Class A Calcium Channel Variants in Pancreatic Islets and Their Role in Insulin Secretion*

Brooke LigonDagger , Aubrey E. Boyd III§dagger , and Kathleen Dunlap

From the Departments of Neuroscience and Physiology, and § Division of Endocrinology, Tufts University School of Medicine, Boston, Massachusetts 02111

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The initiation of insulin release from rat islet beta  cells relies, in large part, on calcium influx through dihydropyridine-sensitive (alpha 1D) voltage-gated calcium channels. Components of calcium-dependent insulin secretion and whole cell calcium current, however, are resistant to L-type channel blockade, as well as to omega -conotoxin GVIA, a potent inhibitor of alpha 1B channels, suggesting the expression of additional exocytotic calcium channels in the islet. We used a reverse transcription-polymerase chain reaction-based strategy to ascertain at the molecular level whether the alpha 1A calcium channel isoform was also present. Results revealed two new variants of the rat brain alpha 1A channel in the islet with divergence in a putative extracellular domain and in the carboxyl terminus. Using antibodies and cRNA probes specific for alpha 1A channels, we found that the majority of cells in rat pancreatic islets were labeled, indicating expression of the alpha 1A channels in beta  cells, the predominant islet cell type. Electrophysiologic recording from isolated islet cells demonstrated that the dihydropyridine-resistant current was sensitive to the alpha 1A channel blocker, omega -agatoxin IVA. This toxin also inhibited the dihydropyridine-resistant component of glucose-stimulated insulin secretion, suggesting functional overlap among calcium channel classes. These findings confirm the presence of multiple high voltage-activated calcium channels in the rat islet and implicate a physiologic role for alpha 1A channels in excitation-secretion coupling in beta  cells.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The metabolism of glucose in beta  cells is linked to membrane excitation through increases in the components that influence the ATP/ADP ratio (1, 2). A local increase in ATP relative to ADP inhibits the ATP-sensitive K+ (KATP) channel, giving rise to oscillations in KATP permeability and consequent fluctuations in membrane potential (3, 4). ATP-induced depolarizations evoke insulin release through the activation of voltage-dependent calcium channels, promoting calcium influx (3-6). If extracellular calcium is eliminated, glucose-stimulated insulin secretion is abolished, highlighting the important role of calcium channels in insulin homeostasis (7-11).

Of the six genes (A-E, S) encoding the pore-forming alpha 1 subunits of high voltage-activated calcium channels (12), all are capable of coupling to exocytotic machinery, although differences in tissue distribution and efficacy with which they stimulate secretion vary among the channel classes (for review, see Ref. 13). Pharmacological and heterologous expression studies have identified selective antagonists for certain of these: omega -agatoxin IV blocks alpha 1A, omega -conotoxin GVIA blocks alpha 1B, and 1,4-dihydropyridines block alpha 1C, alpha 1D, and alpha 1S (13). No selective antagonist has been identified to date for alpha 1E calcium channels, although they are sensitive to nonselective calcium channel antagonists (e.g. omega -grammotoxin SIA and cadmium).

