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L-Type Calcium Channels in Insulin-Secreting Cells: Biochemical Characterization and Phosphorylation in RINm5F Cells

Pharmazeutisches Institut, Lehrstuhl Pharmakologie (H.S., U.K., A.B., M.R., H.P.T.A.) Universität Tübingen D-72076 Tübingen, Germany
Max Delbrück Centrum für Molekulare Medizin (H.H., I.M.) Cardiology D-13125 Berlin, Germany
Pfizer (M.K.A.) Central Research Division Groton, Connecticut 06340
Institut für Biochemische Pharmakologie (J.S., M.F., T.N.C.) Universität Innsbruck A-6020 Innsbruck, Austria

	ABSTRACT

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Opening of dihydropyridine-sensitive voltage-dependent L-type Ca²⁺-channels (LTCCs) represents the final common pathway for insulin secretion in pancreatic ß-cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[³H]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that 1C subunits (199 kDa, short form) contribute only a minor portion of the total 1 immunoreactivity in membranes and partially purified Ca²⁺-channel preparations. However, 1C forms a major constituent of (+)-[³H]isradipine-labeled LTCCs as 54% of solubilized (+)-[³H]isradipine-binding activity was specifically immunoprecipitated by 1C antibodies. Phosphorylation of immunopurified 1C with cAMP-dependent protein kinase revealed the existence of an additional 240-kDa species (long form), that remained undetected in Western blots. Fifty seven percent of labeled LTCCs were immunoprecipitated by an anti-ß-antibody directed against all known ß-subunits. Isoform-specific antibodies revealed that these mainly corresponded to ß1b- and ß3-subunits. We found ß2- and ß4-subunits to be major constituents of cardiac and brain L-type channels, respectively, but not part of L-type channels in RINm5F cells. We conclude that 1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as a substrate for cAMP-dependent protein kinase. ß1b- and ß3-Subunits were also found to associate with L-type channels in these cells. These isoforms may therefore represent biochemical targets for the modulation of LTCC activity in RINm5F cells.

	INTRODUCTION

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Voltage-dependent Ca²⁺ channels mediate depolarization-induced influx of Ca²⁺ into a variety of electrically excitable cells, including pancreatic ß-cells. Opening of these channels represents the final common pathway for insulin secretion triggered by the major insulin secretagogues glucose, amino acids, and sulfonylureas (for review see Ref.1). Voltage-dependent Ca²⁺ channels therefore represent an important physiological target for the initiation and modulation of insulin release (1, 2). Like in neurons and cardiac muscle, major biochemical pathways of channel modulation include the activation of pertussis toxin-sensitive G proteins (3, 4, 5, 6) or phosphorylation/dephosphorylation processes (1, 7, 8).

Based on different biophysical and pharmacological properties, at least six different channel types can be distinguished. Insulin secretion from pancreatic ß-cells is tightly coupled to Ca²⁺ influx through so-called L-type channels (LTCCs) (1). This channel type is activated by strong depolarizations and selectively blocked by low concentrations of Ca²⁺ antagonist drugs, e.g. the dihydropyridines (DHPs) nifedipine or isradipine (9, 10). DHPs reversibly bind to the LTCC complex in various insulin-secreting cell lines with high affinity (11, 12) and are potent blockers of insulin secretion (1, 12, 13).

LTCCs in brain and muscle tissues exist as hetero-oligomeric complexes of at least an 1-, an 2--, and a ß-subunit (9, 14). The 1-subunits form the ion-conducting pore and determine the channel’s sensitivity for DHPs. From the six cloned 1-subunit genes (termed 1A through 1E and 1S) only 1C, 1D and 1S participate in the formation of DHP-sensitive Ca²⁺ channels (for review and nomenclature see Ref.15). From the four known ß-subunit isoforms, only ß1a- (in skeletal muscle) and ß2-subunits (in heart) have so far been found to be associated with LTCCs (16, 17). ß-Subunits are critical for 1 function and determine the properties of the channel in a ß-isoform-specific manner (18, 19, 20).

Although 1C- and 1D-transcripts (21, 22, 23, 24), as well as transcripts from mammalian ß-subunits [mainly, ß1b, ß2, and ß3 (24, 25, 26, 27)], were identified in insulin-secreting cells, no biochemical analysis of the LTCC complex has yet been reported. Knowledge of the subunit composition of LTCCs in insulin-secreting cells is an important prerequisite for the generation of cell lines heterologously expressing channels with the functional and pharmacological properties of LTCCs in pancreatic ß-cells. Such cell lines would be of great value to develop drugs that selectively modulate (neuro)endocrine LTCCs. For example, ß-cell LTCC activators (28) could be very useful in stimulating insulin secretion in type II diabetes.

