Targeting of protein phosphatases PP2A and PP2B to the C-terminus of the L-type calcium channel Ca v1.2

The L-type Ca(2+) channel Ca(v)1.2 forms macromolecular signaling complexes that comprise the β(2) adrenergic receptor, trimeric G(s) protein, adenylyl cyclase, and cAMP-dependent protein kinase (PKA) for efficient signaling in heart and brain. The protein phosphatases PP2A and PP2B are part of this complex. PP2A counteracts increase in Ca(v)1.2 channel activity by PKA and other protein kinases, whereas PP2B can either augment or decrease Ca(v)1.2 currents in cardiomyocytes depending on the precise experimental conditions. We found that PP2A binds to two regions in the C-terminus of the central, pore-forming α(1) subunit of Ca(v)1.2: one region spans residues 1795-1818 and the other residues 1965-1971. PP2B binds immediately downstream of residue 1971. Injection of a peptide that contained residues 1965-1971 and displaced PP2A but not PP2B from endogenous Ca(v)1.2 increased basal and isoproterenol-stimulated L-type Ca(2+) currents in acutely isolated cardiomyocytes. Together with our biochemical data, these physiological results indicate that anchoring of PP2A at this site of Ca(v)1.2 in the heart negatively regulates cardiac L-type currents, likely by counterbalancing basal and stimulated phosphorylation that is mediated by PKA and possibly other kinases.     

Unchanged beta-adrenergic stimulation of cardiac L-type calcium channels in Ca v 1.2 phosphorylation site S1928A mutant mice

Phosphorylation of serine 1928 (Ser(1928)) of the cardiac Ca(v)1.2 subunit of L-type Ca(2+) channels has been proposed as the mechanism for regulation of L-type Ca(2+) channels by protein kinase A (PKA). To test this directly in vivo, we generated a knock-in mouse with targeted mutation of Ser(1928) to alanine. This mutation did not affect basal L-type current characteristics or regulation of the L-type current by PKA and the beta-adrenergic receptor, whereas the mutation abolished phosphorylation of Ca(v)1.2 by PKA. Therefore, our data show that PKA phosphorylation of Ser(1928) of Ca(v)1.2 is not functionally involved in beta-adrenergic stimulation of Ca(v)1.2-mediated Ca(2+) influx into the cardiomyocyte.     

Deletion of the C-terminal phosphorylation sites in the cardiac β-subunit does not affect the basic β-adrenergic response of the heart and the Ca(v)1.2 channel

Phosphorylation of the cardiac β subunit (Ca(v)β(2)) of the Ca(v)1.2 L-type Ca(2+) channel complex has been proposed as a mechanism for regulation of L-type Ca(2+) channels by various protein kinases including PKA, CaMKII, Akt/PKB, and PKG. To test this hypothesis directly in vivo, we generated a knock-in mouse line with targeted mutation of the Ca(v)β(2) gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; βStop mouse). This mutation prevented translation of the Ca(v)β(2) C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac βStop mice were born at Mendelian ratio, had a normal life expectancy, and normal basal L-type I(Ca). The regulation of the L-type current by stimulation of the β-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). βStop mice were cross-bred with mice expressing the Ca(v)1.2 gene containing the mutation S1928A (SAβStop) or S1512A and S1570A (SFβStop) in the C terminus of the α(1C) subunit. The β-adrenergic regulation of the cardiac I(Ca) was unaltered in these mouse lines. In contrast, truncation of the Ca(v)1.2 at Asp(1904) abolished β-adrenergic up-regulation of I(Ca) in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Ca(v)β(2), Ser(1928), Ser(1512), and Ser(1570) of the Ca(v)1.2 protein is functionally not involved in the adrenergic regulation of the murine cardiac Ca(v)1.2 channel.     

Binding of protein phosphatase 2A to the L-type calcium channel Cav1.2 next to Ser1928, its main PKA site, is critical for Ser1928 dephosphorylation

