Abstract 1093: Proteomic Analysis of Chloride Inward Rectifier Channel Complex and its Pacemaker Function in the Heart
Background: A Cl− inward rectifier channel (Cl.ir) has been recently discovered in cardiac myocytes of several species. ClC-2, a member of the ClC voltage-gated Cl− channel gene family, may be responsible for endogenous Cl.ir in the heart. In this study ClCn2−/− mice were used to directly test the hypothesis that Cl.ir may play an important role in cardiac electrophysiology, especially the pacemaker activity.
Methods: A multitude of approaches were used to examine the functional and molecular expression of ClC-2 in sino-atrial (SA) nodal region of guinea pig heart. Telemetry ECG, echocardiograph, and whole-cell voltage- or current-clamp were used to examine the changes in cardiac phenotype in age-matched ClCn2−/− mice and wild-type (WT) littermates. Functional proteomics approaches (co-immunoprecipitation, affinity pull-down, and yeast two-hybrid) were used to identify potential partners of ClC-2 channels in the heart.
Cl.ir was functionally expressed at a significantly higher percentage of SA nodal cells than in atrial and ventricular myocytes of guinea pig heart.
RT-PCR and immunohistological analyses support the molecular expression of ClC-2 in guinea pig SA nodal cells. In addition, Cl.ir currents in guinea pig SA nodal cells are blocked by intracellular dialysis of antibody against ClC-2.
Targeted ClC-2 disruption in mouse caused decreases in cardiac function and a slower heart rate under both rest and exercise conditions, strongly suggesting that ClC-2 may encode endogenous Cl.ir and play an important role in cardiac function, especially the pacemaker activity.
PKA, protein phosphatase 1, myosin binding protein C, and Hsp-40 were identified as potential partners of functional ClC-2 channel complex in the heart.
Conclusion: Cl.ir is important in pacemaker activity of the heart. The functional Cl.ir channels may be a multiprotein complex of ClC-2 channel protein and other regulatory proteins.