Here we present a biochemical analysis of the subunit composition of LTCCs in a (neuro)endocrine, insulin-secreting cell line, RINm5F cells. With the help of subunit-specific antibodies, we demonstrate that 1C-subunits, together with ß- and 2--subunits, represent a major constituent of DHP-labeled LTCCs channels in these cells. From the four ß-subunit isoforms only ß1b and ß3 were found as part of the LTCC complexes.

We also demonstrate the existence of 1C in two lengths. Its long (full- length) form was identified as a substrate for in vitro phosphorylation by cAMP-dependent protein kinase (cA-PK). As in neurons and muscle cells, posttranslational processing of 1C may therefore play an important physiological role for LTCC modulation in insulin-secreting cells. Our results provide the basis for further studies assessing the role of these subunits for channel function and stimulus-secretion coupling in pancreatic ß-cells. ß1b- and ß3-subunits must be considered the major candidates for coexpression with 1-subunits to construct stable cell lines expressing neuroendocrine LTCCs.

	RESULTS

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Different Classes of 1-Subunit Polypeptides Copurify with (+)-[³H]Isradipine-Labeled LTCC Complexes
To identify polypeptides forming LTCCs in RINm5F cells, we prepared sequence-directed antibodies against Ca²⁺ channel 1-, 2--, and ß-subunits. 1-Antibodies were directed against peptides corresponding to a sequence conserved in all known 1-subunits (anti-1) as well as to a sequence unique for 1C (anti-1C). We used these antibodies to identify 1 immunoreactivity copurifying with LTCCs on wheat germ agglutinin (WGA)-Sepharose (29, 30). Membrane-bound channels were prelabeled with (+)-[³H]isradipine, an L-type selective Ca²⁺ channel blocker. In our preparations (+)-[³H]isradipine labeled 513 ± 33 fmol of receptor sites per mg of membrane protein (n = 3) with subnanomolar K_d and a pharmacological profile typical for LTCC (11). Accordingly, the Ca²⁺ antagonist (+)-tetrandrine (31) stimulated binding to 131 ± 7% (n = 4) of control values. For channel purification, (+)-[³H]isradipine- prelabeled membranes were solubilized with 1% (wt/vol) digitonin. Solubilized binding activity was adsorbed to WGA-Sepharose and, after extensive washing, biospecifically eluted with N-acetyl-D-glucosamine. Specific binding activity was absent in the column flow-through but increased 7-to 15-fold (range, n = 6) with respect to the digitonin extract in the column eluate. No specific binding activity was retained on the resin (not shown), suggesting that complete elution was achieved.

Figure 1 shows that the non-subtype-selective antibody anti-1 recognized two diffuse bands in membranes as well as in the digitonin extract. The bands migrated slightly above and below the prestained myosin marker, with apparent molecular masses of 204 ± 3 and 174 ± 3 kDa (n = 6), respectively. This staining pattern was highly reproducible among different membrane preparations (n = 3). Staining was completely eliminated after the affinity-purified antibody was blocked with the antigenic peptide (1 µM, Fig. 1A). The two bands were absent in the column flow-through but enriched in the eluate with respect to the digitonin extract (see legend to Fig. 1).

View larger version (99K): [in this window] [in a new window]   Figure 1. 1-Subunit Immunostaining in RINm5F Cell Membranes (+)-[3H]isradipine-binding activity from RINm5F cells was partially purified by WGA-Sepharose affinity chromatography as described in Materials and Methods. The following protein samples were separated on a 8% polyacrylamide SDS gel and processed for immunostaining with affinity-pufified anti-1 (A) or anti-1C (B) antibodies: RINm5F membranes (MEM, 0.017 mg/lane); solubilized membranes (SOL, 0.012 mg/lane), WGA-Sepharose column flow-through (FT, 0.020 mg/lane), WGA-Sepharose eluate (EL, 0.003 mg/lane). Nonspecific staining (+) was determined after preincubation of antibodies with 1 µM antigenic peptide. The migration of molecular mass markers (200, 94, and 68 kDa) is indicated on the right. The migration of specifically stained bands enriched in the WGA-Sepharose eluates is indicated by arrows.

View larger version (50K): [in this window] [in a new window]   Figure 5. Immunopurification and Phosphorylation of RINm5F 1C Immunopurification using a double-immunoprecipitation protocol was performed as described in Materials and Methods. After a first immunoprecipitation step with anti-1C (left lane) or anti-2 (right lane), protein A-bound proteins were phosphorlyated with cA-PK followed by immunoprecipitation with anti-1. Antibody-bound phosphorylated proteins were then separated on a 7% polyacrylamide gel and visualized by autoradiography of the fixed and dried gel (12 h exposure time). The open arrow illustrates the migration of a phosphorylated chimeric 1-construct with a calculated molecular mass of 240 kDa that was phosphorylated in parallel. It corresponds to a previously characterized 1-chimera (chimera TM-D in Ref. 32). After heterologous expression in Saccharomyces cerevisiae, membranes were prepared (32) and subjected to immunopurification and phosphorylation as described for RINm5F membranes. Molecular mass standards are as in Fig. 1.

