Structures and functions of calcium channel beta subunits

Calcium channel beta subunits have profound effects on how alpha1 subunits perform. In this article we summarize our present knowledge of the primary structures of beta subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with alpha1 subunits, the effects of beta subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by beta subunits, such as long-lasting prepulse facilitation of alpha1C (absent in alpha1E) and inhibition by G protein betagamma dimer of alpha1E, absent in alpha1C. One beta subunit, a brain beta2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of alpha1 in oocytes requires a beta subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca2+ channels to account for "alpha1 alone" channels seen in cells with limited beta subunit expression. In one model, beta dissociates from the mature alpha1 after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of beta would then have subunit composition alpha1beta. In the other model, the "chaperoning" beta remains associated with the mature channel and "alpha1 alone" channels would in fact be alpha1beta channels. Upon co-expression of high levels of beta the regulated channels would have composition [alpha1beta]beta.     

Protein kinase C-mediated regulation of L-type Ca channels from skeletal muscle requires phosphorylation of the alpha 1 subunit

Dihydropyridine-sensitive, L-type Ca channels are hetero-oligomeric proteins that are modulated in certain cell types by protein kinase C (PKC). In native skeletal muscle membranes, PKC phosphorylates the alpha 1 and beta subunits of these Ca channels and modulates channel activity. However, it is unknown if phosphorylation of both subunits is necessary for PKC-mediated channel regulation. Here we report that stoichiometric phosphorylation of the alpha 1 subunit was required for activation of these Ca channels by PKC, while PKC-mediated phosphorylation of the beta subunit alone did not modify channel activity. Furthermore, reversal of the functional effects of PKC by protein phosphatase-1c was quantitatively correlated with dephosphorylation of the alpha 1 subunit.     

Complexes of the alpha1C and beta subunits generate the necessary signal for membrane targeting of class C L-type calcium channels

In the present study, we investigated the role of channel subunits in the membrane targeting of voltage-dependent L-type calcium channel complexes. We co-expressed the calcium channel pore-forming alpha1C subunit with different accessory beta subunits in HEK-tsA201 cells and examined the subcellular localization of the channel subunits by immunohistochemistry using confocal microscopy and whole-cell radioligand binding studies. While the pore-forming alpha1C subunit exhibited perinuclear staining when expressed alone, and several of the wild-type and mutant beta subunits also exhibited intracellular staining, co-expression of the alpha1C subunit with either the wild-type beta2a subunit, a palmitoylation-deficient beta2a(C3S/C4S) mutant or three other nonpalmitoylated beta isoforms (beta1b, beta3, and beta4 subunits) resulted in the redistribution of both the alpha1C and beta subunits into clusters along the cell surface. Furthermore, the redistribution of calcium channel complexes to the plasma membrane was observed when alpha1C was co-expressed with an N- and C-terminal truncated mutant beta2a containing only the central conserved regions. However, when the alpha1C subunit was co-expressed with an alpha1 beta interaction-deficient mutant, beta2aBID-, we did not observe formation of the channels at the plasma membrane. In addition, an Src homology 3 motif mutant of beta2a that was unable to interact with the alpha1C subunit also failed to target channel complexes to the plasma membrane. Interestingly, co-expression of the pore-forming alpha1C subunit with the largely peripheral accessory alpha2 delta subunit was ineffective in recruiting alpha1C to the plasma membrane, while co-distribution of all three subunits was observed when beta2a was co-expressed with the alpha1C and alpha2 delta subunits. Taken together, our results suggested that the signal necessary for correct plasma membrane targeting of the class C L-type calcium channel complexes is generated as a result of a functional interaction between the alpha1 and beta subunits.     

cAMP-dependent phosphorylation of the cardiac L-type Ca channel: a missing link?