Previous work on pancreatic beta  cells demonstrated that calcium influx and depolarization-evoked insulin release are blocked 60-80% by inhibitors of alpha 1D channels (14-16). In addition, omega -conotoxin GVIA blocks a portion of calcium current (17) and 27% of the second phase insulin secretion from rat pancreatic beta  cells under conditions of maximal glucose stimulation (18). In the presence of both omega -conotoxin GVIA and the dihydropyridine antagonist nifedipine, 25% of Ca2+-dependent insulin secretion remains, suggesting the involvement of another calcium channel type. Because alpha 1A and alpha 1B channels are known to colocalize within mammalian central nervous system nerve terminals and play prominent roles in neurosecretion (19-22), we sought to determine whether the alpha 1A isoform was also present in beta  cells and responsible for the current and insulin release that are resistant to dihydropyridines and omega -conotoxin GVIA. Here we report the full-length sequences of two unique alpha 1A splice variants cloned from rat pancreatic islets. We demonstrate their expression in beta  cells and provide an initial characterization of their pharmacologic and electrophysiologic properties using tight seal whole cell recording. These channels play a role in calcium-dependent insulin secretion. Results may have significant implications for understanding, and perhaps treating, beta  cell disorders such as diabetes and hyperinsulinemia.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Islet Isolation-- Islets were isolated from male, 200-225 gm, Spraque-Dawley rats. Animals were anesthetized (Nembutol 50 mg/kg), and the pancreatic bile duct exposed, cannulated, and infused with 2 mg/ml collagenase (Boehringer Mannheim) in medium M199 (Life Technologies, Inc., with 5.6 gm/l HEPES, 2.2 gm/l NaHCO3). The infused pancreas was removed and incubated in a 50-ml tube submerged in a 37 °C water bath for 17 min. Tubes were shaken briefly to dissociate tissue, the digestion reaction was stopped by the addition of cooled M199 with 10% newborn calf serum (Life Technologies, Inc.), and the tissue was shaken again to further dissociate while washing free of collagenase. The tissue was spun for 30 s (speed 3) in an ICN table-top clinical centrifuge and washed in fresh medium M199 with serum. This procedure was repeated twice to remove collagenase. After a final spin (1 min. at speed 3) to pellet the tissue, the medium was discarded, and the tissue was suspended in 10 ml of Histopaque (Sigma) and overlaid with 10 ml of M199 (without serum). The tissue was centrifuged in this gradient at 900 × g in a swinging bucket rotor at 4 °C for 20 min. The supernatant containing the islets was removed and centrifuged (speed 4, ICN centrifuge) to pellet the islets. The supernatant was discarded and the islets washed with 10 ml of M199 (with serum) twice before use. They were used intact for insulin release assays, further dissociated into single cells for electrophysiology, or homogenized for isolation of RNA.

Cloning-- Primers were designed along the full-length of the rat brain class A channel (rbA-1, GenBankTM accession no. M64373). Regions of high homology to the rat brain alpha 1B channel (rbB) were avoided. Sense and antisense oligonucleotides ranged in length from 18 to 24 bases beginning at the following 5' nucleotides (nt)1 of the rbA-1 sequence: -20, 540, 1265, 1610, 2091, 2484, 2879, 3309, 3658, 4104, 4620, 5170, 5607, 5961, 6527, 7010, and 7125. Total RNA purified from homogenized rat islets (Trizol, Life Technologies, Inc.) was DNase treated (Life Technologies, Inc., amplification grade DNase) and reverse-transcribed for 30 min at 42 °C (reverse transcriptase, Perkin Elmer). GC rich regions were reverse transcribed at 50 °C (Superscript, Life Technologies, Inc. or avian myeloblastosis virus reverse transcriptase, Amersham Pharmacia Biotech) and 10% glycerol was used in the PCR amplification. At least two separate PCR amplifications (Taq polymerase, Perkin Elmer, Amersham Pharmacia Biotech) were performed for each primer set and 2 sense and 2 antisense strands sequenced (ABI, fluorescent-labeled automated sequencing) to ensure accuracy of sequence. To verify regions detected to diverge from rbA-1 over a ~2-kilobase region, reverse transcription of islet total RNA was performed at 50 °C for 1 h using avian myeloblastosis virus reverse transcriptase (Amersham Pharmacia Biotech) and Superscript (Life Technologies, Inc.) to generate longer products over GC rich regions. For this "long" PCR, primers at nt 4620 and from the rbA-1 3' UT region were used and generated two ~2 kilobases alternatively spliced carboxyl-terminal fragments as described below.

Generation of cRNA Probe-- Primers were designed to yield a 587-bp product from the 3'-end (5961-6548) of coding sequence of rbA-1 (23). Freshly isolated islet RNA for reverse transcription (Perkin Elmer reverse transcriptase), and Taq polymerase (Perkin Elmer) were used for cDNA amplification. Control samples, in which reverse transcriptase was omitted, were included to ensure that products had not been amplified from genomic DNA. Because the region contained a high percentage of GC-rich sequence, 10% glycerol was added to the reaction mix. The annealing temperature was 60 °C.

