Voltage-dependent facilitation of a neuronal alpha 1C L-type calcium channel.

Abstract

Calcium entry into excitable cells through voltage‐gated calcium channels can be influenced by both the rate and pattern of action potentials. We report here that a cloned neuronal alpha 1C L‐type calcium channel can be facilitated by positive pre‐depolarization. Both calcium and barium were effective as charge carriers in eliciting voltage‐dependent facilitation. The induction of facilitation was shown to be independent of intracellular calcium levels, G‐protein interaction and the level of phosphatase activity. Facilitation was reduced by the injection of inhibitors of protein kinase A and required the coexpression of a calcium channel beta subunit. In contrast, three neuronal non‐L‐type calcium channels, alpha 1A, alpha 1B and alpha 1E, were not subject to voltage‐dependent facilitation when coexpressed with a beta subunit. The results indicate that the mechanism of neuronal L‐type calcium channel facilitation involves the interaction of alpha 1 and beta subunits and is dependent on protein kinase A activity. The selective voltage‐dependent modulation of L‐type calcium channels is likely to play an important role in neuronal physiology and plasticity.

A potential site of functional modulation by protein kinase A in the cardiac Ca2+ channel α1C subunit

“…This assumption is supported by the finding that PKA inhibitors reduce the Ca 2+ current via channels formed by C~lc alone or with some or all of the auxiliary subunits in CHO and HEK293 cells and in Xenopus oocytes [17,[20][21][22], suggesting that Cqc is the main target for this modulation. There is little doubt that the observed decrease in Ca 2--channel current is caused by dephosphorylation of PKA sites due to the activity of endogenous protein phosphatases, because the peptide and proteins used to inhibit PKA in these experiments are highly specific toward PKA [21].…”

“…Since our assay involved the measurement of effects of dephosphorylation (as a result of inhibition of the basal activity of PKA) rather than phosphorylation, it is not clear whether S1928 is the site whose phosphorylation by PKA normally causes the Ca 2+ current increase in cardiac cells. However, if the hypothesis [17,21,22] that the inability of PKA to enhance the channel's activity is due to an abnormally high level of basal phosphorylation in expression systems is correct, then there is a good chance that S1928 is indeed the functionally important site whose phosphorylation by PKA underlies most of the adrenergic regulation of the cardiac Ca 2+ channel. It is noteworthy that channels containing the alc(S1928A) mutant displayed a strongly reduced current amplitude.…”

“…The presence of 13 may be necessary for a voltage-dependent facilitation of Ca 2+ channel current which, in turn, may be modulated by PKA [22]. However, most studies in expression systems do not support a role for the I] subunit, since coexpression of 13 with alc does not restore the positive modulation of the channel by PKA in Xenopus oocytes and in CHO cells and does not significantly alter the reducing effects of PKA inhibitors [20][21][22][23]. Our finding that the removal of a single PKA site in ~lC completely eliminates the effects of PKA inhibitors, and that the presence of the ~2A subunit does not restore these effects, also argues against the involvement of the 13 subunit in PKA modulation in the expression systems.…”

“…However, others [19,20] could not reproduce these results. When expressed in Xenopus oocytes, channels composed of cardiac or neuronal ale or cqc + (x2/~ are insensitive to PKA or PKA-activating treatments [21][22][23]. An early report [24] that coexpression of the [3 subunit with alC renders the expressed channel sensitive to PKA also could not be confirmed [21][22][23].…”

“…It has been proposed that the failure of PKA to enhance the current via the expressed Ca 2+ channel in Xenopus oocytes may be due to an abnormally high level of its basal phosphorylation [21,22], possibly because of a difference in the balance between phosphorylation and dephosphorylation processes in the heart and in the expression systems. This assumption is supported by the finding that PKA inhibitors reduce the Ca 2+ current via channels formed by C~lc alone or with some or all of the auxiliary subunits in CHO and HEK293 cells and in Xenopus oocytes [17,[20][21][22], suggesting that Cqc is the main target for this modulation.…”