TRPC7 antibody raised to the extreme C-terminus [START_REF] Sekaran | 2-Aminoethoxydiphenylborane is an acute inhibitor of directly photosensitive retinal ganglion cell activity In Vitro and In Vivo[END_REF] to assess distribution in a rat pituitary cell line, GH 4 C 1 . Consistent with the perinuclear concentration in COS-7 cells expressing FLAG-hTRPC7, the endogenous protein was detected in a Golgi-like area close to the nucleus (Figure 4A). Double-labelling experiments with the cis-Golgi This supports the finding that the signal detected by immunofluroescence was indeed TRPC7.

Expression of either hTRPC3 or hTRPC7, but not hTRPC1, enhances constitutive secretion

As a central organelle of the secretory pathway, one of the major functions of the Golgi complex is in sorting and modifying proteins destined for either the plasma membrane, endosomal compartments or specialized secretory granules. These processes are dependent on both the formation of vesicles and the fusion of these vesicles with a target membrane. Several studies implicate a role for lumenal calcium in organellar fusion events, particularly endosome/lysosome fusion [32]. We therefore reasoned that one possible consequence of having a calcium channel in the Golgi complex might be to increase vesicle fusion and therefore constitutive secretion. In order to test this hypothesis, COS-7 control or COS-hTRPC3-1 cells were transfected with a plasmid encoding a secreted form of alkaline phosphatase (SEAP) [START_REF] Towler | The manganese cation disrupts membrane dynamics along the secretory pathway[END_REF]. The newly synthesised enzyme is translocated into the lumen of the ER, transported constitutively through the Golgi complex and then targeted for secretion at the plasma membrane. The amount of alkaline phosphatase present in the cell culture medium of untransfected control, untransfected control under inducing conditions (50 M ZnCl 2 ), uninduced or induced COS-hTRPC3 was measured 48 hours after transfection. As indicated in Figure 1C, hTRPC3 is expressed at low levels in this cell line even in the absence of induction. The 2-fold increase in secretion of the enzyme over control even in uninduced cells suggests that the presence of hTRPC3 has a significant effect on transport through the secretory pathway. A 3-fold increase was observed after induction of COS-hTRPC3 cells compared to control cells treated with 50 M ZnCl 2 ; there was also a significant increase when compared to uninduced cells (Figure 5A).

To examine whether this was specific for hTRPC3, we conducted similar experiments using hTRPC1, hTRPC7 and a resident cell surface protein that normally traffics to the plasma membrane through the Golgi complex, CD8. In the absence of stable cell lines expressing these proteins it was necessary to carry out these studies on transiently transfected cells. In our hands, co-transfections often result in cells not only co-expressing but also expressing either one or the other protein. Hence, for all experiments, the number of cells expressing both the protein of interest and SEAP was determined by immunofluorescence to ensure comparable levels of both cotransfected and singly transfected cells (data not shown). Expression of each construct was compared to the expression of eGFP-N1 to control for effects of transfection alone. As can be seen in Figure 5B, expression of neither CD8 nor hTRPC1 had significant effects on the amount of SEAP in the medium; however, the expression of both hTRPC3 and its closely related family member, hTRPC7 increased constitutive secretion by at least 2.5 fold, and almost 3.5 fold in the case of hTRPC3.

These findings suggest that not only are hTRPC3 and hTRPC7 expressed in the Golgi complex but they also have a significant effect on a major function of this organelle, constitutive secretion. and 6 were clearly localised to the plasma membrane, however TRPC3 in particular was found almost exclusively intracellularly in perinuclear structures reminiscent of the Golgi [START_REF] Vandebrouck | Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers[END_REF]. We have characterised the distribution of hTRPC3 using double-label immunofluorescence microscopy in COS-7 cells and demonstrate that: 1) a major pool of hTRPC3 colocalises with markers for both the TGN and the Golgi stack, 2) it differs from hTRPC1 which is also found predominantly intracellularly but remains associated with the ER, and 3) a large proportion of the perinuclear pool redistributes after Brefeldin A treatment suggesting it localises throughout the Golgi stack. A similar distribution pattern is observed for hTRPC7; considering the close sequence homology of these subfamily members and their ability to interact as demonstrated by co-immunoprecipitation experiments this is not surprising [START_REF] Goel | Selective association of TRPC channel subunits in rat brain synaptosomes[END_REF][START_REF] Vannier | The membrane topology of human transient receptor potential 3 as inferred from glycosylation-scanning mutagenesis and epitope immunocytochemistry[END_REF]. Although previous studies include evidence for TRPC1 on the plasma membrane, our results show a completely intracellular distribution which is consistent with results from subcellular fractionation of endogenous TRPC1 in platelets [36]and sperm [19] and overexpressed TRPC1 in HEK293 cells [START_REF] Hofmann | Subunit composition of mammalian transient receptor potential channels in living cells[END_REF]. Potential ER exit motifs are present within the amino acid sequence of TRPC3/7 but are lacking in TRPC1. Putative ER retention signals are also present in TRPC1; these are now being investigated to determine whether the difference in trafficking between these subfamily members is due to specific targeting signals.