In Situ Hybridization-- The original 587-bp PCR product (nt 5961-6548) above was subcloned (vector pCR I, Invitrogen) and sequenced in both directions. Comparison of the sequences revealed 100% nucleotide identity to rbA-1. Using this subclone as a template, digoxigenin-labeled (Boehringer Mannheim) sense and antisense RNA probes were synthesized for in situ hybridization studies of rat pancreas. Paraffin sections were mounted on slides, cleared, and hydrated before treatment with proteinase K. This was followed by equilibration in 0.1 M triethanolamine, pH 8.0, to which acetic anhydride had been added. The probe was added at 4 ng/µl to the hybridization mix. Sections were incubated overnight at 60 °C in a humid chamber. Post hybridization, the slides were soaked in 2× SSC (300 mM sodium chloride, 30 mM sodium citrate), gently agitated in warm 50% formamide in 2× SSC, and transferred to 60 °C. The sections were then treated with RNase A buffer (500 mM NaCl, 10 mM Tris, 1 mM EDTA, and RNase A at 20 µg/ml) to remove unhybridized probe. Slides were washed in 0.1× SSC at 65 °C. For color detection of labeled cells (Boehringer Mannheim Genius System), slides were blocked, washed, and incubated overnight at 4 °C with 200-500 ml of antidigoxigenin-alkaline phosphatase. The subsequent chromagen substrate (Sigma) consisted of nitroblue tetrazolium salt and X-phos (bromo-4-chloro-3-indoyl phosphate toluidinium salt).

Immunohistochemistry-- Experiments employed the antipeptide antibody, CNA-1 (generously provided by Drs. R. Westenbroek and W. A. Catterall). This antibody recognizes amino acids 865-881 in rbA-1, corresponding to nucleotides 2595-2643 (identical amino acids in the islet). 8-µm cryosections of rat pancreas were blocked with Tris-buffered saline, 1% bovine serum albumin, 0.1% Triton, washed, and incubated for 1 h at room temperature with CNA-1 primary antibody diluted 1:25 in Tris-buffered saline. Sections were washed, incubated with rhodamine-labeled anti-rabbit secondary antibody (Sigma, diluted 1:350 in Tris-buffered saline) for 20 min, washed again, then visualized and photographed through a Zeiss Axioplan Universal microscope.

Whole Cell Recording-- Islets were isolated as described above, dissociated in trypsin/EDTA (0.05% trypsin, 0.53 mM EDTA) and cultured 1-3 days in M199 (with serum) prior to the experiments. Voltage-dependent calcium currents were examined in the whole cell configuration using pipettes fabricated from nonheparinized soda lime glass (VWR, 73811). Pipette resistances averaged ~2 megaohms with an internal solution containing 120 mM N-methyl-D-glucamine, 20 mM tetraethylammonium-OH, 10 mM HEPES, 11 mM EGTA, 1 mM CaCl2, and 4 mM MgATP (pH 7.0) and an external solution containing 10 mM BaCl2, 120 mM NaCl, 20 mM triethanolamine, and 1.2 mM MgCl2, 10 mM Na-HEPES, 1 µM tetrodotoxin, 1 µM nimodipine (pH 7.4). omega -Agatoxin IVA was applied by pressure ejection from a wide-bore (~2-3 µm) pipette positioned within 20 µm of the cell. The toxin was stored at -40 °C in 10-µl samples of 10 mM in distilled deionized water and diluted to the final working concentration in external solution immediately before use.