Cardiac inotropic effects of beta adrenergic agonists occur mainly through an increase in L-type (class C) calcium channel activity. This response has been attributed to phosphorylation of the L-type Ca channel, or a closely associated protein, by the cAMP-dependent protein kinase A (PKA). Among the three subunits forming the cardiac L-type Ca channel (alpha 1, beta and alpha 2-delta), biochemical studies have revealed that two subunits, alpha 1 and beta, are phosphorylated in vitro by protein kinase A, the alpha 1 subunit being the primary target. However, attempts to reconstitute the cAMP-dependent regulation of the expressed class C Ca channel, either in Xenopus oocytes or in cell lines, have provided contradictory results. We were unable to detect cAMP-dependent modulation of class C alpha 1 subunit Ca channels expressed in Xenopus oocytes, even when coinjected with auxiliary subunits beta and alpha 2-delta. Nevertheless, activity of Ca channels recorded from cardiac-mRNA injected oocytes was potentiated by injection of cAMP or PKA, even when expression of the beta subunit was suppressed using antisense oligonucleotide. Taken together, these results indicate that cAMP-dependent regulation does not exclusively involve the alpha 1 and the beta subunits of the Ca channel and suggest that unidentified protein(s), expressed in cardiac tissue, are most likely necessary.     

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

Maintenance of recombinant type A gamma-aminobutyric acid receptor function: role of protein tyrosine phosphorylation and calcineurin

In the present study, rundown of gamma-aminobutyric acid (GABA)-activated Cl- channels was studied in recombinant GABAA receptors stably expressed in human embryonic kidney cells (HEK 293), with conventional whole-cell and amphotericin B-perforated patch recording. When [ATP]i was lowered to 1 mM and resting [Ca++]i was buffered to a relatively high level, the response of alpha 3 beta 2 gamma 2 GABAA receptors to relatively low [GABA] (up to 50 microM) did not show rundown in the whole-cell configuration. However, high [GABA] (greater than 200 microM) induced significant rundown, which was observed by decreases in both the maximum GABA-induced current and GABA EC50. Rundown was prevented completely with a solution containing 4 mM Mg(++)-ATP and low resting [Ca++]i, or during perforated patch recording. The magnitude of rundown was comparable in alpha 1 beta 2 gamma 2 and beta 2 gamma 2 receptors. Neither stimulation nor inhibition of protein kinase A or protein kinase C had a significant effect on rundown. However, sodium metavanadate, an inhibitor of protein tyrosine phosphatase, significantly reduced rundown. In addition, inhibition of protein tyrosine kinase activity by either genistein or lavendustin A induced rundown of the GABA response. Inhibition of the Ca++/calmodulin-dependent phosphatase calcineurin with fenvalerate also prevented rundown of the response to GABA. Our results demonstrate that rundown of GABAA receptor function is concentration-dependent, due to depletion of ATP and/or unbuffered [Ca++]i, and does not depend on the presence or subtype of the alpha subunit. We propose that protein phosphorylation at a tyrosine kinase-dependent site, and a distinct unidentified site, which is dephosphorylated by calcineurin, maintains the function of GABAA receptors.     

Run-down of L-type Ca2+ channels occurs on the alpha 1 subunit

Run-down of L-type Ca2+ channels in CHO cells stably expressing alpha 1c, alpha 1c beta 1a, or alpha 1c beta 1a alpha 2 delta gamma subunits was studied using the patch-clamp technique (single channel recording). The channel activity (NPo) of alpha 1c channels was increased 4- and 8-fold by coexpression with beta 1a and beta 1a alpha 2 delta gamma, respectively. When membranes containing channels composed of different subunits were excised into basic internal solution, the channel activity exhibited run-down, the time-course of which was independent of the subunit composition. The run-down was restored by the application of calpastatin (or calpastatin contained in cytoplasmic P-fraction) + H-fraction (a high molecular mass fraction of bovine cardiac cytoplasm) + 3 mM ATP, which has been shown to reverse the run-down in native Ca2+ channels in the guinea-pig heart. The restoration level was 64.7, 63.5, and 66.4% for channels composed of alpha 1c, alpha 1c beta 1a, and alpha 1c beta 1a alpha 2 delta gamma, respectively, and was thus also independent of the subunit composition. We conclude that run-down of L-type Ca2+ channels occurs via the alpha 1 subunit and that the cytoplasmic factors maintaining Ca2+ channel activity act on the alpha 1 subunit.     

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.     

Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen

The interaction of the N-type calcium channel beta3 subunit with the alpha1B subunit alters the activation/inactivation kinetics and the maximal conductance of the channel. The defined protein-protein interaction of the human alpha1B and beta3 subunits provides a target for small-molecule modulation of N-type channel activity. We describe a high throughput screen based on a counterselection yeast two-hybrid assay, which was used to identify small molecules that disrupt alpha1B-beta3 subunit interactions and inhibit N-type calcium channel activity. These small molecules may be a new class of calcium channel antagonists with therapeutic potential.     

