The alpha1-beta-subunit interaction that modulates calcium channel activity is reversible and requires a competent alpha-interaction domain

High voltage-gated calcium channels consist of a pore-forming subunit (alpha(1)) and three nonhomologous subunits (alpha(2)/delta, beta, and gamma). Although it is well established that the beta-subunit promotes traffic of channels to the plasma membrane and modifies their activity, the reversible nature of the interaction with the alpha(1)-subunit remains controversial. Here, we address this issue by examining the effect of purified beta(2a) protein on Ca(V)1.2 and Ca(V)2.3 channels expressed in Xenopus oocytes. The beta(2a)-subunit binds to the alpha(1)-interaction domain (AID) in vitro, and when injected into oocytes, it shifts the voltage dependence of activation and increases charge movement to ionic current coupling of Ca(V)1.2 channels. This increase depended on the integrity of AID but was not abolished by bafilomycin, demonstrating that the alpha(1)-beta interaction through the AID site can take place at the plasma membrane. Furthermore, injection of beta(2a) protein inhibited inactivation of Ca(V)2.3 channels and converted fast inactivating Ca(V)2.3/beta(1b) channels to slow inactivating channels. Inhibition of inactivation required larger concentration of beta(2a) in oocytes expressing Ca(V)2.3/beta(1b) channels than expressing Ca(V)2.3 alone but reached the same maximal level as expected for a competitive interaction through a single binding site. Together, our data show that the alpha(1)-beta interaction is reversible in intact cells and defines calcium channels beta-subunits as regulatory proteins rather than stoichiometric subunits.     

Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14-3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation

Voltage-gated Ca2+ channels (VDCCs) are heteromultimeric proteins that mediate Ca2+ influx into cells upon membrane depolarization. These channels are involved in various cellular events, including gene expression, regulation of hormone secretion and synaptic transmission. Kir/Gem, Rad, Rem, and Rem2 belong to the RGK family of Ras-related small G proteins. RGK proteins interact with the beta-subunits and downregulate VDCC activity. Kir/Gem was proposed to prevent surface expression of functional Ca2+ channels, while for Rem2 the mechanism remains controversial. Here, we have analyzed the mechanism by which Rad and Rem regulate VDCC activity. We show that, similar to Kir/Gem and Rem2, 14-3-3 and CaM binding regulate the subcellular distribution of Rad and Rem, which both inhibit Ca2+ channel activity by preventing its expression on the cell surface. This function is regulated by calmodulin and 14-3-3 binding only for Rad and not for Rem. Interestingly, nuclear targeting of Rad and Rem can relocalize and sequester the beta-subunit to the nucleus, thus providing a novel mechanism for Ca2+ channel downregulation.     

Analysis of the Rem2 - voltage dependant calcium channel beta subunit interaction and Rem2 interaction with phosphorylated phosphatidylinositide lipids

Voltage dependant calcium channels (VDCC) play a critical role in coupling electrical excitability to important physiological events such as secretion by neuronal and endocrine cells. Rem2, a GTPase restricted to neuroendocrine cell types, regulates VDCC activity by a mechanism that involves interaction with the VDCC beta subunit (Ca(V)beta). Mapping studies reveal that Rem2 binds to the guanylate kinase domain (GK) of the Ca(V)beta subunit that also contains the high affinity binding site for the pore forming and voltage sensing VDCC alpha subunit (Ca(V)alpha) interaction domain (AID). Moreover, fine mapping indicates that Rem2 binds to the GK domain in a region distinct from the AID interaction site, and competitive inhibition studies reveal that Rem2 does not disrupt Ca(V)alpha - Ca(V)beta binding. Instead, the Ca(V)beta subunit appears to serve a scaffolding function, simultaneously binding both Rem2 and AID. Previous studies have found that in addition to Ca(V)beta binding, Rem2 must be localized to the plasma membrane to inhibit VDCC function. Plasma membrane localization requires the C-terminus of Rem2 and binding studies indicate that this domain directs phosphorylated phosphatidylinositide (PIP) lipids association. Plasma membrane localization may provide a unique point of regulation since the ability of Rem2 to bind PIP lipids is inhibited by the phosphoserine dependant binding of 14-3-3 proteins. Thus, in addition to Ca(V)beta binding, VDCC blockade by Rem2 is likely to be controlled by both the localized concentration of membrane PIP lipids and direct 14-3-3 binding to the Rem2 C-terminus.     