View larger version (87K): [in this window] [in a new window]   Figure 2. 2-Subunit Immunostaining in RINm5F Cell Membranes (+)-[3H]isradipine-binding activity from rabbit skeletal muscle and RINm5F cells was partially purified by WGA-Sepharose affinity chromatography as described in Materials and Methods. The following protein samples were separated on a 8% polyacrylamide SDS-gel and processed for immunostaining with affinity-purified anti-2 antibodies: WGA-Sepharose eluate/skeletal muscle (EL/S; 0.006 mg/lane); RINm5F membranes (MEM, 0.011 mg/lane); WGA-Sepharose eluate/RINm5F (EL, 0.005 mg/lane). Nonspecific staining (+) was determined after preincubation of antibodies with 1 µM antigenic peptide. The migration of molecular mass markers (200, 94, 68, and 45 kDa) is indicated on the right. The migration of specifically stained bands enriched in the WGA-Sepharose eluates is indicated by arrows.

1C Is a Major Constituent of the (+)-[³H]Isradipine-Labeled LTCC Complexes in RINm5F Cells

View larger version (37K): [in this window] [in a new window]   Figure 3. Immunoprecipitation of (+)-[3H]Isradipine-Binding Activity Solubilized from RINm5F, Heart, and Brain Membranes A and B, Immunoprecipitation by anti-1 (A) and anti-ß (B) antibodies was carried out as described in Materials and Methods. Immunoprecipitation is expressed as percent of the total amount of specific precipitable (+)-[3H]isradipine-binding activity added to each tube. Specific precipitable binding activity was determined by polyethylene glycol-precipitation/filtration as described in Materials and Methods. Data are given as means ± SE (n 3). Panel C shows typical experiments illustrating the concentration dependence of immunoprecipitation by anti-1C and anti-ß. The curves were obtained by fitting the experimental data to a monophasic hyperbolic function, yielding a maximal immunoprecipitation of 55 and 67% of total specific binding for anti-1C and anti-ß, respectively. An affinity-purified antibody against p-glycoprotein (anti-pgp-389, 64) was used as a control antibody. This antibody efficiently immunoprecipitates DHP-photolabeled p-glycoprotein from multidrug-resistant cells (64) but does not recognize Ca2+ channel subunits. The top x-axis indicates the concentration of the anti-ß antibody. D and E, Immunoprecipitation of (+)-[3H]isradipine binding activity by isoform-specific ß-subunit antibodies: labeled channels were immunoprecipitated by saturating concentrations of anti-ß2 (0.002 mg/ml), anti-ß3 (0.11 mg/ml), anti-ß1b (0.083 mg/ml), and anti-ß4 (0.093 mg/ml). Data are given as means ± SE (panel D, n 3) or ± range (panel E, n = 2). Immunoprecipitation by equivalent concentrations of control rabbit IgG was subtracted to yield specific immunoprecipitation. To calculate the percent of ß-subunits associated with a particular isoform, data were expressed as percent of the radioactivity specifically immunoprecipitated by anti-ß in the same tissue.

To determine which ß-isoform is coupled to 1C in RINm5F cells, we used isoform-specific antibodies raised against the C-terminal portions of all known ß-subunit isoforms (ß1b, ß2, ß3, ß4). ß2-Subunits, together with 1C, are the major component of DHP-labeled LTCCs in mammalian heart (16). A ß2 cDNA has recently also been cloned from RINm5F cells. In control experiments, saturating concentrations of affinity-purified anti-ß2 bound 45 ± 8% (n = 5) of ß-subunit-associated (+)-[³H]isradipine binding activity solubilized from guinea pig or rabbit heart membranes (n = 3, Fig. 3D). Under the same experimental conditions, no binding activity above background was recognized by anti-ß2 antibodies in RINm5F cells (Fig. 3D). In brain also, only a small portion of channels was associated with ß2. In contrast, anti-ß3 antibodies recognized no labeled channels solubilized from heart but accounted for 21 ± 6% (n = 5) of channel-associated ß-immunoreactivity in RINm5F membranes (Fig. 3D) and a similar portion (20 ± 3%, n = 8) in brain.

Thus it appeared that the ß-subunit composition of LTCCs in RINm5F cells resembled brain LTCCs. ß1b and ß4 are also preferentially expressed in neuronal cells (37, 38). As shown in Fig. 3E, these two isoforms together comprise more than 50% of the LTCC ß-subunits in extracts of whole mammalian brain. In RINm5F cells, affinity-purified anti-ß1b-antibodies also efficiently immunoprecipitated DHP-binding activity whereas anti-ß4 was ineffective (Fig. 3E). Taken together, the majority of ß-subunits associated with LTCCs in RINm5F cells corresponded to the ß1b- or ß3-isoform.