Targeted disruption of the Ca2+ channel beta3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding alpha1 subunit, considerably less is understood about how beta subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel beta3 subunit, the major Ca2+ channel beta subunit in neurons, by using a gene-targeting strategy. The beta3 deficient (beta3-/-) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic beta3-/- neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the "reluctant" and "willing" states reported for N-type channels. The absence of the beta3 subunit was associated with a hyperpolarizing shift of the "reluctant" component of activation. Norepinephrine inhibited wt and beta3-/- neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the beta3-/- mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

A molecular mechanism for electrical tuning of cochlear hair cells

Cochlear frequency selectivity in lower vertebrates arises in part from electrical tuning intrinsic to the sensory hair cells. The resonant frequency is determined largely by the gating kinetics of calcium-activated potassium (BK) channels encoded by the slo gene. Alternative splicing of slo from chick cochlea generated kinetically distinct BK channels. Combination with accessory beta subunits slowed the gating kinetics of alpha splice variants but preserved relative differences between them. In situ hybridization showed that the beta subunit is preferentially expressed by low-frequency (apical) hair cells in the avian cochlea. Interaction of beta with alpha splice variants could provide the kinetic range needed for electrical tuning of cochlear hair cells.     

Choroidal melanoma: diagnosis and management

We have expressed the alpha 1 A calcium channel subunit alone, and in combination with different beta subunits, and investigated the effect of the external Ba2+ concentration on the voltage dependence of activation. Increasing the external Ba2+ concentration from 2.5 to 40 mM induced in all cases a depolarising shift of the potential for half-activation. The magnitude of this shift however, was different depending on whether the alpha 1 A subunit was expressed alone or with a beta subunit. Consistently, calculated external surface-charge density and potential were larger when a beta subunit was expressed. These results suggest that expression of an auxiliary subunit can influence calcium channel gating by modifying the sensitivity of the voltage sensor to the membrane potential profile.     

Optimization of exopolysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus RR grown in a semidefined medium

The role of K+ channels in the nitric oxide-independent renal vasodilator effect of acetylcholine (Ach) was examined to address the hypothesis that the mechanism underlying this response was different from that of bradykinin, because an earlier study indicated the possibility of different mediators. We used the rat isolated, perfused kidney that was constricted with phenylephrine and treated with nitroarginine and indomethacin to inhibit nitric oxide synthase and cyclooxygenase, respectively. The nonspecific K+ channel inhibitors, procaine and tetraethylammonium (TEA), reduced vasodilator responses to Ach and cromakalim, but not those to nitroprusside. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, reduced vasodilator responses to cromakalim but did not affect those to Ach or nitroprusside. Charybdotoxin, an inhibitor of Ca(++)-activated K+ channels, reduced vasodilator responses to Ach without affecting those to cromakalim or nitroprusside. Iberiotoxin and apamin, inhibitors of large- and small-conductance Ca(++)-activated K+ channels, respectively, did not reduce vasodilation induced by Ach, cromakalim or nitroprusside. The inhibitor of cytochrome P450, clotrimazole, reduced the renal vasodilator effects of Ach and bradykinin but not those of nitroprusside or SCA 40, an agonist for Ca(++)-activated K+ channels. These results suggest that in the rat kidney, Ach, like bradykinin, utilizes a charybdotoxin-sensitive Ca(++)-activated K+ channel of intermediate conductance to elicit vasodilation and that this effect may be dependent on cytochrome P450 activity.     

An isoform of the rod photoreceptor cyclic nucleotide-gated channel beta subunit expressed in olfactory neurons

Sensory transduction in olfactory neurons involves the activation of a cyclic nucleotide-gated (CNG) channel by cAMP. Previous studies identified a CNG channel alpha subunit (CNG2) and a beta subunit (CNG5), which when heterologously expressed form a channel with properties similar but not identical to those of native olfactory neurons. We have cloned a new type of CNG channel beta subunit (CNG4. 3) from rat olfactory epithelium. CNG4.3 derives from the same gene as the rod photoreceptor beta subunit (CNG4.1) but lacks the long, glutamic acid-rich domain found in the N terminus of CNG4.1. Northern blot and in situ hybridization revealed that CNG4.3 is expressed specifically in olfactory neurons. Expression of CNG4.3 in human embryonic kidney 293 cells did not lead to detectable currents. Coexpression of CNG4.3 with CNG2 induced a current with significantly increased sensitivity for cAMP whereas cGMP affinity was not altered. Additionally, CNG4.3 weakened the outward rectification of the current in the presence of extracellular Ca2+, decreased the relative permeability for Ca2+, and enhanced the sensitivity for L-cis diltiazem. Upon coexpression of CNG2, CNG4.3, and CNG5, a conductance with a cAMP sensitivity greater than that of either the CNG2/CNG4.3 or the CNG2/CNG5 channel and near that of native olfactory channel was observed. Our data suggest that CNG4.3 forms a subunit of the native olfactory CNG channel. The expression of various CNG4 isoforms in retina and olfactory epithelium indicates that the CNG4 subunit may be necessary for normal function of both photoreceptor and olfactory CNG channels.     