Downregulation of the voltage-dependent calcium channel (VDCC) beta-subunit mRNAs in pancreatic islets of type 2 diabetic rats

The present study was undertaken to determine whether altered expression of the VDCC beta-subunits in pancreatic beta-cells could play a role in the changes in beta-cell sensitivity to glucose that occur with diabetes. Application of competitive RT-PCR procedure revealed that in normal Wistar rats, LETO and prediabetic OLETF rats, the beta(2)-subunit mRNA levels were 60-200-fold greater than the levels for the beta(3)-subunit. These findings suggest that the beta(2)-subunit as well as the beta-cell type VDCC1 alpha(1)-subunit may be the predominant form of the VDCC expressed in pancreatic beta-cells. The levels of mRNA encoding the beta-subunits and the beta-cell type alpha(1)-subunit as well as insulin were significantly reduced in diabetic rats. Perfusion experiments revealed that diabetic rats showed the higher basal insulin secretion and profoundly impaired insulin secretory responses to glucose compared with non-diabetic rats. Alternatively, impaired insulin secretory responses to glucose in high dose glucose-infused rats were recovered partly with the elevation of mRNA levels of the VDCC beta(2)- and beta(3)-subunits as well as the alpha(1)-subunit by the treatment with diazoxide. Thus, considering the possibility that the most striking effect of the VDCC alpha(1) beta-subunit coexpression in pancreatic beta-cells might occur on activation kinetics like the skeletal muscle, the impairment of further activation of the VDCCs to acute glucose challenge caused by the reduced expressions of the alpha(1) beta-subunits mRNAs in type 2 diabetic animals might be at least partly associated with the alterations in beta-cell sensitivity to glucose.     

Analysis of the complex between Ca2+ channel beta-subunit and the Rem GTPase

Voltage-gated calcium channels are multiprotein complexes that regulate calcium influx and are important contributors to cardiac excitability and contractility. The auxiliary beta-subunit (CaV beta) binds a conserved domain (the alpha-interaction domain (AID)) of the pore-forming CaV alpha1 subunit to modulate channel gating properties and promote cell surface trafficking. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. Here, we describe the Rem-association domain within CaV beta2a. The Rem interaction module is located in a approximately 130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, CaV beta mutants were identified that lost the ability to bind AID but retained their association with Rem, indicating that the AID and Rem association sites of CaV beta2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for CaV beta2a interaction is lower than that of AID for CaV beta2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of CaV beta2a with AID. Instead, CaV beta can simultaneously associate with both Rem and CaV alpha1-AID. Previous studies had suggested that RGK proteins may regulate Ca2+ channel activity by blocking the association of CaV beta subunits with CaV alpha1 to inhibit plasma membrane trafficking. However, surface biotinylation studies in HIT-T15 cells indicate that Rem can acutely modulate channel function without decreasing the density of L-type channels at the plasma membrane. Together these data suggest that Rem-dependent Ca2+ channel modulation involves formation of a Rem x CaV beta x AID regulatory complex without the need to disrupt CaV alpha1 x CaV beta association or alter CaV alpha1 expression at the plasma membrane.     

The Src homology 3 domain of the beta-subunit of voltage-gated calcium channels promotes endocytosis via dynamin interaction

High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary beta-subunit to the alpha-interaction-domain in the pore-forming alpha1-subunit is required to form functional channels. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the alpha-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated beta-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the alpha1-subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing alpha1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length beta-subunit also down-regulates voltage-gated calcium channels but only when lacking the alpha-interaction domain. Moreover, isolated Src homology 3 domain and the full-length beta-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the beta-subunit regulates the turnover of voltage-gated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the beta-subunit to the alpha1-pore-forming subunit. Our findings define a novel function for the beta-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.     