The specificity of our antibodies was verified in control experiments using (+)-[³H]isradipine-labeled skeletal muscle LTCCs, which only contain the ß1a-isoform. Only anti-ß, but none of our isoform-specific antibodies (n = 2), specifically immunoprecipitated the skeletal muscle channel complex. Further evidence for the specific interaction of anti-ß3 and anti-ß1b antibodies was obtained in immunoblots. The ß3 polypeptide (apparent molecular mass, 62 ± 2 kDa, n = 4) was detected in RINm5F, guinea pig brain, and guinea pig cerebellum membranes, but not in rabbit skeletal muscle membranes (Fig. 4A). In some experiments a very faint band was detected in guinea pig heart membranes (not shown). Anti-ß1b was less reactive in immunoblots. However, specific staining of the expected ß1b-polypeptide (85 ± 1 kDa; n = 3) could be demonstrated in guinea pig cerebral cortex after enrichment of ß-subunits by affinity chromatography on a glutathione-S-transferase (GST) fusion protein with the 1 interaction domain of the 1C subunit (AIDA) (Fig. 4B). GST-AIDA binds all ß-subunit isoforms with high affinity and can be used for ß-subunit purification (Ref. 17; see Materials and Methods). Although this procedure allowed the specific enrichment of ß3-subunits (not shown) from RINm5F cells and of ß1b-subunits from brain (Fig. 4B), no specific ß1b staining was found in RINm5F membranes. Therefore, ß1b seems to be expressed at lower densities in RINm5F cells than in brain.

View larger version (61K): [in this window] [in a new window]   Figure 4. ß3- and ß1b-Subunit Immunostaining A, The following protein samples were separated on a 10% polyacrylamide SDS-gel and processed for immunostaining with affinity-purified anti-ß3 antibodies: RINm5F membranes (RIN, 0.07 mg/lane); guinea pig cerebellum membranes (CER, 0.06 mg/lane); guinea pig brain membranes (BR, 0,05 mg/lane); rabbit skeletal muscle membranes (SKM, 0.006 mg/lane). The migration of prestained molecular mass markers (106, 80, 49.5, 32.5, and 27.5 kDa) is indicated on the right. The migration of the specifically stained ß3-band is indicated by an arrow. In skeletal muscle membranes, two bands (indicated by asterisks) were nonspecifically stained by the second antibody. B, ß-Subunits were enriched from brain (BR) or RINm5F (RIN) membranes by affinity chromatography on GST-AIDA as described in Materials and Methods. Purified fractions extracted from about 1 mg of starting membrane protein) were separated on a 10% SDS gel and immunostained with affinity-purified anti-ß1b. The migration of prestained molecular mass markers and of the ß1b-subunit (85 kDa; arrow) is indicated. The asterisk indicates the migration of GST-AIDA, which was nonspecifically stained as was a faint 70-kDa band. Both were also observed in skeletal muscle ß-subunit preparations. ß1b staining in RINm5F membranes was absent in three independent experiments in which ß1b staining in brain was always observed.

Phosphorylation of a Long Form of 1C by cA-PK in RINm5F Membranes

This implies that in RINm5F cells proteolytic removal of the C-terminal portion containing the consensus sites for phosphorylation by cA-PK (45) takes place in most of the 1C-subunits, giving rise to the 199-kDa form detected in immunoblots.

	DISCUSSION

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Our studies provide the first biochemical analysis of an LTCC in a (neuro)endocrine cell type on the protein level. Because biochemical studies in islets of Langerhans would be technically difficult to perform due to low harvest number and cell type heterogeneity, we selected RINm5F cells for our studies. This cell line is widely used to study the role of LTTCs for insulin secretion (see references in Ref.12). Although RINm5F cells have lost some typical features of mouse or human pancreatic ß-cells (e.g. the glucose dependence of insulin secretion (46, 47), many properties of their membrane-signaling and of the Ca²⁺-dependent insulin-secretion process closely resemble those in ß-cells. RINm5F cells respond to insulin secretagogues (such as glucose, glyceraldehyde, alanine, and sulfonylureas) with membrane depolarization, resulting in LTCC activation and spike activity (48). LTCCs in RINM5F cells possess pharmacological and electrophysiological properties, e.g. single-channel conductance and gating kinetics, indistinguishable from those in ß-cells. This also holds true for their modulation by protein kinases A and C (1, 8, 49). Therefore, these cells should serve as a suitable biochemical model of (neuro)endocrine LTCCs coupled to insulin secretion.

