Ion channel function is fundamental to the existence of life. In metazoans, the coordinate activities of voltagegated Na + channels underlie cellular excitability and control neuronal communication, cardiac excitationcontraction coupling, and skeletal muscle function. However, despite decades of research and linkage of Na + channel dysfunction with arrhythmia, epilepsy, and myotonia, little progress has been made toward understanding the fundamental processes that regulate this family of proteins. Here, we have identified β IV -spectrin as a multifunctional regulatory platform for Na + channels in mice. We found that β IV -spectrin targeted critical structural and regulatory proteins to excitable membranes in the heart and brain. Animal models harboring mutant β IV -spectrin alleles displayed aberrant cellular excitability and whole animal physiology. Moreover, we identified a regulatory mechanism for Na + channels, via direct phosphorylation by β IV -spectrin-targeted calcium/calmodulin-dependent kinase II (CaMKII). Collectively, our data define an unexpected but indispensable molecular platform that determines membrane excitability in the mouse heart and brain.

Rationale: Parasympathetic regulation of heart rate is mediated by acetylcholine binding to G protein-coupled muscarinic M2 receptors, which activate heterotrimeric G i/o proteins to promote G protein-coupled inwardly rectifying K ؉ (GIRK) channel activation. Regulator of G protein signaling (RGS) proteins, which function to inactivate G proteins, are indispensable for normal parasympathetic control of the heart. However, it is unclear which of the more than 20 known RGS proteins function to negatively regulate and thereby ensure normal parasympathetic control of the heart.Objective: To examine the specific contribution of RGS6 as an essential regulator of parasympathetic signaling in heart. Methods and Results: We developed RGS6 knockout mice to determine the functional impact of loss of RGS6 on parasympathetic regulation of cardiac automaticity. RGS6 exhibited a uniquely robust expression in the heart, particularly in sinoatrial and atrioventricular nodal regions. Loss of RGS6 provoked dramatically exaggerated bradycardia in response to carbachol in mice and isolated perfused hearts and significantly enhanced the effect of carbachol on inhibition of spontaneous action potential firing in sinoatrial node cells. Consistent with a role of RGS6 in G protein inactivation, RGS6-deficient atrial myocytes exhibited a significant reduction in the time course of acetylcholine-activated potassium current (I KACh ) activation and deactivation, as well as the extent of I KACh desensitization.Conclusions: RGS6 is a previously unrecognized, but essential, regulator of parasympathetic activation in heart, functioning to prevent parasympathetic override and severe bradycardia. These effects likely result from actions of RGS6 as a negative regulator of G protein activation of GIRK channels. (Circ Res. 2010;107:1345-1349.) Key Words: RGS6 Ⅲ SA node Ⅲ Heart rate Ⅲ K ϩ channel Ⅲ G proteins S ince the discovery that acetylcholine (ACh) release from the vagus produces bradycardia, key proteins and mechanisms underlying this action of ACh in heart have been identified. It is now known that ACh binds to muscarinic M2 receptors (M2Rs) that activate heterotrimeric G proteins (G i/o ) in key pacemaking regions of the heart. Activation of these G proteins causes release of G␤␥ subunits that bind to and activate G proteincoupled inwardly rectifying K ϩ (GIRK) channels, which results in a large K ϩ current (acetylcholine-activated potassium current [I KACh ]) and membrane hyperpolarization. 1 RGS proteins function as GTPase-activating proteins (GAPs) for G␣ subunits, accelerating their conversion to the inactive GDP-bound form. 2 This results in their reassembly with G␤␥ to form inactive G protein heterotrimers, thereby terminating signaling by both G␣ and G␤␥ proteins. Heterologous expression of various members of the RGS protein family with GIRK channels and M2Rs are required to reconstitute the normal activation and deactivation kinetics of native atrial GIRK channels. 3 In vivo evidence for this key role of RGS proteins in controlli...

Background Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting over two million patients in the US alone. Despite decades of research, surprisingly little is known regarding the molecular pathways underlying the pathogenesis of AF. ANK2 encodes ankyrin-B, a multifunctional adapter molecule implicated in membrane targeting of ion channels, transporters, and signaling molecules in excitable cells. Methods and Results Here, we report early-onset AF in patients harboring loss-of-function mutations in ANK2. In mice, we show that ankyrin-B-deficiency results in atrial electrophysiological dysfunction and increased susceptibility to AF. Moreover, ankyrin-B+/− atrial myocytes display shortened action potentials, consistent with human AF. Ankyrin-B is expressed in atrial myocytes, and we demonstrate its requirement for the membrane targeting and function of a subgroup of voltage-gated Ca2+ channels (Cav1.3) responsible for low-voltage activated L-type Ca2+current. Ankyrin-B directly associates with Cav1.3, and this interaction is regulated by a short, highly-conserved motif specific to Cav1.3. Moreover, loss of ankyrin-B in atrial myocytes results in decreased Cav1.3 expression, membrane localization, and function sufficient to produce shortened atrial action potentials and arrhythmias. Finally, we demonstrate reduced ankyrin-B expression in atrial samples of patients with documented AF, further supporting an association between ankyrin-B and AF. Conclusions These findings support that reduced ankyrin-B expression or mutations in ANK2 are associated with atrial fibrillation. Additionally, our data demonstrate a novel pathway for ankyrin-B-dependent regulation of Cav1.3 channel membrane targeting and regulation in atrial myocytes.

Rationale: Cardiac membrane excitability is tightly regulated by an integrated network of membrane-associated ion channels, transporters, receptors, and signaling molecules.