Complementary DNAs encoding three novel and distinct beta subunits (CaB2a, CaB2b and CaB3) of the high voltage activated (L‐type) calcium channel have been isolated from rabbit heart. Their deduced amino acid sequence is homologous to the beta subunit originally cloned from skeletal muscle (CaB1). CaB2a and CaB2b are splicing products of a common primary transcript (CaB2). Northern analysis and specific amplification of CaB2 and CaB3 specific cDNAs by polymerase chain reactions showed that CaB2 is predominantly expressed in heart, aorta and brain, whereas CaB3 is most abundant in brain but also present in aorta, trachea, lung, heart and skeletal muscle. A partial DNA sequence complementary to a third variant of the CaB2 gene, subtype CaB2c, has also been cloned from rabbit brain. Coexpression of CaB2a, CaB2b and CaB3 with alpha 1heart enhances not only the expression in the oocyte of the channel directed by the cardiac alpha 1 subunit alone, but also effects its macroscopic characteristics such as drug sensitivity and kinetics. These results together with the known alpha 1 subunit heterogeneity, suggest that different types of calcium currents may depend on channel subunit composition.

Presynaptic voltage-gated K ϩ (Kv) channels play a physiological role in the regulation of transmitter release by virtue of their ability to shape presynaptic action potentials. However, the possibility of a direct interaction of these channels with the exocytotic apparatus has never been examined. We report the existence of a physical interaction in brain synaptosomes between Kv␣1.1 and Kv␤ subunits with syntaxin 1A, occurring, at least partially, within the context of a macromolecular complex containing syntaxin, synaptotagmin, and SNAP-25. The interaction was altered after stimulation of neurotransmitter release. The interaction with syntaxin was further characterized in Xenopus oocytes by both overexpression and antisense knockdown of syntaxin. Direct physical interaction of syntaxin with the channel protein resulted in an increase in the extent of fast inactivation of the Kv1.1/Kv␤1.1 channel. Syntaxin also affected the channel amplitude in a biphasic manner, depending on its concentration. At low syntaxin concentrations there was a significant increase in amplitudes, with no detectable change in cell-surface channel expression. At higher concentrations, however, the amplitudes decreased, probably because of a concomitant decrease in cell-surface channel expression, consistent with the role of syntaxin in regulation of vesicle trafficking. The observed physical and functional interactions between syntaxin 1A and a Kv channel may play a role in synaptic efficacy and neuronal excitability.

Voltage-gated K؉ channels isolated from mammalian brain are composed of ␣ and ␤ subunits. Interaction between coexpressed K v 1.1 (␣) and K v ␤1.1 (␤) subunits confers rapid inactivation on the delayed rectifier-type current that is observed when ␣ subunits are expressed alone. Integrating electrophysiological and biochemical analyses, we show that the inactivation of the ␣␤ current is not complete even when ␣ is saturated with ␤, and the ␣␤ current has an inherent sustained component, indistinguishable from a pure ␣ current. We further show that basal and protein kinase A-induced phosphorylations at Ser-446 of the ␣ protein increase the extent, but not the rate, of inactivation of the ␣␤ channel, without affecting the association between ␣ and ␤. In addition, the extent of inactivation is increased by agents that lead to microfilament depolymerization. The effects of phosphorylation and of microfilament depolymerization are not additive. Taken together, we suggest that phosphorylation, via a mechanism that involves the interaction of the ␣␤ channel with microfilaments, enhances the extent of inactivation of the channel. Furthermore, phosphorylation at Ser-446 also increases current amplitudes of the ␣␤ channel as was shown before for the ␣ channel. Thus, phosphorylation enhances in concert inactivation and current amplitudes, thereby leading to a substantial increase in A-type activity.

Kv channels inhibit release indirectly by hyperpolarizing membrane potential, but the significance of Kv channel interaction with the secretory apparatus is not known. The Kv2.1 channel is commonly expressed in the soma and dendrites of neurons, where it could influence the release of neuropeptides and neurotrophins, and in neuroendocrine cells, where it could influence hormone release. Here we show that Kv2.1 channels increase dense-core vesicle (DCV)-mediated release after elevation of cytoplasmic Ca 2ϩ . This facilitation occurs even after disruption of pore function and cannot be explained by changes in membrane potential and cytoplasmic Ca 2ϩ . However, triggering release increases channel binding to syntaxin, a secretory apparatus protein. Disrupting this interaction with competing peptides or by deleting the syntaxin association domain of the channel at the C terminus blocks facilitation of release. Thus, direct association of Kv2.1 with syntaxin promotes exocytosis. The dual functioning of the Kv channel to influence release, through its pore to hyperpolarize the membrane potential and through its C-terminal association with syntaxin to directly facilitate release, reinforces the requirements for repetitive firing for exocytosis of DCVs in neuroendocrine cells and in dendrites.

L.Type calcium chalm¢l was expressed in Xem~pus hteris oocytes injected with RNAs coding Ibr different cardiac Ca;" channel subunits, or with total heart RNA. The el'fects of activation of protein kin:tse C (PKC) by the phorbol ester PMA (4fl.phorbol 12-mvristate 13.acetate) were studied, Currel~ts through chantaels eot'nposed of the re:tin (rt 0 subanit alone were initiall>, increased and Ihen decreased by PMA. A similar biphasie modul.'ttion was observed when the rt~ stLbunit w:ts expressed in combin:ttion with er../d~. ,B and/or ~' subunits, and when the channels were expressed following, injection or total rat heart RNA. No el'l~ets on the volta~.e dependence of activation were observed. The el'f~ts of PMA were blocked by staurosporine, a protein kinase inhibitor. ,a subunit moderated the enhancement caused by PMA. We conclude that both enhancement and inhibition of c~irdiae L.t~,'pe Ca"" currents by PKC are mediated via an effect orl the et z subunit, while th¢,O subunit may play a tllild modulatory role.