The best characterised trafficking routes for proteins recycling through the plasma membrane back to an intracellular store is to and from the TGN or from the TGN through the plasma membrane to recycling endosomes or specialised pools such as for the glucose transporter, GLUT4, or neurotransmitters. Hence, our first hypothesis as to a possible function for the TGN/Golgi-localised pool of hTRPC3 was as a 'readily releasable' storage pool. A number of studies have recently demonstrated translocation of a variety of TRP family members from an intracellular pool to the plasma membrane including a C. elegans TRPC homolog, TRP-3 [START_REF] Xu | A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization[END_REF], TRPV1 [START_REF] Morenilla-Palao | Regulated exocytosis contributes to protein kinase C potentiation of vanilloid receptor activity[END_REF],

TRPC3 [START_REF] Singh | Trp1-dependent enhancement of salivary gland fluid secretion: role of store-operated calcium entry[END_REF] and TRPC5 [START_REF] Bezzerides | Rapid vesicular translocation and insertion of TRP channels[END_REF]. Most of these studies postulate that the translocation occurs from a vesicular pool close to the plasma membrane. Our finding that the majority of the intracellular pool was localised in a perinuclear localisation overlapping the Golgi stack was therefore surprising. An intriguing possibility, however, is that the translocatable pool is at least partially Golgi-derived. [START_REF] Singh | VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx[END_REF] clearly demonstrate a decrease in the agonist stimulated plasma membrane localisation of TRPC3 after treatment of cells for 30-60 minutes with Brefeldin A, a compound which disrupts the Golgi stack and intra-Golgi transport but does not inhibit post-Golgi transport to the cell surface [START_REF] Klausner | Brefeldin A: insights into the control of membrane traffic and organelle structure[END_REF]. Since the pool of TRPC3 that was being translocated was reported to be a subplasmalemmal vesicular pool, BFA should not have inhibited this translocation step but it would inhibit translocation from the Golgi stack. Precedence for transport occurring along this trafficking route in response to external stimuli comes from studies on the stimulation of Fas from the Golgi to the cell surface which occurs upon stimulation by p53 [START_REF] Bennett | Cell surface trafficking of Fas: a rapid mechanism of p53-mediated apoptosis[END_REF]. Although this could be one possibility for our finding that a large pool of TRPC3 resides in the Golgi stack, it raised the question as to whether TRPC3 may have functions in the Golgi in addition to, or instead of, acting as a calcium entry channel at the plasma membrane.

A variety of Ca 2+ -dependent functions occur within the Golgi which include posttranslational modifications of secretory proteins [START_REF] Austin | Prosomatostatin processing in permeabilized cells. Calcium is required for prohormone cleavage but not formation of nascent secretory vesicles[END_REF], formation of COPI coats on Golgi-ER transport vesicles [START_REF] Ahluwalia | A role for calcium in stabilizing transport vesicle coats[END_REF] and both anterograde and retrograde vesicular transport through and to the Golgi complex [START_REF] Porat | Regulation of intra-Golgi membrane transport by calcium[END_REF][START_REF] Chen | Selective effects of calcium chelators on anterograde and retrograde protein transport in the cell[END_REF]. In addition to having a role in coat formation, release of lumenal calcium from vesicles has also been suggested to play a role in vesicle fusion as shown for insulin containing secretory granules with the plasma membrane [START_REF] Mitchell | Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicleassociated membrane protein aequorin chimaera[END_REF]. Thus, we hypothesised that an alternative function for TRPC3 could be mediating vesicle fusion, either between the ER and the cis-Golgi, transport from the trans-Golgi/TGN and/or increasing fusion at the plasma membrane.