Differential Phosphorylation of Two Size Forms of 1C in RINm5F Cells
Agents that increase cytosolic cAMP enhance stimulus-secretion coupling in ß-cells. This is attributed to cA-PK-mediated stimulation of Ca²⁺-influx through LTCCs as well as to direct effects on the secretory machinery itself (1, 43). Ca²⁺ influx is increased by potentiation of LTCC activity due to a phosphorylation-induced slowing of channel inactivation (43). cA-PK-mediated LTCC modulation in brain, skeletal muscle, and cardiac muscle could be mediated by direct phoshorylation of the C-terminal portions of the channels’ 1-subunits (32, 41, 50, 51, 52, 53). Using sequence-directed 1-subunit antibodies, we demonstrate the existence of at least two different classes of 1-polypeptides. One class was unequivocally identified as 1C. The apparent molecular mass of 1C in immunoblots of 199 kDa indicated that it does not represent its full-length form. This was confirmed by phosphorylation of immunopurified fractions disclosing a long form that serves as the substrate for cA-PK in vitro. This form must therefore contain the C-terminal site phosphorylated by cA-PK [serine 1928 of the class C sequence (32, 41, 53)]. Such posttranslational proteolytic processing of Ca²⁺ channel 1 C-termini has also been described in skeletal muscle (50), brain (51, 52), and heart (32, 53). Here we demonstrate that it also occurs in a (neuro)endocrine cell, indicating that the protease responsible for this cleavage is also expressed in RINm5F cells. It was proposed that this cleavage would allow the differential modulation of the two size forms of 1 by cA-PK-dependent phosphorylation (52, 54). This could be especially important if the two forms are targeted to different regions within the plasma membrane. Specific targeting of DHP-sensitive Ca²⁺ channels to specific plasmalemmal compartments has not only been reported for skeletal muscle myocytes (55) and neurons (34), but also, more recently, for insulin-secreting ß-cells (56). In these cells LTCCs are preferentially located on the secretory vesicle-containing side of the plasma membrane, where their activity is tightly coupled to insulin secretion (56).

It is also possible that C-terminal truncation and phosphorylation affect the interaction of the 1-subunit with ß-, 2--, or G protein subunits, thereby modulating channel function. Further biochemical studies are required to address this important question.

1C-, ß1b-, and ß3-Subunits Are Major Constituents of (+)-[³H]isradipine-Labeled LTCC in RINm5F Cells
Immunostaining of channels partially purified by lectin affinity chromatography revealed that 1C represents only a fraction of the total 1-subunit immunoreactivity as detected in RINm5F cells with our nonselective antibody (anti-1). These 1-subunits most likely represent non-LTCC 1-subunits, which are also expressed in these cells (8, 33), or translation products of the class D gene. 1D Transcripts have been found to be abundant in insulin-secreting cells (21, 22, 23, 24). Similar to 1C and 1S, 1D-subunits were shown to mediate DHP agonist- and antagonist-sensitive L-type Ca²⁺ currents after heterologous expression (24, 57) and are believed to bind (+)-[³H]isradipine with similar affinity as 1C (51). Whether about one half of (+)-[³H]isradipine-binding activity not recognized by anti-1C in our experiments is associated with an LTCC complex formed by 1D or is sterically not accessible for our anti-1C antibodies remains to be determined. In rat pancreatic islets, 1C and 1D represent about one and two thirds, respectively, of L-type 1-transcripts (21). This is similar to our biochemical finding of approximately 50% 1C, suggesting that the relative expression of 1C vs. 1D is similar in rat RINm5F cells and rat pancreatic islets. This again argues for the validity of these cells as a biochemical model for ß-cell LTCCs.

We also provide evidence that all four known ß-subunit isoforms participate in the formation of LTCCs in brain to a different extent (Fig. 3, D and E). However, in RINm5F cells we only found a substantial association of ß1b- or ß3-subunits with channel complexes. This is in good agreement with the preliminary finding that ß3- and ß1-transcripts can be detected in insulin-secreting cells at higher levels (25) than ß2 (25, 26) and ß4 (25, 38). Although the possibility exists that the relative abundance of the two isoforms differs in RINm5F cells from that in pancreatic ß-cells, our data clearly emphasize that future studies should focus on the role of these two subunits for LTCC function in pancreatic ß-cells. It will be important to determine whether these isoforms confer different biophysical or pharmacological properties to coexpressed 1C- or 1D-subunits in heterologous systems.

We would like to point out that our data cannot predict to which extent LTCC complexes formed by 1C-, ß1b-, or ß3-subunits contribute to voltage-gated Ca²⁺ entry in RINm5F or ß-cells. However, they provide important biochemical information that will help to address this question, e.g. by employing antisense-techniques (58) in these cells.

Based on our data, ß1b- and ß3-subunits will be the ß-subunits of choice for the generation of cell lines stably expressing (neuro)endocrine LTCCs. Such cell lines could prove to be important tools for the development of drugs selectively modulating LTCCs in neuroendocrine cells such as pancreatic ß-cells. In a recent report, 1D-subunits were expressed together with ß2- subunits cloned from RINm5F cells in an attempt to establish such a cell line (24). However, based on our data, the physiological significance of this subunit combination remains questionable.

