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, I(K1). To investigate the functional effects of silencing SAP97 on I(K1) in adult rat ventricular myocytes, SAP97 was silenced using an adenoviral short hairpin RNA vector. Western blot analysis showed that SAP97 was silenced by approximately 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 I(K1) whole cell density by approximately 55%. I(K1) 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 I(K1) were unaffected by SAP97 silencing. The major mechanism for the reduction of I(K1) density appears to be a decrease in Kir2.x channel abundance. Furthermore, SAP97 silencing impaired I(K1) regulation by beta(1)-adrenergic receptor (beta1-AR) stimulation. In control, isoproterenol reduced I(K1) amplitude by approximately 75%, an effect that was blunted following SAP97 silencing. Our co-IP data show that beta1-AR associates with SAP97 and Kir2.1 and also that Kir2.1 co-IPs with protein kinase A and beta1-AR. SAP97 immunolocalizes with protein kinase A and beta1-AR in the cardiac myocytes. Our results suggest that in cardiac myocytes SAP97 regulates surface expression of channels underlying I(K1), as well as assembles a signaling complex involved in beta1-AR regulation of I(K1).