Role of Ca _V β Subunits, and Lack of Functional Reserve, in Protein Kinase A Modulation of Cardiac Ca _V 1.2 Channels

Abstract

Abstract-Protein kinase A (PKA)-mediated enhancement of L-type calcium currents (I Ca,L ) is essential for sympathetic regulation of the heartbeat and is the classic example of channel regulation by phosphorylation, and its loss is a common hallmark of heart failure. Mechanistic understanding of how distinct Ca V channel subunits contribute to PKA modulation of I Ca,L has been intensely pursued yet remains elusive. Moreover, critical features of this regulation such as its functional reserve (the surplus capacity available for modulation) in the heart are unknown. Here, we use an overexpression paradigm in heart cells to simultaneously identify the impact of auxiliary Ca V ␤s on PKA modulation of I Ca,L and to gauge the functional reserve of this regulation in the heart. Ca V 1.2 channels containing wild-type ␤ 2a or a phosphorylationdeficient mutant (␤ 2a,AAA ) were equally upregulated by PKA, discounting a necessary role for ␤ phosphorylation. Nevertheless, channels reconstituted with ␤ 2a displayed a significantly diminished PKA response compared with other ␤ isoforms, an effect explainable by a uniquely higher basal P o of ␤ 2a channels. Overexpression of all ␤s increased basal current density, accompanied by a concomitant decrease in the magnitude of PKA regulation. Scatter plots of fold increase in current against basal current density revealed an inverse relationship that was conserved across species and conformed to a model in which a large fraction of channels remained unmodified after PKA activation. These results redefine the role of ␤ subunits in PKA modulation of Ca V 1.2 channels and uncover a new design principle of this phenomenon in the heart, vis à vis a limited functional reserve. (Circ Res. 2008;102:e54-e64.)2) channels are important determinants of cardiac excitability and indispensable for excitationcontraction coupling in the heart. 1 Fitting with their central role in cardiac biology, tuning Ca V 1.2 channel activity is a prominent method for physiological and therapeutic regulation of the heartbeat. 2 A classic example of physiological modulation is the upregulation of cardiac Ca V 1.2 channel activity in response to ␤-adrenergic receptor stimulation, an effect mediated through activation of protein kinase A (PKA). [2][3][4] This phenomenon is an important contributor to sympathetic regulation of the heartbeat, and its loss is a harmful hallmark of heart failure. 2,5 PKA-mediated regulation of cardiac Ca V 1.2 channels represents the original example of channel modulation by phosphorylation, and mechanistic understanding of this phenomenon has been widely pursued for more than 3 decades. 3,4,6 Despite years of intense study, many key features of how activated PKA upregulates Ca V 1.2 channel activity remain unknown. Native Ca V 1.2 channels are comprised minimally of a pore-forming ␣ 1C protein assembled in a 1:1 ratio with auxiliary Ca V ␤ and ␣ 2 ␦ subunits. 2,7 Ca V ␤ subunits are necessary for trafficking Ca V ␣ 1 to the plasma membrane and also greatly influence channel gating prope...

Regulation of Cardiac L-Type Ca 2+ Channel Ca V 1.2 Via the β-Adrenergic-cAMP-Protein Kinase A Pathway

“…77 In contrast, subsequent studies were unsuccessful in reproducing the robust PKA-induced regulation of either full-length or dCT-truncated α 1C , even in the presence of AKAPs, in HEK cells or Xenopus oocytes. At best, small increases (15%-20%) in I Ca were observed 42,160,171,172 (and our unpublished results). Despite these reservations, the 2 studies 77,164 have led to a prevailing concept of a molecular mechanism in which PKA, anchored at the CT of α 1C via an AKAP, regulates Ca V 1.2 by phosphorylating Ser1928 in α 1C and 2 serines in Ca V β after activation by cAMP 18,21 .…”

“…First, PKA-induced enhancement of I Ca was preserved in cardiomyocytes infected with a Ca V β 2 lacking the crucial phosphorylation sites Ser459, Ser478, and Ser479 171,173 . Second, transgenic mice with Ca V β 2 truncated to remove PKA phosphorylation sites Ser478 and Ser479 (according to rabbit β 2b count) demonstrated an unaltered β-adrenergic stimulation of cardiomyocytes by Iso.…”

“…173 Notably, the Iso-induced increase in I Ca was ≈50%, substantially smaller than the >3 fold increase of the endogenous I Ca recorded in the same cells. This difference could be because of overexpression of the Ca V β subunit 171 or the use of Ca V β 2a instead of Ca V β 2b , which is the dominant Ca V β in the heart.…”

Manipulating L-type calcium channels in cardiomyocytes using split-intein protein transsplicing

“…Reconstituting LTCCs in heterologous cells to address such questions has proven inadequate because these cells are deficient in essential structural elements such as transverse (t) tubules (Fig. 1B), and also lack a permissive environment for PKA modulation (11,12). Moreover, because resolving these structurefunction questions will likely require many different constructs, it is impractical to use knock-in mice for such investigative purposes.…”

Unchanged β-Adrenergic Stimulation of Cardiac L-type Calcium Channels in Cav1.2 Phosphorylation Site S1928A Mutant Mice

“…In contrast, the necessity of coexpression and phosphorylation of the Ca v ␤2a subunit was reported (13). Recently, the functional importance of Ca v ␤2a phosphorylation for ␤-adrenergic regulation of I CaL has been questioned (14). Regardless of these findings, mutation of Ca v 1.2 Ser 1928 to alanine prevented phosphorylation and regulation of the channel by PKA in heterologous expression systems (15)(16)(17).…”