Phosphorylation-dependent modulation of cardiac calcium current by intracellular free magnesium

Abstract

We compared the effects of cytosolic free magnesium (Mg(2+)(i)) on L-type Ca(2+) current (I(Ca,L)) in patch-clamped guinea pig ventricular cardiomyocytes under basal conditions, after inhibition of protein phosphorylation, and after stimulation of cAMP-mediated phosphorylation. Basal I(Ca,L) density displayed a bimodal dependence on the concentration of Mg(2+)(i) ([Mg(2+)](i); 10(-6)-10(-2) M), which changed significantly as cell dialysis progressed due to a pronounced and long-lasting rundown of I(Ca,L) in low-Mg(2+) dialysates. Ten minutes after patch breakthrough, I(Ca,L) density (at +10 mV) in Mg(2+)(i)-depleted cells ([Mg(2+)](i) approximately 1 microM) was elevated, increased to a maximum at approximately 20 microM [Mg(2+)](i), and declined steeply at higher [Mg(2+)](i). Treatment with the broad-spectrum protein kinase inhibitor K252a (10 microM) reduced I(Ca,L) density and abolished these effects of Mg(2+)(i) except for a negative shift of I(Ca,L)-voltage relations with increasing [Mg(2+)](i). Maximal stimulation of cAMP-mediated phosphorylation occluded the Mg(2+)(i)-induced stimulation of I(Ca,L) and prevented inhibitory effects of the ion at [Mg(2+)](i) <1 mM but not at higher concentrations. These results show that the modulation of I(Ca,L) by Mg(2+)(i) requires protein kinase activity and likely originates from interactions of the ion with proteins involved in the regulation of protein phosphorylation/dephosphorylation. Stimulatory effects of Mg(2+)(i) on I(Ca,L) seem to increase the cAMP-mediated phosphorylation of Ca(2+) channels, whereas inhibitory effects of Mg(2+)(i) appear to curtail and/or reverse cAMP-mediated phosphorylation.

Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes

“…In any case, the affinity of Mg 2+ for the low affinity site in the L-type channel permeation pathway would seem to indicate that the highest [Mg 2+ ] i used in the present study, 1.8 mM, would not have a significant effect on ion permeation. Nonetheless, given the assumptions and extrapolations needed to arrive at this conclusion, it is important to experimentally test whether [Mg 2+ ] i could be blocking ion permeation under the conditions of our experiments [3,4], that are similar to those of other reports in which [Mg 2+ ] i has been reported to decrease whole-cell Ca 2+ currents [1,5,22,23,25].…”

“…This finding could be consistent with Mg 2+ acting as an open channel blocker. The findings of White & Hartzell [1] and Pelzer et al [25] that channel phosphorylation promotes the reduction of Ca 2+ current by cytosolic Mg 2+ current might also be consistent with Mg 2+ block of ion permeation through the open channel. This possibility motivated the present experiments to determine if [Mg 2+ ] i used here could decrease current in a manner consistent with permeation block.…”

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

“…This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. (1,18,31,(41)(42)(43). Our recent study (37) also showed large changes in I Ca around physiologically relevant [Mg 2ϩ ] i .…”

“…[Mg 2ϩ ] i could affect the level of channel phosphorylation by altering the activity of Mg 2ϩ -dependent kinases and phosphatases, and it could also modulate the effect of channel phosphorylation on gating kinetics. Previous studies (31) have shown that the former mechanism is possible. In addition, the latter has been discussed as an explanation for the results of Yamaoka and Seyama (44); however, direct experimental support for this mechanism is lacking. ]…”

“…[Mg 2ϩ ] i effects on I Ca are very much dependent on channel phosphorylation state (1,31,37,41,44,45). Although the mechanism by which channel phosphorylation affects [Mg 2ϩ ] i actions on I Ca is not clear, two alternatives have been proposed.…”