Indeed, TRPC3 has already been shown to interact by yeast two hybrid with VAMP2, NSF and SNAP [START_REF] Singh | VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx[END_REF] which normally reside in vesicles and play a role in vesicle fusion. TRPM7 has also recently been localised to synaptic vesicle membranes where it forms complexes with SNAREs and their associated proteins, e.g. snapin, as well as the scaffolding protein synapsin. Synapsin has been postulated to anchor vesicles in a

DISCUSSION

Despite an increasing number of studies focusing on hTRPC3 function and localisation, an in-depth analysis of its subcellular distribution has not previously been conducted. The initial hypothesis that TRPCs function as store-operated calcium channels predicts a plasma membrane localisation for the TRPC family. However a large intracellular pool of hTRPC3 is apparent in many immunofluorescence micrographs [19][START_REF] Mckay | Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3[END_REF][START_REF] Vandebrouck | Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers[END_REF][START_REF] Wang | Cloning of Trp1beta isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein[END_REF]. Most dramatically, in immunofluorescence studies on the expression of TRPC isoforms in mouse skeletal muscle fibers, endogenous TRPC1,4 and 6 were clearly localised to the plasma membrane, however TRPC3 in particular was found almost exclusively intracellularly in perinuclear structures reminiscent of the Golgi [START_REF] Vandebrouck | Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers[END_REF]. We have characterised the distribution of hTRPC3 using double-label immunofluorescence microscopy in COS-7 cells and demonstrate that: 1) a major pool of hTRPC3 colocalises with markers for both the TGN and the Golgi stack, 2) it differs from hTRPC1 which is also found predominantly intracellularly but remains associated with the ER, and 3) a large proportion of the perinuclear pool redistributes after Brefeldin A treatment suggesting it localises throughout the Golgi stack. A similar distribution pattern is observed for hTRPC7; considering the close sequence homology of these subfamily members and their ability to interact as demonstrated by co-immunoprecipitation experiments this is not surprising [START_REF] Goel | Selective association of TRPC channel subunits in rat brain synaptosomes[END_REF][START_REF] Vannier | The membrane topology of human transient receptor potential 3 as inferred from glycosylation-scanning mutagenesis and epitope immunocytochemistry[END_REF]. Although previous studies include evidence for TRPC1 on the plasma membrane, our results show a completely intracellular distribution which is consistent with results from subcellular fractionation of endogenous TRPC1 in platelets [36]and sperm [19] and overexpressed TRPC1 in HEK293 cells [START_REF] Hofmann | Subunit composition of mammalian transient receptor potential channels in living cells[END_REF]. Potential ER exit motifs are present within the amino acid sequence of TRPC3/7 but are lacking in TRPC1. Putative ER retention signals are also present in TRPC1; these are now being investigated to determine whether the difference in trafficking between these subfamily members is due to specific targeting signals.

The best characterised trafficking routes for proteins recycling through the plasma membrane back to an intracellular store is to and from the TGN or from the TGN through the plasma membrane to recycling endosomes or specialised pools such as for the glucose transporter, GLUT4, or neurotransmitters. Hence, our first hypothesis as to a possible function for the TGN/Golgi-localised pool of hTRPC3 was as a 'readily releasable' storage pool. A number of studies have recently demonstrated translocation of a variety of TRP family members from an intracellular pool to the effective than EGTA at inhibiting many of the organellar fusion and transport events within the cell most likely due to a much faster on rate for calcium binding, it has been proposed that calcium transients or gradients are more important than steady state calcium levels. This would also suggest the necessity for either a vesicular or organellar calcium release channel which could mediate rapid but localised increases in calcium; TRPC3 could fulfil this function. Since a number of agents which affect the ability of TRPC3 to mediate calcium entry at the plasma membrane are also integral to the Golgi complex, e.g. DAG, PLC , and PLD, an intriguing possibility is that these agents may affect channel function at both locations. It has recently been

shown that diacylgycerol levels in the Golgi are critical for Golgi secretory function [START_REF] Litvak | Maintenance of the diacylglycerol level in the Golgi apparatus by the Nir2 protein is critical for Golgi secretory function[END_REF]. DAG also activates mitochondrial cationic channels and elicits release of sequestered calcium in this organelle [48] so it is possible that this may also occur in the Golgi mediated via TRP channels. Indeed, release of Golgi calcium in response to menthol, an activator of TRPM8, has recently been reported but surprisingly this response could also be elicited in cells without endogenous TRPM8 suggesting that other TRP channels may be responsible for this release from Golgi stores [49].

TRPC3/7 could be ideal candidates.