Mutations of nonconserved residues within the calcium channel alpha1-interaction domain inhibit beta-subunit potentiation

Voltage-dependent calcium channels consist of a pore-forming subunit (Ca(V)alpha(1)) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary beta-subunit (Ca(V)beta) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. Ca(V)beta binds reversibly to a conserved consensus sequence in Ca(V)alpha(1), the alpha(1)-interaction domain (AID), which forms an alpha-helix when complexed with Ca(V)beta. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on Ca(V)beta. Here, we studied the effect of mutating residues located opposite to the AID-Ca(V)beta contact surface in Ca(V)1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other Ca(V)alpha(1) subunits (E462R, K465N, D469S, and Q473K) hinders Ca(V)beta's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for Ca(V)beta. At the single channel level, these Ca(V)1.2 mutants coexpressed with Ca(V)beta(2a) visit high open probability mode less frequently than wild-type channels. On the other hand, Ca(V)1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs Ca(V)beta binding), or a deletion of the whole AID sequence, does not exhibit Ca(V)beta-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of Ca(V)beta, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that Ca(V)beta-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that Ca(V)beta spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by Ca(V)beta.     

Structural analysis of the voltage-dependent calcium channel beta subunit functional core and its complex with the alpha 1 interaction domain

Voltage-dependent calcium channels (VDCC) are multiprotein assemblies that regulate the entry of extracellular calcium into electrically excitable cells and serve as signal transduction centers. The alpha1 subunit forms the membrane pore while the intracellular beta subunit is responsible for trafficking of the channel to the plasma membrane and modulation of its electrophysiological properties. Crystallographic analyses of a beta subunit functional core alone and in complex with a alpha1 interaction domain (AID) peptide, the primary binding site of beta to the alpha1 subunit, reveal that beta represents a novel member of the MAGUK protein family. The findings illustrate how the guanylate kinase fold has been fashioned into a protein-protein interaction module by alteration of one of its substrate sites. Combined results indicate that the AID peptide undergoes a helical transition in binding to beta. We outline the mechanistic implications for understanding the beta subunit's broad regulatory role of the VDCC, particularly via the AID.     

Folding of active calcium channel beta(1b) -subunit by size-exclusion chromatography and its role on channel function

Voltage-gated calcium channels mediate the influx of Ca(2+) ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming alpha(1)-subunit and the auxiliary beta- and alpha(2)delta-subunits. The beta-subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity. Four beta-subunit genes have been cloned, beta(1-4), with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The beta(1b)-subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming alpha(1)- and the regulatory beta-subunit. However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria. We applied an on-column refolding procedure based on size exclusion chromatography to fold the beta(1b)-subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The beta(1b)-subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the alpha(1)-subunit (the alpha-interaction domain). Using the cut-open oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac alpha(1)-subunit (alpha(1C)) co-injected with folded-beta(1b)-protein or beta(1b)-cRNA. We demonstrate that the co-expression of the alpha(1C)-subunit with either folded-beta(1b)-protein or beta(1b)-cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the beta(1b)-subunit primarily modulates channel activity, rather than expression.     

Voltage-gated rearrangements associated with differential beta-subunit modulation of the L-type Ca(2+) channel inactivation

Auxiliary beta-subunits bound to the cytoplasmic alpha(1)-interaction domain of the pore-forming alpha(1C)-subunit are important modulators of voltage-gated Ca(2+) channels. The underlying mechanisms are not yet well understood. We investigated correlations between differential modulation of inactivation by beta(1a)- and beta(2)- subunits and structural responses of the channel to transition into distinct functional states. The NH(2)-termini of the alpha(1C)- and beta-subunits were fused with cyan or yellow fluorescent proteins, and functionally coexpressed in COS1 cells. Fluorescence resonance energy transfer (FRET) between them or with membrane-trapped probes was measured in live cells under voltage clamp. It was found that in the resting state, the tagged NH(2)-termini of the alpha(1C)- and beta-subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between alpha(1C) and beta(1a) suggesting mutual reorientation of the NH(2)-termini, but their distance vis-à-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the beta(1a)-subunit was replaced by beta(2). Differential beta-subunit modulation of inactivation and of FRET between alpha(1C) and beta were eliminated by inhibition of the slow inactivation. Thus, differential beta-subunit modulation of inactivation correlates with the voltage-gated motion between the NH(2)-termini of alpha(1C)- and beta-subunits and targets the mechanism of slow voltage-dependent inactivation.     

