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

Mutations in CAV3 cause LQT syndrome 9 (LQT9). A previously reported LQT9 patient had prominent U waves on ECG, a feature that has been correlated with Kir2.1 loss of function. Our objective was to determine whether caveolin 3 (Cav3) associates with Kir2.1 and whether LQT9-associated CAV3 mutations affect the biophysical properties of Kir2.1. Kir2.1 current (I K1 ) density was measured using the whole-cell voltage clamp technique. WT-Cav3 did not affect I K1 . However, F97C-Cav3 and T78M-Cav3 decreased I K1 density significantly by ∼60%, and P104L-Cav3 decreased I K1 density significantly by ∼30% at −60 mV. Immunostained rat heart cryosections and HEK293 cells cotransfected with Kir2.1 and WT-Cav3 both demonstrated colocalization of Kir2.1 and WT-Cav3 by confocal imaging. Cav3 coimmunoprecipitated with Kir2.1 in human ventricular myocytes and in heterologous expression systems. Additionally, FRET efficiency was highly specific, with a molecular distance of 5.6 ± 0.4 nm, indicating close protein location. Colocalization experiments found that Cav3 and Kir2.1 accumulated in the Golgi compartment. On-cell Western blot analysis showed decreased Kir2.1 cell surface expression by 60% when expressed with F97C-Cav3 and by 20% when expressed with P104L-Cav3 compared with WT-Cav3. This is the first report of an association between Cav3 and Kir2.1. The Cav3 mutations F97C-Cav3, P104L-Cav3, and T78M-Cav3 decreased I K1 density significantly. This effect was related to a reduced cell surface expression of Kir2.1. Kir2.1 loss of function is additive to the increase described previously in late I Na , prolonging repolarization and leading to arrhythmia generation in Cav3-mediated LQT9. Background: Regulation of Kir2.1 by WT caveolin 3 and LQT9-causing mutants of caveolin3 is unknown. Results: WT caveolin 3 and mutants of caveolin 3 associate with Kir2.1. Caveolin 3 mutants reduce Kir2.1 current density. Surface expression of Kir2.1 is decreased by caveolin 3 mutations. Conclusion: Caveolin 3 mutations affect Kir2.1 current density by decreasing cell surface expression of Kir2.1. Significance: Kir2.1 loss of function may contribute to the mechanism of arrhythmia generation in caveolin 3-mediated LQT9.