Insulin Secretion Evaluated by RIA-- Islets were isolated from exocrine pancreas as described above. They were selected for uniform size (150-200 µm), washed in Krebs-Ringer buffer (KRB, equilibrated with O2 for 30 min), evenly divided into KRB with 2.8 mM glucose (with or without 500 nM omega -agatoxin IVA) and allowed to recover from isolation. The islets were incubated at 37 °C (95% O2, 5% CO2) during equilibration. After 90 min of preincubation in low glucose KRB, the islets were divided into groups of 10 islets/vial in 0.5 ml of fresh KRB, three vials for each condition (2.8 or 11 mM glucose with or without omega -agatoxin IVA). All samples were incubated in a 37 °C water bath for 1 h to promote glucose-stimulated insulin secretion. Samples were centrifuged at 700 rpm, and 300 µl of supernatant was removed and viewed under a dissecting microscope to ensure no islet tissue was present. This sample was assayed for insulin content using ICN insulin radioimmunoassay reagents. Briefly, 100 µl of each sample or standards were added to duplicate anti-insulin antibody-coated tubes followed by 900 µl of 125I-labeled insulin diluted into buffer. In addition, control samples of toxin alone were included to ensure that the omega -agatoxin IVA did not interfere with insulin antibody binding. The samples and standards were incubated for 18 h at room temperature. The supernatant was removed, and tubes were washed with double-distilled H2O and counted in a gamma counter. Sample counts were compared with the standard curve for determination of insulin concentrations. Results are reported as mean ± S.E. of the mean, and statistical significance was evaluated by Student's t test.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cloning of Islet alpha 1A Splice Variants-- Given our goal of identifying the class A (alpha 1A) variants in pancreatic islets, a PCR strategy was followed using primers designed along the full-length of the cloned alpha 1A sequence from rat brain, rbA-1 (23, 24). Each product was generated twice, and both the sense and antisense strands were sequenced to ensure accuracy. Reverse transcriptase was eliminated from control samples to ensure that amplification products were from islet mRNA and not of genomic origin. The entire coding sequence was determined. From the 5'-UT to base 4806, the sequence was 100% identical to the type "a" splice variant (alpha 1A-a) of rbA-1 (25). At nt 4807, in a putative extracellular domain, there is a six base in-frame insertion in both variants described below cloned from rat islets. This insertion results in the addition of amino acids asparagine and proline (Figs. 1A and 2), as previously reported for alpha 1A in rat kidney cortex (26) and alpha 1A-b in rat brain (25). In addition, two carboxyl-terminal variants are present in the islet that have not been reported elsewhere. One, riA-1, diverges over the bases 5385-5477, altering 10 amino acids throughout the region (Figs. 1 and 2). Further 3', the bases corresponding to 6160-6195 (in rbA-1) are deleted in riA-1. The riA-1 variant is also significantly longer than rbA-1 due to a 5-base (GCCAG) insertion before the stop codon, resulting in a frameshift (Figs. 1 and 2). This same insertion has also been described in human brain alpha 1A channels (27). The contiguity of the observed variations in riA-1 was confirmed by generating and sequencing >2 kilobase fragments using primers at 4620 and in the 3'-untranslated region. Although combinations of these carboxyl-terminal modifications have been described in human brain partial cDNA clones (27), riA-1 is unique among known full-length clones.


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Fig. 1.   Comparison of alpha 1A splice variants. A, primary sequences of the two islet alpha 1A variants are aligned with those of rbA-1, beginning at nt 4700 where divergence begins. The approximate locations of amino acid insertions (solid bars), deletions (cross-hatched bars), and divergence (stippled bars) are noted. The precise locations (relative to rbA-1) of the variations found in riA-1 and riA-2 are as follows: A, 6-base insertion at nt 4807; B, divergence between nt 5385-5477; C, deletion of nt 6160-6195; D, 5-base insertion at nt 6625 (causing frameshift); E, new amino acid sequence due to frameshift; F, deletion of nt 4922-4987; G, deletion of nt 5256-5480. B, predicted membrane topology of riA-1 and riA-2 beginning at homologous repeat IV and ending at the carboxyl terminus. Insertions and divergent regions marked as in A; asterisks (*) mark approximate locations of deletions.