We cannot rule out that an even higher percentage of LTCCs are associated with ß3- and ß1b-subunits in RINm5F cell and brain. In contrast to our nonselective anti-ß-antibody, our isoform-specific antibodies were raised against C-terminal portions of these subunits for several reasons. This region is not conserved among subunit isoforms, seems not to undergo alternative splicing [as does, for example, the N-terminal region (37)] and is more likely to be accessible for the antibody under nondenaturating conditions. However, our antibodies would not detect ß-subunits with a proteolytically degraded C terminus. Although we do not have evidence for major proteolytic degradation of ß3- and ß1b-subunits (Fig. 4, A and B) in immunoblots, this possibility cannot be completely excluded.

Taken together, we demonstrate that 1C-, ß3-, and ß1b-subunits are major constituents of (+)-[³H]isradipine-labeled LTCCs in RINm5F cells. The full length form of 1C serves as an in vitro substrate for cAMP-dependent phosphorylation. Backphosphorylation experiments of these subunits in RINm5F cells with different protein kinases should provide a powerful biochemical tool with which to study the effects of hormones, drugs, and glucose metabolism on Ca²⁺ channel modulation in insulin-secreting cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Materials
Materials were obtained from the following sources: ATP, sheep anti-rabbit-IgG (conjugated to alkaline phosphatase), catalytic subunit of cA-PK, protein A-Sepharose, protease inhibitors, and N-acetyl-glucosamine from Sigma (St. Louis, MO); CNBr-activated Sepharose 4B from Pharmacia (Vienna, Austria); digitonin from Biosynth AG (Basel, Switzerland); [³²P]ATP from Chemomedica (Vienna, Austria); (+)-[³H]isradipine (80 Ci/mmol) from New England Nuclear (Vienna, Austria); okadaic acid and soybean trypsin inhibitor from Boehringer (Vienna, Austria); X-Omat AR5 films from Kodak (Rochester, NY); Immobilon-P transfer membranes from Millipore (Bedford, MA); and prestained molecular weight markers from Bio-Rad (Vienna, Austria) and GIBCO BRL (Vienna, Austria). Unlabeled isradipine was a gift from Sandoz AG (Basel, Switzerland).

Cell Culture
The insulin-secreting RINm5F insulinoma cell line was originally provided by C.B. Wollheim (Geneva). Cell culture was carried out as described in detail elsewhere (11).

Membrane Preparation
RINm5F cells were resuspended in 50 mM Tris-HCl supplemented with 2 mM MgCl₂, 2 mM EDTA, 0.5 mM phenylmethylsulfonylfluoride (PMSF), and 1 µg/ml soybean trypsin inhibitor, pH 7.6, and lysed by ten strokes in a Dounce homogenizer. Membranes were obtained by sequential centrifugation at 700 x g and 40,000 x g for 15 and 35 min (4 C), respectively. The 40,000 x g pellet was resuspended in binding buffer (50 mM Tris-HCl, pH 7.4, 0.1 mM PMSF) at a final protein concentration of 5–14 mg of protein/ml). Rabbit and guinea pig cardiac and brain membranes were prepared as described (59).

Sequence-Directed Antibody Production and Immunoblotting
Sequence-directed antibodies were raised by immunization of New-Zealand White rabbits with synthetic peptides coupled to BSA as described (35). Antibody anti-1 was raised against a synthetic peptide corresponding to residues 1382–1400 of the skeletal muscle 1-subunit (35). This sequence is highly conserved among all Ca²⁺ channel 1-subunits. Antibody anti-1C is directed against a synthetic peptide corresponding to residues 818–835 of the 1C-subunit (45). Antibody anti-2 was raised against a synthetic peptide corresponding to residues 721–738 of the skeletal muscle 2-subunit (60). Non-isoform-specific anti-ß-subunit antibodies were generated against peptides corresponding to residues 61–79 of ß1a (anti-ß). This sequence is highly conserved among all four ß-subunit genes (38). Isoform-selective antibodies were generated against the following residues: ß1b, 516–530; ß2, 595–604; ß3, 470–483; ß4, 460–474 (16, 38). Immunoblotting was carried out as described previously (61) employing affinity-purified (35) antibodies. Nonspecific staining was assessed by preincubation of antibodies with the antigenic peptide (1 µM). Affinity-purification was carried out as described (35).

Lectin Sepharose Affinity Chromatography
Ca²⁺ channels in digitonin extracts of the RINm5F cell membrane fraction were partially purified by WGA-Sepharose chromatography as described previously (62). In typical experiments about 10 mg of membrane protein were prelabeled with a saturating concentration of (+)-[³H]isradipine (2–3 nM) in 5–10 ml of 50 mM Tris-HCl, pH 7.4, 1 mM CaCl₂, 0.1 mM PMSF (37 C, 45 min). Membranes were collected by centrifugation (40,000 x g) and resuspended in the same buffer containing 0.15 M NaCl and 1% (wt/vol) digitonin. After incubation for 30 min on ice, nonsoluble material was removed by centrifugation (30 min, 100 000 x g). Twenty to 30% (range, n = 6) of DHP-binding activity was recovered in the extract together with 50–60% of the total membrane protein. Solubilized binding activity was purified on 2 ml WGA-Sepharose (10 mg of wheat germ lectin immobilized per 1 ml of Sepharose 4B Cl) as described for skeletal muscle Ca²⁺ channels.