Functional modularity of the beta-subunit of voltage-gated Ca2+ channels

The beta-subunit of voltage-gated Ca(2+) channels plays a dual role in chaperoning the channels to the plasma membrane and modulating their gating. It contains five distinct modular domains/regions, including the variable N- and C-terminus, a conserved Src homology 3 (SH3) domain, a conserved guanylate kinase (GK) domain, and a connecting variable and flexible HOOK region. Recent crystallographic studies revealed a highly conserved interaction between the GK domain and alpha interaction domain (AID), the high-affinity binding site in the pore-forming alpha(1) subunit. Here we show that the AID-GK domain interaction is necessary for beta-subunit-stimulated Ca(2+) channel surface expression and that the GK domain alone can carry out this function. We also examined the role of each region of all four beta-subunit subfamilies in modulating P/Q-type Ca(2+) channel gating and demonstrate that the beta-subunit functions modularly. Our results support a model that the conserved AID-GK domain interaction anchors the beta-subunit to the alpha(1) subunit, enabling alpha(1)-beta pair-specific low-affinity interactions involving the N-terminus and the HOOK region, which confer on each of the four beta-subunit subfamilies its distinctive modulatory properties.     

Cytoplasmic and transmembrane domain deletions of Na,K-ATPase beta-subunit. Effects on subunit assembly and intracellular transport.

cDNAs encoding Na,K-ATPase beta-subunits containing deletions in the cytoplasmic domain or in the single membrane-spanning domain of the molecule were constructed and expressed in mouse L cells to determine the effect(s) of deletions in these domains on alpha/beta-subunit assembly and intracellular targeting. Avian beta-subunits lacking some or all of the cytoplasmic domain (endodomain) assemble with the endogenous mouse alpha-subunit and are correctly transported to the plasma membrane. Mutants containing deletions in the transmembrane domain were constructed by fusing portions of cDNAs encoding the amino-terminal one-third of human beta-subunit deletion mutants with avian beta-subunit cDNA encoding the carboxyl two-thirds of the molecule. A deletion of 3 amino acids in transmembrane domain resulted in correct alpha/beta-subunit assembly and localization to the plasma membrane. In contrast, deletions of 5 or more amino acids in the transmembrane domain prevented expression of the beta-subunit at the cell surface and resulted in the accumulation of these molecules in the ER. In spite of these targeting differences, all beta-subunit mutants capable of membrane insertion were also able to assemble with the alpha-subunit. These results suggest that the specificity for alpha/beta assembly resides in the ectodomains of the subunits.     

Functional alteration of dihydropyridine-sensitive Ca(2+) channels in the adrenal glomerulosa of pregnant rats

Our previous work on aldosterone secretion suggested that dihydropyridine-sensitive calcium channels, one type of voltage-dependent calcium channels (VDCC), are functionally impaired in adrenal capsule preparations from the pregnant rat. The aim of this study was to determine whether, during pregnancy, the density and/or activity of these channels is altered in the adrenal zona glomerulosa. These VDCC measured with [(3)H]nitrendipine binding were not different between membrane preparations of nonpregnant and pregnant rats. Western blots were performed using two different antibodies, a polyclonal (PcAb) directed against the alpha(1)-subunit of VDCC and a monoclonal (McAb) that recognizes an intracellular domain of that protein. McAb immunoreactivity showed a significant decrease in preparations from pregnant rats, whereas no difference was observed with PcAb. VDCC activity was estimated by (45)Ca(2+) uptake in isolated adrenal cortex and by intracellular calcium concentration ([Ca(2+)](i)) in adrenal glomerulosa cells with the Ca(2+) probe fura PE3. These measurements revealed that KCl stimulation produced greater Ca(2+) influx in nonpregnant than in pregnant rats. Nifedipine (a blocker of VDCC) inhibited this stimulation only in nonpregnant rats, whereas BAY K 8644 (an activator of VDCC) increased Ca(2+) influx in pregnant rats only. These data suggest that, during pregnancy, the altered regulation of calcium homeostasis in adrenal glomerulosa is linked to a conformational alteration of VDCC.     

Specific translocation of protein kinase Calpha to the plasma membrane requires both Ca2+ and PIP2 recognition by its C2 domain

The C2 domain of protein kinase Calpha (PKCalpha) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCalpha and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCalpha Ca2+-binding loops (CBLs) and PIP2 binding site (beta-strands 3-4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCalpha C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on beta-strands 3-4, which bind to plasma membrane PIP2.     

