Abstract 1073: Cardiac Voltage-gated Nav channel Nav1.5 Requires An AnkyrinG-dependent Pathway For Targeting in Cardiomyocytes
Membrane localization of ion channels is very important for normal function in excitable cells. In heart, voltage-gated Na+ channels are necessary for the rapid upstroke of the cardiomyocyte action potential, and variants in SCN5A (encodes Nav1.5) are associated with fatal arrhythmias. We have identified ankyrin family proteins as critical components for normal ion channel and transporter targeting in cardiomyocytes. Humans with ANK2 (encodes ankyrin-B) loss of function variants display abnormal cardiac phenotypes and risk for sudden cardiac death. Mice that lack ankyrin-B expression display a similar phenotype. Our most recent results demonstrate that a second ankyrin gene product, ankyrin-G (encoded by ANK3) is critical for targeting Nav1.5 to specific cardiomyocyte membrane domains. We assessed the hypothesis that Nav1.5 membrane expression and localization is controlled by an ankyrin-G-dependent pathway and disruption of ankyrin-G/Nav1.5 interactions lead to human cardiac disease in this study. We used a combination of techniques including biochemistry, confocal microscopy, lentiviral expression, and electrophysiology to evaluate the functional relationship between ankyrin-G and Nav1.5. We defined the structural elements on ankyrin-G and Nav1.5 for their interaction using site-directed mutagenesis and in vitro binding assays. Lentiviral expression of shRNA targeted to rat 190 kD ankyrin-G effectively reduced the expression of ankyrin-G with a concomitant reduction of Nav1.5 in immunofluorescence and immunoblot assays. Further-more, primary cardiomyocytes with reduced ankyrin-G expression have a significant reduction in Na+ current density with no evident biophysical effects on Ca2+ current or inactivation-gating of Nav1.5 These results confirm the importance of ankyrin polypeptides for normal cardiac function and shed new light on the importance of intracellular trafficking pathways for the delivery and stability of critical ion channels and transporters in excitable cells.