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Fig. 2.   Amino acid sequences of alpha 1A variants. Primary sequences of riA-1 and riA-2 are compared with those of rbA-1a (GenBankTM accession no. M64373) and a human brain alpha 1A (GenBankTM accession no. U79666) beginning at amino acid 1400 where divergence begins. Amino acids identical to those of rbA-1 are shaded. Deletions are noted with dashes (-). Asterisks (*) denote locations of stop codons.

In the other product, riA-2, the reading frame and stop codon of rbA-1 are maintained, but bases 4922-4987 and 5256-5480 are deleted (Fig. 1). The latter deletion corresponds to the region of nucleotide divergence found in riA-1 and is homologous to the proposed calcium-binding, EF-hand motif in alpha 1C (25, 28). Thus, rat pancreatic islets appear to express two unique forms of class A channel. This represents the first molecular evidence of class A channels in islets. We have explored their expression and localization at the levels of RNA and protein, provided an initial description of their pharmacology and electrophysiology, and evaluated their role in insulin secretion.

Cellular Localization of Rat Islet alpha 1A Channels-- In situ hybridization methods were employed to examine the cellular localization of alpha 1A mRNA in islets. A riboprobe was synthesized by PCR of reverse transcribed islet total RNA with class A calcium channel primers, generating a 587-bp carboxyl-terminal fragment (bp 5961-6548). No product was obtained if reverse transcriptase was omitted. This fragment, 100% identical to rbA-1 (23), was subcloned and labeled with digoxigenin-UTP to generate both sense and antisense RNA probes. The majority of the cells, representing the islet core, were labeled with the antisense probe. This demonstrates that the alpha 1A calcium channel mRNA is present in beta  cells, which comprise the core and ~80% of islet cells (Fig. 3A). Islets remained unlabeled when the digoxigenin-sense probe was used (Fig. 3B).


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Fig. 3.   Localization of alpha 1A mRNA in islets. Photograph of paraffin sections of rat pancreas exposed to digoxigenin-labeled RNA probes. A, large islet labeled with antisense probe (corresponding nucleotides 5961-6548 of rbA-1 carboxyl terminus). Smaller islet (B) remains unlabeled with sense probe.

To determine whether alpha 1A channel protein was also present in beta  cells, immunohistochemistry was employed with an antipeptide antibody, CNA-1 (29). This antibody recognizes amino acids 865-881 of rbA-1 in the putative second intracellular loop. As the islet clones code for identical amino acids in this domain, we used CNA-1 to detect the expression of class A channels in islets. Intense staining was observed on the majority (>75%) of cells in the core of the islet, again indicating the presence of class A channel protein in beta  cells (Fig. 4, A and B). No staining was seen in control samples in which CNA-1 antibody had been omitted (data not shown).


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Fig. 4.   Immunohistochemical localization of alpha 1A protein in islets. Fluorescence photographs of two islets labeled with primary anti-alpha 1A antibody, CNA-1 (and secondary rhodamine-labeled anti-rabbit IgG) (A, B). Note that the majority of cells in both islets are labeled with CNA-1 antibody, indicating the presence of alpha 1A protein in beta  cells. Calibration bar = 50 µm.

Whole Cell Recording of beta  Cell Calcium Channel Currents-- To test whether the rat islet class A transcripts give rise to functional channels in beta  cells, we recorded from dissociated islet cells using tight seal whole cell methods. Calcium channel currents were isolated with standard intra- and extracellular solutions (30, 31). Nimodipine (1 µM) was added to all extracellular solutions in order to suppress dihydropyridine-sensitive (class C/D) currents. Ba2+ (10 mM) was used as a charge carrier to enhance calcium channel currents over those observed in 1.2 mM Ca2+ (the normal extracellular concentration). beta  cells could be identified by the presence of small processes, as has been reported elsewhere (32).