The role of temporal relationships in the transfer of conditioned inhibition

We have cloned and molecularly and functionally characterized the first human member of the family of Ca2+-activated Cl- channels, human (h) CLCA1. The 31,902-bp gene is located on chromosome 1p22-31 and is preceded by a canonic promoter region that contains an L1 transposable element. In contrast to all previously known homologs in other species, hCLCA1 is exclusively expressed in intestinal basal crypt epithelia and goblet cells, suggesting that it does not represent the human counterpart of any of them. Expression of the 914-amino-acid hCLCA1 protein in HEK 293 cells yielded a 125-kDa precursor that was processed to yield two cell-surface-associated subunits, a 90-kDa protein and a group of 37- to 41-kDa proteins. Four transmembrane domains were established within the 90-kDa subunit. HEK 293 cells transfected with CLCA1 exhibited an increase in whole-cell Ca2+-sensitive Cl- currents that were outwardly rectified and inhibited by 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid, dithiothreitol, and niflumic acid. Cell-attached patch recordings of transfected cells revealed single channels with a slope conductance of 13.4 pS. These findings suggest that human CLCA1 mediates a Ca2+-activated Cl- conductance in the human intestine and make it an interesting candidate as a modulating factor in the pathogenesis of cystic fibrosis.     

Expression and transcriptional control of the Salmonella typhimurium Ipf fimbrial operon by phase variation

Functional effects of human alpha 5 nicotinic ACh receptor (AChR) subunits coassembled with alpha 3 and beta 2 or with alpha 3 and beta 4 subunits, were investigated in Xenopus oocytes. The presence of alpha 5 subunits altered some properties of both alpha 3 AChRs and differentially altered other properties of alpha 3 beta 2 AChRs vs. alpha 3 beta 4 AChRs. alpha 5 subunits increased desensitization and Ca++ permeability of all alpha 3 AChRs. The Ca++ permeabilities of both alpha 3 beta 2 alpha 5 and alpha 3 beta 4 alpha 5 AChRs were comparable to that of alpha 7 AChRs. As we have shown previously, alpha 5 subunits increased the ACh sensitivity of alpha 3 beta 2 AChRs 50-fold but had little effect on alpha 3 beta 4 AChRs. alpha 5 caused only subtle changes in the activation potencies of alpha 3 AChRs for nicotine, cytisine and 1,1-dimethyl-4-plenylpiperazinium (DMPP). However, alpha 5 increased the efficacies of nicotine and DMPP on alpha 3 beta 2 AChRs but decreased them on alpha 3 beta 4 AChRs. Immunoisolation of cloned human AChRs expressed in oocytes showed that alpha 5 efficiently coassembled with alpha 3 plus beta 2 and/or beta 4 subunits. As expected, human AChRs immunoisolated from SH-SY5Y neuroblastoma cells showed that AChRs containing alpha 3 and probably alpha 5 subunits were present, but alpha 4 AChRs were not. In brain, by contrast, alpha 4 beta 2 AChRs were shown to predominate over alpha 3 AChRs. Some of the brain alpha 4 beta 2 AChRs were found to contain alpha 5 subunits.     