Biochemical

  Journal Immediate Publication. Published on 02 May 2008 as manuscript BJ20071488 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071488 S t a g e 2 ( a ) P O S T -P R I N T 100 U/ml penicillin-streptomycin. Transfections were performed using Gene Juice transfection reagent (Novagen, UK) using 1 g of construct DNA except for cotransfections wherein 0.5 g of each plasmid was used. Further manipulations of the cells were carried out 24 hours post-transfection unless otherwise specified. For the generation of stable cell lines, COS-7 cells were transfected with the inducible expression vector pMep4-hTRPC3 and stable cell lines isolated after selection in 50 ug/ml hygromycin B (Sigma) for approximately two weeks. Three clones were chosen for further study based on differential levels of expression in uninduced versus induced conditions. For inducible expression, 50 ZnCl 2 was added to the media and cells incubated for a further 24-48 hours prior to processing for immunocytochemistry or immunoblotting. The rat pituitary cell line, GH4C1, was a kind gift of Professor Priscilla Dannies, Department of Pharmacology, Yale University and was grown in a 1:1 mixture of DMEM and Ham's F-10 media supplemented with 15% horse serum. Protein Extraction and Immunoblotting Confluent cells were harvested by scraping on ice into lysis buffer consisting of 100 mM Tris HCl, pH 8.0, 1 mM MgCl 2, 0.1 mM PMSF and a protease inhibitor cocktail (Roche, Complete Mini, EDTA-free) (Buffer A) and frozen at -80 o C until use. Frozen cells were thawed on ice, homogenized in a Dounce homogenizer and diluted in 0.25M sucrose, 10 mM Tris-HEPES, pH 7.4 containing protease inhibitors. The

  Photoshop 5.0 (Adobe Systems, Mountain View, CA).

  It was therefore of interest to determine whether the perinuclear localisation of hTRPC3 and hTRPC7 corresponded to the TGN using doubleimmunofluorescence labelling of transiently transfected COS-7 cells with a marker for the TGN, TGN46 [29]. For comparison, cells were also transfected with hTRPC1 and processed for IF using a monoclonal anti-FLAG antibody and sheep anti-TGN46. Limited if any co-localisation was detected for hTRPC1 and TGN46 10 Biochemical Journal Immediate Publication. Published on 02 May 2008 as manuscript BJ20071488 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071488 S t a g e 2 ( a ) P O S T -P R I N T (Figure 2B, top panel), however a high degree of colocalisation was seen between both hTRPC3 and hTRPC7 and TGN46 in the perinuclear region (Figure 2B, middle and lower panel). This data along with the co-transfection experiment supports the conclusion that these TRPC3/7 and TRPC1 isoforms reside in separate intracellular compartments.

Biochemical

  Journal Immediate Publication. Published on 02 May 2008 as manuscript BJ20071488 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071488 S t a g e 2 ( a ) P O S T -P R I N T marker -mannosidase II, indicate a high degree of colocalisation although not complete overlap. Immunoblotting of whole cell lysate revealed a positive signal at approximately 100kD corresponding to the same band detected in HEK293 cells overexpressing hTRPC7 which is not detected in untransfected cells (Figure 4B).

Biochemical

  Journal Immediate Publication. Published on 02 May 2008 as manuscript BJ20071488 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071488 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 02 May 2008 as manuscript BJ20071488 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071488 S t a g e 2 ( a ) P O S T -P R I N T DISCUSSION Despite an increasing number of studies focusing on hTRPC3 function and localisation, an in-depth analysis of its subcellular distribution has not previously been conducted. The initial hypothesis that TRPCs function as store-operated calcium channels predicts a plasma membrane localisation for the TRPC family. However a large intracellular pool of hTRPC3 is apparent in many immunofluorescence micrographs [19-21, 34]. Most dramatically, in immunofluorescence studies on the expression of TRPC isoforms in mouse skeletal muscle fibers, endogenous TRPC1,4

Figure 1 .

 1 Figure 1. Expression of FLAG-hTRPC1, Myc-hTRPC3 and FLAG-hTRPC7 in COS-7 cells. A) COS-7 cells transiently expressing FLAG-hTRPC1, Myc-hTRPC3 or FLAG-hTRPC7 were processed for immunofluorescence microscopy using antibodies to the epitope tags. B) A clonal cell line expressing myc-hTRPC3 downstream of the metallothionein promoter (COS-hTRPC3-1) was grown for 48 hours either in the absence (uninduced) or presence of 10 M or 50 M ZnCl 2 to induce increasing levels of expression. Cells were fixed and processed for immunofluorescence microscopy using a monoclonal antibody to the myc epitope tag. C) Untransfected COS-7 and COS-hTRPC3-1 cells were treated as in (B) and processed for immunoblotting. The immunoblot obtained using a monoclonal anti-myc antibody is shown on the left. The filled arrow indicates the position of hTRPC3 which is absent in the untransfected cells. The dashed arrow indicates the position of a cross-reacting band which appears in the untransfected cells. Increases in expression levels were quantitated and are expressed as arbitraty units in the right hand panel. Bars, 10 m.