Following whole cell access, the cells were held at -80 mV, and 20 ms rectangular test pulses were applied. Inward Ba2+ current activated near -30 mV and was maximal at +10 mV. Peak currents (at +10 mV) varied from 50 to 400 pA. Activation was rapid (reaching peak in ~5 ms at 0 mV), inactivation was slow (with no decay in 20 ms), and deactivation was rapid (complete in ~300 µs). In these ways, the current resembled alpha 1A currents expressed in other cell types (33, 34).

To determine whether these currents were, in fact, generated through class A calcium channels, we applied omega -agatoxin IVA (the selective class A channel antagonist). In 8 of 12 cells tested, omega -agatoxin IVA (1 µM) produced a significant reduction in inward current (Fig. 5, A and C). Inhibition ranged from 15 to 80% in the responsive cells with an average reduction of 48.4 ± 8.5%. The onset of toxin-induced blockade was slow (tau  = 62.5 s) even at 1 µM; the dissociation rate was also slow with no recovery in 3 min (Fig. 5B). These results are consistent with the action of omega -agatoxin IVA on class A calcium currents in other cell types (35-37) and demonstrate the presence of functional class A channels in beta  cells. In addition, the percentage of responsive cells in the sample tested electrophysiologically is similar to that of the islet cells shown to express alpha 1A mRNA and protein (Figs. 3 and 4).


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Fig. 5.   omega -Agatoxin IVA-sensitive calcium channel current in beta  cells. A, superimposed Ba2+ currents in a beta  cell evoked by 20 ms step depolarizations to 10 mV from a holding potential of -80 mV, before (control) and during application of 1 µM omega -agatoxin IVA (as marked). Calibration bar: 100 pA, 5 ms. B, time course for omega -agatoxin IVA-induced inhibition; toxin applied for time indicated by horizontal bar. C, current-voltage relationship from another beta  cell before (open circle ) and during (bullet ) exposure to 1 µM omega -agatoxin IVA. All currents recorded in the presence of 1 µM nifedipine to block dihydropyridine-sensitive current.

A Portion of Insulin Secretion Is Inhibited by omega -Agatoxin IVA-- The presence of significant class A current in beta  cells suggested the possibility that these channels play a role in exocytosis. To test this, we employed a radioimmunoassay to measure glucose-stimulated insulin release from islets, and evaluated the involvement of class A calcium channels using omega -agatoxin IVA. Islets were equilibrated for 90 min at 37 °C in KRB ± 500 nM omega -agatoxin IVA. Addition of 11 mM glucose evoked insulin release to a level of 1127% of that seen in basal glucose (2.8 mM) during a 60-min incubation period. omega -Agatoxin IVA (500 nM) diminished this glucose-stimulated insulin release to 811% of basal secretion (Fig. 6A). The inclusion of nimodipine (1 µM) in all solutions to block dihydropyridine-sensitive calcium channels reduced 11 mM glucose-evoked insulin release to a level 140% of that seen in basal glucose (2.8 mM). Thus, a portion of glucose-stimulated insulin release is dihydropyridine-resistant. All of the latter is mediated by Ca2+ influx through class A channels, since it is eliminated by 500 nM omega -agatoxin IVA (Fig. 6B). Control samples without islets, with omega -agatoxin IVA alone, had no counts above background, indicating the toxin does not interact with anti-insulin antibodies. These results demonstrate that class A calcium channels are effective co-regulators of insulin secretion.


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Fig. 6.   Class A channels regulate insulin secretion. A, glucose-stimulated insulin release measured by radioimmunoassay in control cells or cells exposed to 500 nM omega -agatoxin IVA (as marked). Release in 11 mM glucose normalized to that in 2.8 mM glucose (basal). B, identical experiments except all solutions contained 1 µM nimodipine to block dihydropyridine-sensitive release. Values represent means ± S.E. of the mean for n = 3 (A, * p = .05) and n = 7 (B, ** p < 0.005). Significance determined by unpaired Student's t test.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our results demonstrate two new variants of the class A calcium channel in pancreatic islets and establish the presence of both mRNA and protein in beta  cells. We have begun to characterize the electrophysiology and demonstrate that currents through these channels resemble currents recorded through class A channels in other cells (33-38). Additionally, we show that, as in the nervous system, calcium influx through islet class A channels is coupled to exocytosis. Despite identification of several alpha 1A splice variants and missense mutations (described below), little is known about the biophysical properties conferred by these changes. With the molecular information described here, we can begin to ascertain how specific amino acid changes influence electrical and functional properties of alpha 1A channels.

