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, 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 ϳ85% 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 ϳ55%. 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 ␤ 1 -adrenergic receptor (␤1-AR) stimulation. In control, isoproterenol reduced I K1 amplitude by ϳ75%, an effect that was blunted following SAP97 silencing. Our co-IP data show that ␤1-AR associates with SAP97 and Kir2.1 and also that Kir2.1 co-IPs with protein kinase A and ␤1-AR. SAP97 immunolocalizes with protein kinase A and ␤1-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 ␤1-AR regulation of I K1 .In the heart, inward rectifier potassium current (I K1 ) plays a key role in stabilizing the resting membrane potential, determining the excitation threshold, and initiating the final repolarization phase of the action potential. Inward rectifier potassium channel subfamily 2 members (Kir2.1, Kir2.2, and Kir2.3 (Kir2.x) 3 ) are the molecular correlates of I K1 in the heart (1, 2). In contrast to other cardiac ion channels, relatively little information is available on protein-protein interactions involving Kir2.x channels that are important for their function. We have shown recently that Kir2.3 is regulated by synapse-associated protein 97 (SAP97) in a heterologous expression system, but it is not known if cardiac Kir2.x channels are regulated in a similar manner in situ (3).SAP97 belongs to the membrane-associated guanylate kinase family of proteins and is ubiquitously expressed in the heart (4). The membrane-associated guanylate kinase family of proteins are involved in the trafficking and assembly of proteins into macromolecular signaling complexes (5). They have protein-protein interaction domains such as PDZ (PSD-95, disclarge, ZO-1) domains, guanylate k...

The Interaction of Caveolin 3 Protein with the Potassium Inward Rectifier Channel Kir2.1

“…The antibodies were raised in rabbits and purified as described previously (15). Results from in-house anti-Kir2.1 antibody were confirmed by using commercially available Santa Cruz rabbit anti-Kir2.1 antibody (data not shown), which was shown previously to detect Kir2.1 in rat tissue (28). Other antibodies included mouse anti-Cav3 antibody (BD Biosciences), rabbit anti-HA (Applied Biological Materials, Inc.) and mouse anti-HA (Covance).…”

“…This is derived from this work and recent work by Abriel and colleagues (27), who demonstrated that Na V 1.5 associates with syntrophin and SAP97, Makielski and colleagues (5,23), who established that Na V 1.5 coimmunoprecipitates with Cav3 and syntrophin, Vandenberg and colleagues, who showed that Kir2.1 coimmunoprecipitates with syntrophin (11), and Anumonwo and colleagues (28), who assert that Kir2.1 associates with SAP97. This is also in agreement with a recent study by Jalife and colleagues (29), who propose reciprocal modulation of Kir2.1 and Na V 1.5 and suggest that these ion channels are in the same macromolecular complex.…”

“…Immunostaining-Immunostaining of tissue and single cells was performed as described previously (28). The rabbit antiKir2.1 antibody was generated by using a peptide sequence (388 -401, DDSENGVPESTSTD) from the C terminus of hKir2.1.…”

“…As a negative control, isotype control IgG was used. Cell lysates were separated on precast 10 -20% SDS-PAGE gels and immunoblotted as described previously (28).…”

Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation

“…It has been shown that interaction of cardiac ion channels with SAP97 occurs with a number of potassium channels including Kv1.5, Kv4, Kir2.2 and 2.3, and the hyperpolarization-activated, cyclic nucleotidegated channels (1,7,27,45,46,61,81). These ion channels interact with SAP97 through standard PDZ-domain binding to the terminal most amino acids of the ion channel (1,27,45,46,53,61,81) and appear to play an important role in regulation of channel density at the cardiac myocyte membranes (1,79). We show here direct interaction between Panx1 and SAP97, which is important for Panx1 channel density in the membrane.…”