A specific tryptophan in the I-II linker is a key determinant of beta-subunit binding and modulation in Ca(V)2.3 calcium channels

The ancillary beta subunits modulate the activation and inactivation properties of high-voltage activated (HVA) Ca(2+) channels in an isoform-specific manner. The beta subunits bind to a high-affinity interaction site, alpha-interaction domain (AID), located in the I-II linker of HVA alpha1 subunits. Nine residues in the AID motif are absolutely conserved in all HVA channels (QQxExxLxGYxxWIxxxE), but their contribution to beta-subunit binding and modulation remains to be established in Ca(V)2.3. Mutations of W386 to either A, G, Q, R, E, F, or Y in Ca(V)2.3 disrupted [(35)S]beta3-subunit overlay binding to glutathione S-transferase fusion proteins containing the mutated I-II linker, whereas mutations (single or multiple) of nonconserved residues did not affect the protein-protein interaction with beta3. The tryptophan residue at position 386 appears to be an essential determinant as substitutions with hydrophobic (A and G), hydrophilic (Q, R, and E), or aromatic (F and Y) residues yielded the same results. beta-Subunit modulation of W386 (A, G, Q, R, E, F, and Y) and Y383 (A and S) mutants was investigated after heterologous expression in Xenopus oocytes. All mutant channels expressed large inward Ba(2+) currents with typical current-voltage properties. Nonetheless, the typical hallmarks of beta-subunit modulation, namely the increase in peak currents, the hyperpolarization of peak voltages, and the modulation of the kinetics and voltage dependence of inactivation, were eliminated in all W386 mutants, although they were preserved in part in Y383 (A and S) mutants. Altogether these results suggest that W386 is critical for beta-subunit binding and modulation of HVA Ca(2+) channels.     

Evidence for two concentration-dependent processes for beta-subunit effects on alpha1B calcium channels

beta-Subunits of voltage-dependent Ca(2+) channels regulate both their expression and biophysical properties. We have injected a range of concentrations of beta3-cDNA into Xenopus oocytes, with a fixed concentration of alpha1B (Ca(V)2.2) cDNA, and have quantified the corresponding linear increase of beta3 protein. The concentration dependence of a number of beta3-dependent processes has been studied. First, the dependence of the a1B maximum conductance on beta3-protein occurs with a midpoint around the endogenous concentration of beta3 (approximately 17 nM). This may represent the interaction of the beta-subunit, responsible for trafficking, with the I-II linker of the nascent channel. Second, the effect of beta3-subunits on the voltage dependence of steady-state inactivation provides evidence for two channel populations, interpreted as representing alpha1B without or with a beta3-subunit, bound with a lower affinity of 120 nM. Third, the effect of beta3 on the facilitation rate of G-protein-modulated alpha1B currents during a depolarizing prepulse to +100 mV provides evidence for the same two populations, with the rapid facilitation rate being attributed to Gbetagamma dissociation from the beta-subunit-bound alpha1B channels. The data are discussed in terms of two hypotheses, either binding of two beta-subunits to the alpha1B channel or a state-dependent alteration in affinity of the channel for the beta-subunit.     

The role of ion channels in insulin secretion.

Ion channels in beta cells regulate electrical and secretory activity in response to metabolic, pharmacologic, or neural signals by controlling the permeability to K+ and Ca2+. The ATP-sensitive K+ channels act as a switch that responds to fuel secretagogues or sulfonylureas to initiate depolarization. This depolarization opens voltage-dependent calcium channels (VDCC) to increase the amplitude of free cytosolic Ca2+ levels ([Ca2+]i), which triggers exocytosis. Acetyl choline and vasopressin (VP) both potentiate the acute effects of glucose on insulin secretion by generating inositol 1,4,5-trisphosphate to release intracellular Ca2+; VP also potentiates sustained insulin secretion by effects on depolarization. In contrast, inhibitors of insulin secretion decrease [Ca2+]i by either hyperpolarizing the beta cell or by receptor-mediated, G-protein-coupled effects to decrease VDCC activity. Repolarization is initiated by voltage- and Ca(2+)-activated K+ channels. A human insulinoma voltage-dependent K+ channel cDNA was recently cloned and two types of alpha 1 subunits of the VDCC have been identified in insulin-secreting cell lines. Determining how ion channels regulate insulin secretion in normal and diabetic beta cells should provide pathophysiologic insight into the beta cell signal transduction defect characteristic of non-insulin dependent diabetes (NIDDM).