The cAMP-dependent protein kinase (PKA) controls a large number of cellular functions. One critical PKA substrate in the brain and heart is the L-type Ca(2+) channel Ca(v)1.2, the activity of which is upregulated by PKA. The main PKA phosphorylation site is serine 1928 in the central pore forming alpha(1)1.2 subunit of Ca(v)1.2. PKA is bound to Ca(v)1.2 within a macromolecular signaling complex consisting of the beta(2) adrenergic receptor, trimeric G(s) protein, and adenylyl cyclase for fast, localized, and hence specific signaling [Davare, M. A., Avdonin, V., Hall, D. D., Peden, E. M., Buret, A., Weinberg, R. J., Horne, M. C., Hoshi, T., and Hell, J. W. (2001) Science 293, 98-101]. Protein phosphatase 2A (PP2A) serves to effectively balance serine 1928 phosphorylation by PKA through its association with the Ca(v)1.2 complex [Davare, M. A., Horne, M. C., and Hell, J. W. (2000) J. Biol. Chem. 275, 39710-39717]. We now show that native PP2A holoenzymes, as well as the catalytic subunit itself, bind to alpha(1)1.2 immediately downstream of serine 1928. Of those holoenzymes, only heterotrimeric PP2A containing B' and B' ' subunits copurify with alpha(1)1.2. Preventing the binding of PP2A by truncating alpha(1)1.2 28 residues downstream of serine 1928 hampers its dephosphorylation in intact cells. Our results demonstrate for the first time that a stable interaction of PP2A with Ca(v)1.2 is required for effective reversal of PKA-mediated channel phosphorylation. Accordingly, PKA as well as PP2A are constitutively associated with Ca(v)1.2 for its proper regulation by phosphorylation and dephosphorylation of serine 1928.     

Ser1928 is a common site for Cav1.2 phosphorylation by protein kinase C isoforms

Voltage-dependent Ca(2+) channel (Ca(v)1.2, L-type Ca(2+) channel) function is highly regulated by hormones and neurotransmitters in large part through the activation of kinases and phosphatases. Regulation of Ca(v)1.2 by protein kinase C (PKC) is of significant physiologic importance, mediating, in part, the cardiac response to hormonal regulation. Although PKC has been reported to mediate activation and/or inhibition of Ca(v)1.2 function, the molecular mechanisms mediating the response have not been definitively elucidated. We show that PKC forms a macromolecular complex with the alpha(1c) subunit of Ca(v)1.2 through direct interaction with the C terminus. This interaction leads to phosphorylation of the channel in response to activators of PKC. We identify Ser(1928) as the residue that is phosphorylated by PKC in vitro and in vivo. Ser(1928) has been identified previously as the site mediating, in part, the protein kinase A up-regulation of channel activity. Thus, the protein kinase A and PKC signaling pathways converge on the Ca(v)1.2 complex at Ser(1928) to increase channel activity. Our results identify two mechanisms leading to regulation of Ca(v)1.2 activity by PKC: pre-association of the channel with PKC isoforms and phosphorylation of specific sites within the alpha(1c) subunit.     

Facilitation and Ca2+-dependent inactivation are modified by mutation of the Ca(v)1.2 channel IQ motif

The heart muscle responds to physiological needs with a short-term modulation of cardiac contractility. This process is determined mainly by properties of the cardiac L-type Ca(2+) channel (Ca(v)1.2), including facilitation and Ca(2+)-dependent inactivation (CDI). Both facilitation and CDI involve the interaction of calmodulin with the IQ motif of the Ca(v)1.2 channel, especially with Ile-1624. To verify this hypothesis, we created a mouse line in which Ile-1624 was mutated to Glu (Ca(v)1.2(I1624E) mice). Homozygous Ca(v)1.2(I1624E) mice were not viable. Therefore, we inactivated the floxed Ca(v)1.2 gene of heterozygous Ca(v)1.2(I1624E) mice by the α-myosin heavy chain-MerCreMer system. The resulting I/E mice were studied at day 10 after treatment with tamoxifen. Electrophysiological recordings in ventricular cardiomyocytes revealed a reduced Ca(v)1.2 current (I(Ca)) density in I/E mice. Steady-state inactivation and recovery from inactivation were modified in I/E versus control mice. In addition, voltage-dependent facilitation was almost abolished in I/E mice. The time course of I(Ca) inactivation in I/E mice was not influenced by the use of Ba(2+) as a charge carrier. Using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid as a chelating agent for intracellular Ca(2+), inactivation of I(Ca) was slowed down in control but not I/E mice. The results show that the I/E mutation abolishes Ca(2+)/calmodulin-dependent regulation of Ca(v)1.2. The Ca(v)1.2(I1624E) mutation transforms the channel to a phenotype mimicking CDI.     

Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP-dependent protein kinase

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates a vast number of cellular functions. An important target for PKA in brain and heart is the class C L-type Ca(2+) channel (Ca(v)1.2). PKA phosphorylates serine 1928 in the central, pore-forming alpha(1C) subunit of this channel. Regulation of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.     