Multiple alpha 1A Isoforms-- Certain of the variable regions reported here occur in clones described elsewhere, but the islet clones as a whole are unique. Zhuchenko et al. (27) demonstrated five partial human clones from brain containing the GCCAG insertion, producing a frameshift and a longer carboxyl terminus. Unlike the islet clones described here, the human variants contain a CAG repeat in the extended carboxyl terminus that is responsible for a form of familial ataxia, SCA6 (spinocerebellar ataxia). This repeat is not expressed in the riA-1 carboxyl terminus (Fig. 2). Two partial clones from human had the 36-base pair deletion, and two others had the 94-base pair divergent sequence present in riA-1 (27). In addition, the 6-base insertion at nt 4607 in both islet isoforms has been described in a alpha 1A PCR cDNA fragment from rat kidney (26) and in rat brain alpha 1A-b (25). The two deletions in riA-2 (nt 4922-4987 and 5256-5480) have not been described previously. Interestingly, the deletion beginning at nt 5256 in riA-2 corresponds to the 94-bp COOH-terminal region of divergence found in riA-1 and in two of the partial human clones. This region has homology to EF hand motifs in dihydropyridine-sensitive calcium channels demonstrated to bind calcium and promote inactivation (25, 28), but also see Ref. 39. Rabbit BI-I alpha 1A calcium channels are 92% identical to riA-1 but differ substantially in the intracellular loop between repeats II and III. Of note, there are also several 3 and 6 base insertions in BI-I alpha 1A isoform not seen in riA-1 (40).

The functional consequences of these structural variations in alpha 1A are largely unexplored, but even small changes in sequence can lead to significant biophysical differences among the channel variants. For example, four missense mutations in a human alpha 1A calcium channel gene are responsible for hemiplegic migraine (41) and a single nonconservative amino acid substitution in a mouse alpha 1A gene causes seizures and ataxia in the tottering mutant (42). The alterations in calcium channel function produced by these mutations, as well as by the structural variations described for the islet class A calcium channels described here, remain to be examined. Work on voltage-dependent sodium channels suggests that such studies on calcium channels will be fruitful. For example, single amino acid substitutions in sodium channels have dramatic effects on inactivation, giving rise to such disorders as paramyotonia congenita and hyperkalemic periodic paralysis (43-45).

Pharmacological properties of class A channels differ among the variants. Zamponi et al. (25) reported that single amino acid differences in the I-II linker regions of alpha 1A-a, alpha 1A-b, and alpha 1A-c bring about alterations in channel blockade by local anesthetic and antipsychotic drugs. Such naturally occurring structural changes may explain some of the variability reported for omega -agatoxin IVA-sensitive currents in different cell types or when different alpha 1A isotypes are expressed in heterologous systems (13, 46-48). Estimates of omega -agatoxin IVA binding affinity also vary, ranging from the low nM (for cerebellar Purkinje neurons) (35) to high nM for class A channels in Xenopus oocytes (46). Unique pharmacological phenotypes among the variants offer a means to test their differential involvement in calcium-dependent physiological processes and to determine the consequences of the structural changes for functional phenotype.

Heterogeneous beta  Cell Responses-- The density of omega -agatoxin IVA-sensitive current in beta  cells varied considerably from cell to cell. The majority of the cells expressed detectable toxin-sensitive currents (as well as alpha 1A mRNA and protein detected by in situ and immunochemical methods); in those sensitive to toxin blockade, the inhibition varied from 15 to 80%. As omega -agatoxin IVA has been demonstrated to inhibit a variety of alpha 1A calcium channels, both in primary and heterologous cells, the heterogeneity found for the beta  cells likely reflects variations in expression levels for both class A channel types. Quantitating this relationship will require single-cell comparisons between the mRNA or protein levels for the calcium channel subunits and amplitude of omega -agatoxin IVA-sensitive currents. In undifferentiated PC12 cells, both the sensitivity of the calcium current to omega -agatoxin-IVA and the rate of toxin-induced inhibition vary from cell to cell, as observed for neurons (37, 38), raising the possibility that pharmacological differences between class A channel variants might underlie the heterogeneity of beta  cell responses to omega -agatoxin IVA.

