β subunits (Cavβ) increase macroscopic currents of voltage-dependent Ca2+ channels (VDCC) by increasing surface expression and modulating their gating, causing a leftward shift in conductance–voltage (G-V) curve and increasing the maximal open probability, Po,max. In L-type Cav1.2 channels, the Cavβ-induced increase in macroscopic current crucially depends on the initial segment of the cytosolic NH2 terminus (NT) of the Cav1.2α (α1C) subunit. This segment, which we term the “NT inhibitory (NTI) module,” potently inhibits long-NT (cardiac) isoform of α1C that features an initial segment of 46 amino acid residues (aa); removal of NTI module greatly increases macroscopic currents. It is not known whether an NTI module exists in the short-NT (smooth muscle/brain type) α1C isoform with a 16-aa initial segment. We addressed this question, and the molecular mechanism of NTI module action, by expressing subunits of Cav1.2 in Xenopus oocytes. NT deletions and chimeras identified aa 1–20 of the long-NT as necessary and sufficient to perform NTI module functions. Coexpression of β2b subunit reproducibly modulated function and surface expression of α1C, despite the presence of measurable amounts of an endogenous Cavβ in Xenopus oocytes. Coexpressed β2b increased surface expression of α1C approximately twofold (as demonstrated by two independent immunohistochemical methods), shifted the G-V curve by ∼14 mV, and increased Po,max 2.8–3.8-fold. Neither the surface expression of the channel without Cavβ nor β2b-induced increase in surface expression or the shift in G-V curve depended on the presence of the NTI module. In contrast, the increase in Po,max was completely absent in the short-NT isoform and in mutants of long-NT α1C lacking the NTI module. We conclude that regulation of Po,max is a discrete, separable function of Cavβ. In Cav1.2, this action of Cavβ depends on NT of α1C and is α1C isoform specific.

Human L-type voltage-dependent Ca2؉ channels (␣ 1C , or Ca v 1.2) are up-regulated by protein kinase C (PKC) in native tissues, but in heterologous systems this modulation is absent. In rat and rabbit, ␣ 1C has two N-terminal (NT) isoforms, long and short, with variable initial segments of 46 and 16 amino acids, respectively. The initial 46 amino acids of the long-NT ␣ 1C are crucial for PKC regulation. However, only a short-NT human ␣ 1C is known. We assumed that a long-NT isoform of human ␣ 1C may exist. By homology screening of human genomic DNA, we identified a stretch (termed exon 1a) highly homologous to rabbit long-NT, separated from the next known exon of ␣ 1C (exon 1b, which encodes the alternative, short-NT) by an ϳ80 kb-long intron. The predicted 46-amino acid protein sequence is highly homologous to rabbit long-NT. Reverse transcriptase PCR showed the presence of exon 1a transcript in human cardiac RNA. Expression of human long-NT ␣ 1C in Xenopus oocytes produced Ca 2؉ channel enhanced by a PKC activator, whereas the short-NT ␣ 1C was inhibited. The long-NT isoform may be the Ca 2؉ channel enhanced by PKCactivating transmitters in human tissues.Voltage-dependent L-type Ca 2ϩ channels are crucial for cardiac and smooth muscle contraction and hormone secretion, and they regulate gene expression in the brain (1-3). Their function is highly regulated by hormones and neurotransmitters, largely via activation of protein kinases (3,4). Regulation by PKC 1 is believed to be of substantial physiological importance, mediating all or part of the effects of several hormones and intracellular messengers (4). PKC enhances L-type Ca 2ϩ currents in diverse human tissues and cell lines: heart, neuroblastoma, T-cells, and endocrine cells (5-10). Dual modulation by PKC is often observed with activation followed by, or concomitant with, inhibition (5, 10). Similar enhancement by PKC, sometimes followed by inhibition, has been described in other mammals (11,12) and was reproduced in Xenopus oocytes expressing the cloned rabbit cardiac L-type Ca 2ϩ channels (13,14). However, expression of human L-type channels, encoded by all cDNA cloned to date, yielded Ca 2ϩ channels that were only inhibited by PKC; the enhancement could not be reconstituted (15). The reason for the inability to reproduce the PKC modulation of human L-type channels remained unknown. The main, pore-forming subunit of cardiac/smooth muscle L-type channel (␣ 1C or Ca v 1.2), also present in the brain, is the product of the ␣ 1C gene, CACNA1C (16). Several splice variants of CACNA1C are known (17,18). The resulting isoforms of human ␣ 1C protein show differential distribution in human tissues, and in failing versus normal myocardium. They play important roles in Ca 2ϩ -dependent inactivation, oxygen sensing, and drug sensitivity (18 -22). However, the genomic structure of the beginning of N-terminal region of human ␣ 1C is not entirely clear. In the two best studied mammalian species, rat and rabbit, two N-terminal isoforms of ␣ 1C cDNA are known, which mos...

N-type voltage-dependent Ca2+ channels (N-VDCCs) play important roles in neurotransmitter release and certain postsynaptic phenomena. These channels are modulated by a number of intracellular factors, notably by Gβγ subunits of G proteins, which inhibit N-VDCCs in a voltage-dependent (VD) manner. Here we show that an increase in intracellular Na + concentration inhibits N-VDCCs in hippocampal pyramidal neurones and in Xenopus oocytes. In acutely dissociated hippocampal neurones, Ba 2+ current via N-VDCCs was inhibited by Na + influx caused by the activation of NMDA receptor channels. In Xenopus oocytes expressing N-VDCCs, Ba 2+ currents were inhibited by Na + influx and enhanced by depletion of Na + , after incubation in a Na + -free extracellular solution. The Na + -induced inhibition was accompanied by the development of VD facilitation, a hallmark of a Gβγ-dependent process. Na + -induced regulation of N-VDCCs is Gβγ dependent, as suggested by the blocking of Na + effects by Gβγ scavengers and by excess Gβγ, and may be mediated by the Na + -induced dissociation of Gαβγ heterotrimers. N-VDCCs may be novel effectors of Na + ion, regulated by the Na + concentration via Gβγ.

L-type calcium currents (ICa) are influenced by changes in extracellular chloride, but sites of anion effects have not been identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellular chloride with gluconate or perchlorate. Variance-mean analysis of ICa and cell-attached patch single channel recordings indicate that gluconate-induced inhibition is due to intracellular anion effects on Ca2+ channel open probability, not conductance. Inhibition of CaV1.2 currents produced by replacing chloride with gluconate was reduced from ∼75%–80% to ∼50% by omitting β subunits but unaffected by omitting α2δ subunits. Similarly, gluconate inhibition was reduced to ∼50% by deleting an α1 subunit N-terminal region of 15 residues critical for β subunit interactions regulating open probability. Omitting β subunits with this mutant α1 subunit did not further diminish inhibition. Gluconate inhibition was unchanged with expression of different β subunits. Truncating the C terminus at AA1665 reduced gluconate inhibition from ∼75%–80% to ∼50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacement with glutamines reduced gluconate inhibition to ∼60% indicating these residues are particularly important for anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to ∼25% consistent with additive interactions between the two tail regions. Our results suggest that modest changes in intracellular anion concentration can produce significant effects on CaV1.2 currents mediated by changes in channel open probability involving β subunit interactions with the N terminus and a short C terminal region.