Calmodulin effect on purified rat cortical plasma membrane Ca(2+)-ATPase in different phosphorylation states

The plasma membrane Ca(2+)-ATPase in neuronal tissue plays an important role in fine tuning of the intracellular Ca(2+) concentration. The enzyme exhibits a high degree of tissue specificity and is regulated by several mechanisms. Here we analysed the relationship between separate modes of Ca(2+)-ATPase regulation, i.e., reversible phosphorylation processes mediated by protein kinases A and C, protein phosphatases PP1 and PP2A, and stimulation by calmodulin. The activity of PKA- or PKC-phosphorylated Ca(2+)-ATPase was influenced by the further addition of calmodulin, and this effect was more pronounced for PKC-phosphorylated calcium pump. In both cases the fluorescence study revealed the increased calmodulin binding, and for PKA-mediated phosphorylation it was correlated with a higher affinity of calcium pump for calmodulin. The incubation of Ca(2+)-ATPase with CaM prior to protein kinases action revealed that CaM presence counteracts the stimulatory effect of PKA and PKC. Under the in vitro assay cortical Ca(2+)-ATPase was a substrate for PP1 and PP2A. Protein phosphatases decreased both the basal activity of Ca(2+)-ATPase and its affinity for calmodulin. Fluorescence analysis confirmed the lowered ability of dephosphorylated Ca(2+)-ATPase for calmodulin binding. These results may suggest that interaction of CaM with calcium pump and its stimulatory action could be a partly separate phenomenon that is dependent on the phosphorylation state of Ca(2+)-ATPase.     

Mechanisms underlying heterogeneous Ca2+ sparklet activity in arterial smooth muscle

In arterial smooth muscle, single or small clusters of Ca(2+) channels operate in a high probability mode, creating sites of nearly continual Ca(2+) influx (called "persistent Ca(2+) sparklet" sites). Persistent Ca(2+) sparklet activity varies regionally within any given cell. At present, the molecular identity of the Ca(2+) channels underlying Ca(2+) sparklets and the mechanisms that give rise to their spatial heterogeneity remain unclear. Here, we used total internal reflection fluorescence (TIRF) microscopy to directly investigate these issues. We found that tsA-201 cells expressing L-type Cavalpha1.2 channels recapitulated the general features of Ca(2+) sparklets in cerebral arterial myocytes, including amplitude of quantal event, voltage dependencies, gating modalities, and pharmacology. Furthermore, PKCalpha activity was required for basal persistent Ca(2+) sparklet activity in arterial myocytes and tsA-201 cells. In arterial myocytes, inhibition of protein phosphatase 2A (PP2A) and 2B (PP2B; calcineurin) increased Ca(2+) influx by evoking new persistent Ca(2+) sparklet sites and by increasing the activity of previously active sites. The actions of PP2A and PP2B inhibition on Ca(2+) sparklets required PKC activity, indicating that these phosphatases opposed PKC-mediated phosphorylation. Together, these data unequivocally demonstrate that persistent Ca(2+) sparklet activity is a fundamental property of L-type Ca(2+) channels when associated with PKC. Our findings support a novel model in which the gating modality of L-type Ca(2+) channels vary regionally within a cell depending on the relative activities of nearby PKCalpha, PP2A, and PP2B.     

Reconstituted slow muscarinic inhibition of neuronal (Ca(v)1.2c) L-type Ca2+ channels

Ca(2+) influx through L-type channels is critical for numerous physiological functions. Relatively little is known about modulation of neuronal L-type Ca(2+) channels. We studied modulation of neuronal Ca(V)1.2c channels heterologously expressed in HEK293 cells with each of the known muscarinic acetylcholine receptor subtypes. Galphaq/11-coupled M1, M3, and M5 receptors each produced robust inhibition of Ca(V)1.2c, whereas Galphai/o-coupled M2 and M4 receptors were ineffective. Channel inhibition through M1 receptors was studied in detail and was found to be kinetically slow, voltage-independent, and pertussis toxin-insensitive. Slow inhibition of Ca(V)1.2c was blocked by coexpressing RGS2 or RGS3T or by intracellular dialysis with antibodies directed against Galphaq/11. In contrast, inhibition was not reduced by coexpressing betaARK1ct or Galphat. These results indicate that slow inhibition required signaling by Galphaq/11, but not Gbetagamma, subunits. Slow inhibition did not require Ca(2+) transients or Ca(2+) influx through Ca(V)1.2c channels. Additionally, slow inhibition was insensitive to pharmacological inhibitors of phospholipases, protein kinases, and protein phosphatases. Intracellular BAPTA prevented slow inhibition via a mechanism other than Ca(2+) chelation. The cardiac splice-variant of Ca(V)1.2 (Ca(V)1.2a) and a splice-variant of the neuronal/neuroendocrine Ca(V)1.3 channel also appeared to undergo slow muscarinic inhibition. Thus, slow muscarinic inhibition may be a general characteristic of L-type channels having widespread physiological significance.     