Our results suggest the possibility that variations among beta  cells may allow them to perform different roles within the context of integrated islet function. In this way, our results support those of Moitoso de Vargas et al. (49), which demonstrate that glucose-stimulated activation of insulin gene expression varies among beta  cells in intact islets. Such differences signal the presence of functional subsets of beta  cells that are differentially active as physiological conditions vary. It will be important for future work to test such ideas.

Class A Channels in Other Tissues-- Exocytosis from mammalian central neurons relies heavily on class A and B channels and very little on dihydropyridine-sensitive class C/D channels (13). By contrast, secretion from neuroendocrine cells is largely dihydropyridine-sensitive. Our results demonstrate that such differences are not absolute. That is, although the majority of insulin release is dependent on dihydropyridine-sensitive channels, a portion is mediated through class A channels. Class A and B channels have been demonstrated by pharmacology and electrophysiology to be present in adrenal chromaffin cells, (50-52), carcinoid cells of the gut (53), pituitary corticotropes (54), the pituitary cell line AtT20 (55), GH4C1 pituitary cells (56), and in RINm5f and HIT insulinoma cells (57, 58). Dihydropyridine-resistant channels play a role in exocytosis for certain of these cells (e.g. carcinoid and HIT cells) (51, 58) but not all. Release of adrenocorticotrophic hormone from AtT20 cells, for example, is evoked entirely through dihydropyridine-sensitive channels, despite the fact that >50% of the calcium current in the cells is resistant to blockade by dihydropyridines (55). The mechanisms that determine specificity of coupling between calcium channels and the exocytotic machinery remain to be defined.

Significance of alpha 1A Calcium Channels in the Islet-- Recent statistical analyses suggest that more than 15 million people in the United States suffer from diabetes (59). A population-based retrospective study indicates the age-adjusted prevalence of diabetes cases rose 65% for men and 37% for women between 1970 and 1990 (60), and a "massive increase" in the prevalence of noninsulin-dependent diabetes is predicted globally, as countries succumb to the "Westernization" of diet and activity patterns (61). It is important, therefore, to continue to expand our understanding of the mechanisms by which the beta  cell controls the secretion of insulin. Knowing the molecular structure of the alpha 1A isoforms present in the islet and the roles that they play within the context of the functioning islet may allow the development of clinically useful agents to modulate calcium entry into beta  cells through these channels, providing another tool to regulate insulin release. In addition, defining the entire sequence of splice variants will facilitate investigations of the ways in which changes in primary structure alter the pharmacology, biophysical, and/or exocytotic properties of class A voltage-gated calcium channels.

    ACKNOWLEDGEMENTS

We are grateful to L. Moss for support of this project, Joe Dillon and Xudong Liang for assistance with automated sequencing, Jeff Tatro and Jack Leahy for advice on radioimmunoassays, W.A. Catterall and R. Westenbroek for CNA-1 antibody, Ron Lechan and Barbara Corkey for helpful comments on the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health MCSDA KO8 NS01923 (to B. L.), and National Institutes of Health Grants DK34447 (to A. E. B., III and Larry Moss), NS16483 (to K. D., Jacob Javits Award), and National Institutes of Health GRASP Center Grant DK34928.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Neuroscience, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6938; Fax: 617-636-0576.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF051526, AF051527.

dagger Deceased.

1 The abbreviations used are: nt, nucleotide(s); PCR, polymerase chain reaction; bp, base pair(s); KRB, Krebs-Ringer buffer.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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