Double Immunoprecipitation of Phosphorylated 1-Subunits
Immunoprecipitation and phosphorylation of RINm5F Ca²⁺ channels were carried out as described by Hell et al. (63) with minor modifications. Briefly, 0.2 mg membrane protein was solubilized in 0.25–0.4 ml RIA buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% BSA; pH 7.4, 1% [vol/vol] Triton X-100 for 30 min on ice followed by centrifugation for 5 min at 10,000 x g at 4 C. Solubilized protein was preadsorbed (30 min at 4 C) to a mixture of protein A-Sepharose (5 µl) and Sepharose CL-4B (75 µl). After centrifugation for 30 sec, 200 µl of the supernatant were added to a solution (10 µl-50 µl) of affinity-purified antibodies in TBS (anti-1C or the control antibodies anti-2 or anti-pgp-389, see below) and incubated for 2 h at 4 C. Washed protein A-Sepharose (20 µl swollen gel in RIA buffer) was added to the samples and further incubated for 2 h at 4 C. The protein A Sepharose was then washed three times with 1.25 ml RIA buffer and once with phosphorylation buffer (20 mM HEPES, 4 mM MgCl, 0.4 mM EGTA, 0.04% Triton X-100, 0.1 mM PMSF, 1 µM pepstatin A, 12.5 mM NaF, and 20 mM ß-glycerolphosphate). Antibody-bound channel was phosphorylated (30 C for 20 min) in the presence of 3 µM [³²]ATP (specific activity, 100–500 dpm/fmol) and cA-PK (10 U). The resin was washed three times with RIA buffer containing 20 mM EDTA, 20 mM ß-glycerolphosphate, and 12.5 mM NaF (RIA-P buffer) and two times with RIA buffer followed by a wash in TBS. Precipitated protein was eluted from the first-step antibody by denaturation in 1% (wt/vol) SDS, 50 mM Tris-HCl, pH 7.4, 5 mM dithiotreitol, 20 mM ß-glycerolphosphate, 0.1 mM PMSF, and 1 µM pepstatin A (30 min, 56 C). The eluate was diluted 10-fold in RIA-P buffer before the second-step antibody (anti-1) was added. After incubation for 2 h (4 C) the antibody-antigen complex was again recovered on Protein A-Sepharose as above. The resulting protein A-Sepharose pellet was washed three times with RIA buffer and once with TBS. SDS-PAGE sample buffer was added to the washed pellet and subjected to SDS-PAGE (SDS gel electrophoresis, 8% polyacrylamide) under reducing conditions (sample buffer containing 10 mM dithiothreitol).

Immunoprecipitation of (+)-[³H]Isradipine-Prelabeled Channels
Ten to 20 mg of RINm5F membrane protein were prelabeled with (+)-[³H]isradipine (1.5–11 nM) in 5–10 ml of 50 mM Tris-HCl, pH 7.4, 1 mM CaCl₂, 0.1 mM PMSF (37 C, 45 min). Membranes were collected by centrifugation, and digitonin extracts were prepared after resuspension in 5–10 ml solubilization buffer as described above. Aliquots (0.3–0.75 ml) of the extract were added to affinity-purified antibodies immobilized on Protein A-Sepharose (0.03–0.05 ml) equilibrated in TBS containing 0.1% digitonin. After incubation for 3–4 h at 4 C, unbound radioactivity was removed by extensive washing (4 x 1.5 ml) with ice-cold TBS containing 0.1% digitonin. Bound radioactivity was determined by liquid scintillation counting of the washed resin. An affinity-purified, sequence-directed antibody that efficiently immunoprecipitates p-glycoprotein [anti-pgp-389 (64)] was used to determine nonspecific immunoprecipitation.

Note that anti-1 could not be used for these immunoprecipitation experiments. It binds to 1-subunits only after denaturation in detergents such as Triton X-100 or SDS but does not recognize digitonin-solubilized Ca²⁺ channels with high affinity (35).

The amount of precipitable (+)-[³H]isradipine binding was determined on ice as follows: 0.1–0.3 ml digitonin extract was added to 0.1 ml of an ice-cold solution of 50 mM Tris-HCl, pH 7.4, containing 5 mg BSA/ml and 5 mg -globulin/ml. The mixture was rapidly diluted in 10 mM Tris-HCl, pH 7.4, 10% (wt/vol) polyethylene glycol 6000, and 10 mM MgCl₂ and incubated on ice for 2 min. Precipitable (+)-[³H]isradipine-binding activity was determined by filtration over GF/C Whatman glass fiber filters as described previously (59) followed by liquid scintillation counting. To measure nonspecific (+)-[³H]isradipine binding, prelabeling was carried out in the presence of 1 µM (±)-isradipine. Nonspecifically bound (+)-[³H]isradipine was not immunoprecipitated by our antibodies (n = 2).

