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ABSTRACT␤ subunits of voltage-gated Ca 2؉ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for ␤2a, which differs in that it does not support prepulse facilitation of ␣ 1C Ca 2؉ channels, inhibits voltage-induced inactivation of neuronal ␣ 1E Ca 2؉ channels, and is more effective in blocking inhibition of ␣ 1E channels by G protein-coupled receptors. We show that the distinguishing properties of ␤2a, rather than interaction with a distinct site of ␣ 1 , are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of ␤2a were lost in a mutant that could not be palmitoylated [␤2a(Cys 3,4 Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-dependent neuronal and synaptic plasticity. Evidence is presented that there may exist as many as three ␤2 splice variants differing only in their N-termini.Voltage-gated Ca 2ϩ channels are multiprotein complexes made up of at least three distinct types of subunits: an ␣ 1 , which senses voltage changes and spans the membrane multiple times to form the pore and the ␤ and ␣ 2 ␦ subunits, which modulate almost all aspects of ␣ 1 function (1). In addition, ␤ and ␣ 2 ␦ play structural roles that are important, but not well understood, in channel maturation and accumulation at the cell surface. Voltage-gated Ca 2ϩ channels are molecularly diverse. Six ␣ 1 and four ␤ genes are known, and many genes exhibit additional heterogeneity in their translated proteins because of alternative splicing. In contrast, only one gene encoding the ␣ 2 ␦ complex has been found so far, but it also yields transcripts that are spliced alternatively to give slightly differing proteins (2, 3)Although in most cases it has been difficult to ascribe a functional correlate to specific Ca 2ϩ channel splice variants, there is one striking exception: the a-type splice variant of the ␤2 subunit from rat brain (␤2a) acts differently on inactivation of ␣ 1E , on prepulse-induced long lasting facilitation of ␣ 1C , and also, to some extent, on G protein-mediated inhibition of neuronal Ca 2ϩ channels. In ␣ 1E , brain ␤2a reduces the rate at which ␣ 1E inactivates in response to depolarization and causes a right shift in the steady-state inactivation curve. All other ␤ subunits, including the b-type splice variant of ␤2, accelerate channel inactivation and cause steady-state inactivation curves to be left-shifted along the voltage axis (4, 5). In contrast, ␤2a is indistinguishable from its homologs in terms of ␣ 1E activation (5).Prepulse facilitation is a phenomenon in which a train of depolarizations, or a long and strong depolarizing pulse, induces a form of the Ca 2ϩ channel that exhibits an increased opening probability in response to a given test potential that persist...

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GT1-1 cells exhibit spontaneous action potentials and transient increases in intracellular calcium concentration ([Ca2+]i) that occur in individual cells and as spatially propagated intercellular Ca2+ waves. In this study, simultaneous cell-attached patch-clamp recording of action currents (indicative of action potentials) and fluorescence imaging of [Ca2+]i revealed that Ca2+ transients in GT1-1 cells were preceded by a single action current or a burst of action currents. Action currents preceded Ca2+ transients in a similar pattern regardless of whether the Ca2+ transients were limited to the individual cell or occurred as part of an intercellular Ca2+ wave. Both the action currents and Ca2+ transients were abolished by 1 microM tetrodotoxin. Removal of extracellular Ca2+ abolished all spontaneous Ca2+ transients without inhibiting the firing of action currents. Nimodipine, which blocks L-type Ca2+ currents in GT1-1 cells, also abolished all spontaneous Ca2+ signaling. Delivery of small voltage steps to the patch pipette in the cell-attached configuration elicited action currents the latency to firing of which decreased with increasing amplitude of the voltage step. These results indicate that spontaneous intercellular Ca2+ waves are generated by a propagated depolarization, the firing of action potentials in individual cells, and the resulting influx of Ca2+ through L-type Ca2+ channels. These patterns of spontaneous activity may be important in driving the pulsatile release of GnRH from networks of cells.

AB S T R A C T The hypothesis of Cl -/HCO s exchange across the apical membrane of the epithelial cells of Necturus gallbladder was tested by means of measurements of extracellular pH (pH.), intracellular pH (pHi), and Cl -activity (aCli) with ion-sensitive microelectrodes. Luminal pH changes were measured after stopping mucosal superfusion with a solution of low buffering power. Under control conditions, the luminal solution acidifies when superfusion is stopped. Shortly after addition of the Na'/H' exchange inhibitor amiloride (10-3 M) to the superfusate, alkalinization was observed . During prolonged (10 min) exposure to amiloride, no significant pH . change occurred . Shortly after amiloride removal, luminal acidification increased, returning to control rates in 10 min. The absence of Na' in the superfusate (TMA' substitution) caused changes in the same direction, but they were larger than those observed with amiloride. Removal of CI -(cyclamate or sulfate substitution) caused a shortlived increase in the rate of luminal acidification, followed by a return to control values (10-30 min) . Upon re-exposure to Cl -, there was a transient reduction of luminal acidification . The initial increase in acidification produced by CIremoval was partially inhibited by SITS (0 .5 mM). The pH i increased rapidly and reversibly when the Cl-concentration o£ the mucosal bathing solution was reduced to nominally 0 mM . The pH; changes were larger in 10 mM HC03-Ringer's than in 1 mM HEPES-Ringer's, which suggests that HCO-3 is transported in exchange for CI -. In both HEPES-and HC03-Ringer's, SITS inhibited the pHi changes. Finally, intracellular acidification or alkalinization (partial replacement of NaCl with sodium propionate or ammonium chloride, respectively) caused a reversible decrease or increase of aCli. These results support the hypothesis of apical membrane CI -/HC03 exchange, which can be dissociated from Na''/H+ exchange and operates under control conditions . The coexistence at the apical membrane of Na'/H' and CI -/HCO3 antiports suggests that NaCl entry can occur through these transporters .