Preservation of visual sensitivity of older subjects: association with macular pigment density

We examined the actions of ethanol on the single channel properties of large conductance Ca2+-activated K+ (BK) channels isolated from skeletal muscle T-tubule membranes and incorporated into planar lipid bilayer membranes. We have taken advantage of this preparation, because it lacks most elements of cellular complexity, including cytoplasmic constituents and complex membrane lipid composition and architecture, to examine the minimum requirements for the effects of alcohol. Clinically relevant concentrations (25-200 mM) of ethanol increased the activity of BK channels incorporated into bilayers composed of phosphatidylethanolamine (PE) alone or PE and phosphatidylserine. The potentiation of channel activity by ethanol was attributable predominantly to a decrease in the average amount of time spent in closed states. Ethanol did not significantly affect the current amplitude-voltage relationship for BK channels, indicating that channel conductance for K+ was unaffected by the drug. Although base-line characteristics of BK channels incorporated into bilayers composed only of PE differed from those of channels in PE/ phosphatidylserine in a manner expected from the change in bilayer charges, the actions of ethanol on channel activity were qualitatively similar in the different lipid environments. The effects of ethanol on single channel properties of BK channels in the planar bilayer are very similar to those reported for the action of ethanol on neurohypophysial BK channels studied in native membrane, and for cloned BK channels expressed in Xenopus laevis oocytes, which suggests that ethanol's site and mechanism of action are preserved in this greatly simplified preparation.     

Independent trafficking of KATP channel subunits to the plasma membrane

KATP channels are unique in requiring two distinct subunits (Kir6.2, a potassium channel subunit) and SUR1 (an ABC protein) for generation of functional channels. To examine the cellular trafficking of KATP channel subunits, green fluorescent protein (GFP) was tagged to the cytoplasmic N or C terminus of SUR1 and Kir6. 2 subunits and to the C terminus of a dimeric fusion between SUR1 and Kir6.2 (SUR1-Kir6.2). All tagged constructs generated functional channels with essentially normal properties when coexpressed with the relevant other subunit. GFP-tagged Kir6.2 (Kir6.2-GFP) showed perinuclear and plasma membrane fluorescence patterns when expressed alone or with SUR1, and a very similar pattern was observed when channel-forming SUR1-Kir6.2-GFP was expressed on its own. In contrast, whereas SUR1 (SUR1-GFP) also showed a perinuclear and plasma membrane fluorescence pattern when expressed alone, an apparently cytoplasmic fluorescence was observed when coexpressed with Kir6.2 subunits. The results indicate that Kir6.2 subunits traffic to the plasma membrane in the presence or absence of SUR1, in contradiction to the hypothesis that homomeric Kir6.2 channels are not observed because SUR1 is required as a chaperone to guide Kir6.2 subunits through the secretory pathway.     

Subunit stoichiometry of the epithelial sodium channel

The epithelial Na+ Channel (ENaC) mediates Na+ reabsorption in a variety of epithelial tissues. ENaC is composed of three homologous subunits, termed alpha, beta, and gamma. All three subunits participate in channel formation as the absence of any one subunit results in a significant reduction or complete abrogation of Na+ current expression in Xenopus oocytes. To determine the subunit stoichiometry, a biophysical assay was employed utilizing mutant subunits that display significant differences in sensitivity to channel blockers from the wild type channel. Our results indicate that ENaC is a tetrameric channel with an alpha2 beta gamma stoichiometry, similar to that reported for other cation selective channels, such as Kv, Kir, as well as voltage-gated Na+ and Ca2+ channels that have 4-fold internal symmetry.     

Calcium signalling in bovine adrenal chromaffin cells: additive effects of histamine and nicotine

In a previous report, we described the ability of two secretogogues, histamine and nicotine, to stimulate additive effects on catecholamine (CA) release and synapsin II phosphorylation in bovine adrenal chromaffin cells (BACC) [Firestone and Browning (1992), J. Neurochem., 58:441-447]. We hypothesized that these results were due to the combined effects on cytosolic Ca++ of the two distinct signalling pathways. We therefore examined the intracellular Ca++ signals stimulated by histamine and nicotine, alone and together. In Ca(++)-deficient medium, nicotine-stimulated signals were abolished, whereas histamine-stimulated signals were maintained, demonstrating that nicotine depended entirely on Ca++ influx for its effects. Indeed, the nicotine-stimulated signal could also be prevented using a Ca++ channel blocker, nicardipine. Further, the observation that exposure of BACC to thapsigargin reduced histamine-stimulated Ca++ signals verified that histamine mobilizes Ca++ from intracellular stores. Thus, the two secretogogues mobilize Ca++ from distinct pools. When BACC were stimulated with the two secretogogues together, the resulting Ca++ signal was greater than that from either alone. These data are consistent with a model in which two distinct sources of Ca++ can summate within the cell, producing a greater Ca++ signal and, hence, a greater effect on neurotransmitter release.