cAMP-Dependent Regulation of Cardiac L-Type Ca2+ Channels Requires Membrane Targeting of PKA and Phosphorylation of Channel Subunits

Abstract

The cardiac L-type Ca2+ channel is a textbook example of an ion channel regulated by protein phosphorylation; however, the molecular events that underlie its regulation remain unknown. Here, we report that in transiently transfected HEK293 cells expressing L-type channels, elevations in cAMP resulted in phosphorylation of the alpha1C and beta2a channel subunits and increases in channel activity. Channel phosphorylation and regulation were facilitated by submembrane targeting of protein kinase A (PKA), through association with an A-kinase anchoring protein called AKAP79. In transfected cells expressing a mutant AKAP79 that is unable to bind PKA, phosphorylation of the alpha1C subunit and regulation of channel activity were not observed. Furthermore, we have demonstrated that the association of an AKAP with PKA was required for beta-adrenergic receptor-mediated regulation of L-type channels in native cardiac myocytes, illustrating that the events observed in the heterologous expression system reflect those occurring in the native system. Mutation of Ser1928 to alanine in the C-terminus of the alpha1C subunit resulted in a complete loss of cAMP-mediated phosphorylation and a loss of channel regulation. Thus, the PKA-mediated regulation of L-type Ca2+ channels is critically dependent on a functional AKAP and phosphorylation of the alpha1C subunit at Ser1928.

Striatal proenkephalin gene induction: coordinated regulation by cyclic AMP and calcium pathways

“…Because we do not add depolarizing agents with forskolin, our data suggest furthermore that the cyclic AMP pathway increases neuronal excitability to agents already present in the medium, such as KCl (5 mM) and glutamate (1-5 μM) [16]. A possible mechanism might be the phosphorylation of PKA sites on ion channels such as the L-type Ca 2+ channel [2,9]. The notion that PKA contributes to neuronal excitability via modulation of ion channels is supported by our observation that inhibition of PKA prevents L-type Ca 2+ channel-mediated induction of proenkephalin.…”

Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors

“…Using heterologous expression systems, prior structure-function studies have extensively characterized regions of the channel involved in selectivity, permeation, gating, and Ca 2+ /calmodulin-dependent inactivation, but many fundamental questions remain regarding the behavior of the Ca 2+ channel in the native environment of the dyad. A key example is whether modulation of I CaL by cyclic AMPdependent protein kinase requires direct phosphorylation of the α 1 subunit [6,28,29], the β subunit, or a combination of both. Similarly, interactions between L-type Ca 2+ channel domains and the ryanodine receptor, or other dyadic proteins have been proposed [7,30], but there has been no direct method for testing these hypotheses.…”

Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L‐type Ca2+ current in rat ventricular myocytes

“…In particular, at the sarcolemmal membrane, this balance between adenylyl cyclase and PDE activities will control the degree of I Ca stimulation upon hormonal activation Hove-Madsen et al, 1996). Other factors are involved, such as cyclic AMP compartmentation , PKA tethering to the membrane (Gao et al, 1997), or phosphatase activity (Wiechen et al, 1995).…”