Regulation of cardiac inward rectifier potassium current (IK1) by synapse-associated protein-97

Abstract

Synapse-associated protein-97 (SAP97) is a membrane-associated guanylate kinase scaffolding protein expressed in cardiomyocytes. SAP97 has been shown to associate and modulate voltage-gated potassium (Kv) channel function. In contrast to Kv channels, little information is available on interactions involving SAP97 and inward rectifier potassium (Kir2.x) channels that underlie the classical inward rectifier current, IK1. To investigate the functional effects of silencing SAP97 on IK1 in adult rat ventricular myocytes, SAP97 was silenced using an adenoviral short hairpin RNA vector. Western blot analysis showed that SAP97 was silenced by ∼85% on day 3 post-infection. Immunostaining showed that Kir2.1 and Kir2.2 co-localize with SAP97. Co-immunoprecipitation (co-IP) results demonstrated that Kir2.x channels associate with SAP97. Voltage clamp experiments showed that silencing SAP97 reduced IK1 whole cell density by ∼55%. IK1 density at -100 mV was -1.45 ± 0.15 pA/picofarads (n = 6) in SAP97-silenced cells as compared with -3.03 ± 0.37 pA/picofarads (n = 5) in control cells. Unitary conductance properties of IK1 were unaffected by SAP97 silencing. The major mechanism for the reduction of IK1 density appears to be a decrease in Kir2.x channel abundance. Furthermore, SAP97 silencing impaired IK1 regulation by β1-adrenergic receptor (β1-AR) stimulation. In control, isoproterenol reduced I K1 amplitude by ∼75%, an effect that was blunted following SAP97 silencing. Our co-IP data show that β1-AR associates with SAP97 and Kir2.1 and also that Kir2.1 co-IPs with protein kinase A and β1-AR. SAP97 immunolocalizes with protein kinase A and β1-AR in the cardiac myocytes. Our results suggest that in cardiac myocytes SAP97 regulates surface expression of channels underlying IK1, as well as assembles a signaling complex involved in β1-AR regulation of IK1. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.