Modulation of cardiac Ca²⁺ channels in Xenopus oocytes by protein kinase C

Abstract

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.

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

“…1). The extent of reduction was similar in channels of these two subunit combinations, in accord with our previous findings [21] (see Fig. 3A).…”

“…In a previous work [21], we have shown that, in Xenopus oocytes, intracellular injection of protein phosphatase 1, or of specific peptide and protein inhibitors of PKA, reduces the current carried by Ba 2+ (IBa) via the rabbit cardiac L-type Ca 2+ channel of various subunit combinations by 30-40% at 0 mV. This tedious and time-consuming procedure proved inefficient for the screening of a large amount of cells that was necessary in order to test a number of mutant ~1c channels.…”

“…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]. If the target sites for dephosphorylation are located in the ale subunit, their elimination should abolish the effect of PKA inhibitors.…”

“…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.…”