Abstract 419: The Anchoring Protein SAP97 Promotes Cell Surface Expression of Cardiac Kv4.x Channels
The Shal-type Kv channels (Kv4.x) are believed to underlie a large part of the native transient outward current Ito in the heart. These K1channels operate as supramolecular complexes, comprising pore-forming alpha-subunits and a number of proteins such as KChIP (Kv Channel Interacting Protein). The protein SAP97, a member of the membrane associated guanylate kinase (MAGUK) proteins, is a key player in organizing ion channels in the plasma membrane of various cell types including cardiac myocytes. However, little is known on the interaction between SAP97 and Kv4.x channels.
Aim: To study if SAP97 modulates cardiac Kv4.x channels.
Methods: The SAP97 was transiently transfected in a CHO stable cell line expressing Kv4.3 and KChIP2a, or co-transfected with Kv4.x channels in CHO cells. Isolated rat neonatal cardiomyocytes were transduced in culture with a SAP97-recombinant adenovirus. Currents were recorded with the patch-clamp technique in the whole-cell configuration. Co-immunoprecipitation was performed with membrane proteins from adult rat ventricular myocardium.
Results: In CHO cells expressing Kv4.3 without KChIP2a, a small transient outward current was recorded (at +60 mV: 43±8 pA/pF, n=9) which was increased by a factor 1.8 when SAP97 was co-expressed (79±9 pA/pF, n=8; p<0.01). In the absence of KChIP2a, Kv4.2 was also poorly expressed in the membrane (12±2 pA/pF, n=9) but SAP97 increased current density by a factor 1.8 (22±4 pA/pF, n=10; p<0.05). CHO cells co-expressing Kv4.3 and KChIP2a showed a huge transient outward current (177±24 pA/pF, n=11; p<0.001) with a fast recovery from inactivation. In these cells, SAP97 increased the current density (547±107 pA/pF, n=4; p<0.001) by a factor 3 (on 44 cells from 3 series of experiments) without modifying recovery from inactivation. In cardiac myocytes, there was a marked increased of the outward current after adenovirus-mediated overexpression of SAP97, notably of its transient component Ito (3±0.4 pA/pF, n=8 vs 0.8±0.3 pA/pF, n=5 in controls; p<0.01). Finally the two proteins SAP97 and Kv4.3 could be co-immunoprecipitated from cardiac membrane proteins.
Conclusion: SAP97 is a new partner of Kv4.x channels that promotes their surface expression by a mechanism likely distinct from the one of KChIP2a.