Enrichment of ß-Subunits by Affinity Chromatography on GST-AIDA-Sepharose
The 1-interaction domain of the 1A-subunit (AIDA) was overexpressed as a fusion protein (GST-AIDA) with glutathione-S-transferase (GST) in Escherichia coli and purified and coupled to glutathione-Sepharose beads as described previously (17). Ten milligrams of membrane protein were solubilized (30 min, 4 C) in 4.5 ml buffer A (50 mM Tris-HCl, pH 7.4, 0.1 mM benzamidine, 1.0 mM iodoacetamide, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptine, 1 µM pepstatin A, 20 µg/ml calpain inhibitors I, and II) containing 1% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate and 1 M NaCl. Insoluble material was removed by centrifugation (45,000 x g, 30 min, 4 C), the supernatant was diluted 10-fold with buffer A, and 4 ml were subjected to purification on GST-AIDA Sepharose beads (0.03 ml, 0.01 mg GST-AIDA) and SDS-PAGE as described (17).

SDS-PAGE
Membrane proteins were separated on 7–10% straight polyacrylamide gels in a Bio-Rad Minigel apparatus under reducing conditions (10 mM dithiothreitol included in the sample buffer). Apparent molecular masses of Ca²⁺ channel subunits were determined by linear regression analysis from standard curves obtained with prestained marker proteins separated on the same gels.

Protein Assay
Protein was quantified according to the method of Bradford (65) using BSA as a standard.

Statistics
Except when stated otherwise, data are given as means ± SE from the indicated number of experiments.

	ACKNOWLEDGMENTS

 
We thank D. Ostler, E. Penz, A. Lemmermöhle, D. Reimer, and E. Emberger for expert technical assistence, Drs. M. Grabner and J. Mitterdorfer for helpful comments, and Professor H. Glossmann for providing unlabeled Ca²⁺ antagonists and continuous support.

	FOOTNOTES

 
Address requests for reprints to: Jörg Striessnig, Institut für Biochemische Pharmakologie, Peter-Mayrstraße 1, A-6020 Innsbruck, Austria.

This work was supported by the Fonds zur Förderung der Wissenschaftlichen Forschung (S6602 to J.S.), a research grant from the Deutsche Diabetes-Gesellschaft, the Deutsche Froschungsgemeinschaft (to H.H.), and research fellowships from the Graduiertenförderung des Landes Baden-Württemberg (to U.K. and M.R.). T.N.C was supported by a Fullbright Fellowship.

Received for publication February 16, 1996. Revision received January 13, 1997. Accepted for publication February 3, 1997.

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				P. Pinton, T. Tsuboi, E. K. Ainscow, T. Pozzan, R. Rizzuto, and G. A. Rutter Dynamics of Glucose-induced Membrane Recruitment of Protein Kinase C beta II in Living Pancreatic Islet beta -Cells J. Biol. Chem., September 27, 2002; 277(40): 37702 - 37710. [Abstract] [Full Text] [PDF]

				C. Andreolas, G. da Silva Xavier, F. Diraison, C. Zhao, A. Varadi, F. Lopez-Casillas, P. Ferre, F. Foufelle, and G. A. Rutter Stimulation of Acetyl-CoA Carboxylase Gene Expression by Glucose Requires Insulin Release and Sterol Regulatory Element Binding Protein 1c in Pancreatic MIN6 {beta}-Cells Diabetes, August 1, 2002; 51(8): 2536 - 2545. [Abstract] [Full Text] [PDF]

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				U. Gerster, B. Neuhuber, K. Groschner, J. Striessnig, and B. E Flucher Current modulation and membrane targeting of the calcium channel {alpha}1C subunit are independent functions of the {beta} subunit J. Physiol., June 1, 1999; 517(2): 353 - 368. [Abstract] [Full Text] [PDF]

				L. B. Lester, L. K. Langeberg, and J. D. Scott Anchoring of protein kinase A facilitates hormone-mediated insulin secretion PNAS, December 23, 1997; 94(26): 14942 - 14947. [Abstract] [Full Text] [PDF]

				A. Koschak, D. Reimer, I. Huber, M. Grabner, H. Glossmann, J. Engel, and J. Striessnig alpha 1D (Cav1.3) Subunits Can Form L-type Ca2+ Channels Activating at Negative Voltages J. Biol. Chem., June 15, 2001; 276(25): 22100 - 22106. [Abstract] [Full Text] [PDF]

				K. J. Mitchell, P. Pinton, A. Varadi, C. Tacchetti, E. K. Ainscow, T. Pozzan, R. Rizzuto, and G. A. Rutter Dense core secretory vesicles revealed as a dynamic Ca2+ store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera J. Cell Biol., October 1, 2001; 155(1): 41 - 52. [Abstract] [Full Text] [PDF]

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