Functional embryonic cardiomyocytes after disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse

The L-type alpha(1C) (Ca(v)1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated the Ca(v)1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Ca(v)1. 2-/-) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/-), and homozygous (-/-) Ca(v)1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 microm nisoldipine. In (+/+), (+/-), and (-/-) cardiomyocytes, an L-type Ba(2+) inward current (I(Ba)) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of -80 mV, nisoldipine blocked I(Ba) of day 12.5 p.c. (+/+) and (+/-) cells with two IC(50) values of approximately 0.1 and approximately 1 microm. Inhibition of I(Ba) of (-/-) cardiomyocytes was monophasic with an IC(50) of approximately 1 microm. The low affinity I(Ba) was also present in cardiomyocytes of homozygous alpha(1D) (Ca(v)1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.     

Cardiac-specific overexpression of the alpha(1) subunit of the L-type voltage-dependent Ca(2+) channel in transgenic mice. Loss of isoproterenol-induced contraction.

The L-type voltage-dependent calcium channel (L-VDCC) regulates calcium influx in cardiac myocytes. Activation of the beta-adrenergic receptor (betaAR) pathway causes phosphorylation of the L-VDCC and that in turn increases Ca(2+) influx. Targeted expression of the L-VDCC alpha(1) subunit in transgenic (Tg) mouse ventricles resulted in marked blunting of the betaAR pathway. Inotropic and lusitropic responses to isoproterenol and forskolin in Tg hearts were significantly reduced. Likewise, Ca(2+) current augmentation induced by iso- proterenol and forskolin was markedly depressed in Tg cardiomyocytes. Despite no change in betaAR number, isoproterenol-stimulated adenylyl cyclase activity was absent in Tg membranes and NaF and forskolin responses were reduced. We postulate an important pathway for regulation of the betaAR by Ca(2+) channels.     

Lack of muscarinic regulation of Ca(2+) channels in G(i2)alpha gene knockout mouse hearts

The purpose of the present study was to examine the role of G(i2)alpha in Ca(2+) channel regulation using G(i2)alpha gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the beta-adrenergic agonist isoproterenol (Iso) on cardiac L-type Ca(2+) currents in both 129Sv wild-type (WT) and G(i2)alpha gene knockout (G(i2)alpha-/-) mice. Perfusion with CCh significantly inhibited the Ca(2+) current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca(2+) currents in G(i2)alpha-/- ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated Ca(2+) current in WT cardiomyocytes but not in G(i2)alpha-/- cells. These findings demonstrate that, whereas the Iso-G(s)alpha signal pathway is intact in G(i2)alpha gene knockout mouse hearts, these cells lack the inhibitory regulation of Ca(2+) channels by CCh. Therefore, G(i2)alpha is necessary for the muscarinic regulation of Ca(2+) channels in the mouse heart. Further studies are needed to delineate the possible interaction of G(i) and other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type Ca(2+) current in the heart.     

COOH-terminal association of human smooth muscle calcium channel Ca(v)1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Ca(v)) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa(v)1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCa(v)1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y(2134) prevented SH2 binding and resulted in reduced phosphorylation of hCa(v)1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y(1837), Y(1861), and Y(2134) were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y(1837-2134F). These data demonstrate that the COOH terminus of hCa(v)1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.     

Multifaceted roles of Arabidopsis PP6 phosphatase in regulating cellular signaling and plant development

Reversible protein phosphorylation catalyzed by kinases and phosphatases is a major form of posttranslational regulation that plays a central role in regulating many signaling pathways. While large families of both protein kinases and protein phosphatases have been identified in plants, kinases outnumber phosphatases. This raises the question of how a relatively limited number of protein phosphatases can maintain protein phosphorylation homeostasis in a cell. Recent studies have shown that Arabidopsis FyPP1 (Phytochrome-associated serine/threonine protein phosphatase 1) and FyPP3 encode the catalytic subunits of protein phosphatase 6 (PP6), and that they directly binds to the A subunits of protein phosphatase 2A (PP2AA proteins), and SAL (SAPS domain-like) proteins to form the heterotrimeric PP6 holoenzyme complex. Emerging evidence is suggesting that PP6, acts in opposition with multiple classes of kinases, to regulate the phosphorylation status of diverse substrates and subsequently numerous developmental processes and responses to environmental stimuli.     

Caveolin-3 regulates protein kinase A modulation of the Ca(V)3.2 (alpha1H) T-type Ca2+ channels

Voltage-gated T-type Ca(2+) channel Ca(v)3.2 (α(1H)) subunit, responsible for T-type Ca(2+) current, is expressed in different tissues and participates in Ca(2+) entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca(v)3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca(2+) in different cells. We examined the localization and regulation of the Ca(v)3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca(v)3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca(v)3.1 and Ca(v)3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca(v)3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca(v)3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca(v)3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I(Ca,T)) and adenovirus (AdCa(v)3.2)-mediated increase in peak Ca(v)3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I(Ca,T) by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca(v)3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca(2+) signaling during normal and pathological processes.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.