Figure 2 .

 2 Figure 2. (A) Co-expression of TRPC1 and TRPC3 do not alter their subcellular distribution. COS-7 cells were co-transfected with both Flag-hTRPC1 and Myc-hTRPC3 constructs. Distribution of the tagged proteins was determined using a monoclonal anti-Flag antibody and a rabbit polyclonal antiserum to TRPC3. Coexpression of the two constructs did not result in redistribution of either isoform. (B) hTRPC3 and hTRPC7, but not hTRPC1 colocalise with TGN46. Double-label immunofluorescence studies were performed on COS-7cells transiently transfected with hTRPC1 (top panel) using a monoclonal antibody to the FLAG epitope tag and a sheep polyclonal antibody to a marker of the trans-Golgi network (TGN), TGN46. Minimal overlap is seen as represented by a lack of yellow in the merged image. Cells transiently expressing hTRPC3 (middle panel) and hTRPC7 (bottom panel) were processed for immunofluorescence microscopy using a mouse anti-myc epitope antibody 9E10 (hTRPC3) or monoclonal anti-FLAG (hTRPC7) and sheep anti-TGN46 (middle panels). Overlap between hTRPC3 and both TGN46 and Gpp130 is seen in yellow in the merged images. Bar, 10 m

Figure 3 .Figure 4 .Figure 5 .

 345 Figure 3. hTRPC3 and hTRPC7 localise to the Golgi stack. COS cells transiently transfected with Myc-hTRPC3 or FLAG-hTRPC7 were treated with (bottom 3 panels, 'Brefeldin A') or without (top panel, 'control') 5 g/ml BFA for 2 hours, fixed and processed for immunofluorescence microscopy using monoclonal antibodies to either the myc-or FLAG-epitopes respectively and either rabbit anti-Gpp130 or sheep anti-TGN46 as indicated. Overlap between the redistributed Myc-hTRPC3 and Gpp130 is shown in yellow in the merged image. Bar, 10 mFigure 4. Endogenous TRPC7 colocalises with a marker for the cis-Golgi, mannosidase II, in a rat pituitary cell line GH 4 C 1 . A) GH 4 C 1 cells were fixed and processed for immunocytochemistry using a polyclonal rabbit anti-hTRPC7 antibody detected by a goat anti-rabbit Alexa 488 (green) and a monoclonal anti-mannosidase II antibody detected by a goat anti-mouse Alexa 594 (red). Overlap is depicted by yellow (merge). B) Immunoblot demonstrating the specificity of the polyclonal rabbit antisera raised to TRPC7. Whole cell lysate from either untransfected (negative control) or hTRPC7-transfected HEK (positive control) and GH 4 C 1 cells were subjected to SDS-PAGE and then immunoblotted using the affinity-purified anti-TRPC7 antiserum. A band of approximately 100kD is detected in both the hTRPC7 transfected HEK cells and the GH 4 C 1 cells but is absent in the untransfected controls. Bar, 10 m Figure 5. hTRPC3 and hTRPC7, but not hTRPC1, increase constitutive secretion. A) Control untransfected and COS-hTRPC3-1 cells were transfected with a construct which expresses a soluble secreted form of alkaline phosphatase. At 24 hours posttransfection they were cultured in the presence or absence of 50 ZnCl 2 for a further 48 hours and assayed in triplicate for SEAP activity. The data are presented as a percentage of enzyme activity (luminescence) in cells expressing SEAP alone. The results are means +/-S.E.M. for three independent experiments. B) COS-7 cells were co-transfected with the SEAP vector and EGFP-N1, CD8, FLAG-hTRPC1, eEGFP-TRPC3 or FLAG-hTRPC7 and grown 48 hours prior to assaying for SEAP activity. Values are presented as a percentage of the total enzyme activity (luminescence) in the cells expressing eGFP. The results are means +/-S.E.M. for three independent experiments.

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Biochemical Journal Immediate Publication. Published on 02 May 2008 as manuscript BJ20071488 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071488 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

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ACKNOWLEDGEMENTS

The authors would like to thank Dr. Craig Montell for the generous gift of the hTRPC1 and hTRPC3 constructs and Professor J. Paul Luzio for kindly supplying the 9E10 ascites, helpful discussions and much appreciated support. This work was supported by The Wellcome Trust, Grant #069934.