Cardiac Specific Leucine-Rich Repeat Containing 10 (LRRC10) Protein Interacts with and Regulates the Cav1.2 L-Type Ca2+ Channels

Cardiac L-type Ca2+ channels (LTCC) play essential role in multiple cellular processes including excitation-contraction coupling, signaling and gene regulation. Diverse families of regulatory and scaffolding proteins regulate the LTCCs in the cardiomyocytes. Leucine-rich repeat containing 10 (LRRC10) is a cardiac-specific scaffolding protein that plays a critical role in heart development and function. Recently we have demonstrated that the Lrrc10-null (Lrrc10-/-) mice develop dilated cardiomyopathy and Lrrc10-/- cardiomyocytes exhibit reduced LTCC current (ICa,L). However, it is unclear how LRRC10 regulates LTCC function in the heart. To investigate the role of LRRC10 in the regulation of LTCCs, we co-expressed the WT LRRC10 with LTCC channel complex comprising of the Cav1.2, β2CN2, α2δ subunits in HEK293 cells and performed whole-cell patch clamp experiments. Co-expression of LRRC10 significantly enhanced (225%) the peak LTCC current (IBa,L −75±7 pA/pF) density compared to LTCC alone (−33±3 pA/pF). We then introduced a single point mutation (substituted alanine for histidine) in the putative functional interaction site at amino acid position 150 (H150A) of LRRC10. Co-expression of LRRC10H150A caused significant reduction (27%) in IBa,L (-57±4 pA/pF) compared to WT LRRC10 co-expression. Furthermore, G/Gmax analysis revealed a significant shift in the voltage dependence of activation to more negative potentials with the co-expression of either the WT LRRC10 (V1/2,-21.7mV) or the H150A mutation (V1/2, −25.1mV) compared to LTCC alone (V1/2, −16.4mV). Finally, co-immunoprecipitation and western blot analysis in HEK293 cells demonstrated that LRRC10 associates with Cav1.2 subunit but not the Cav2 subunit. Moreover, co-expression of the LRRC10 H150A mutation disrupted the association of Cav1.2 with LRRC10. We conclude that LRRC10 may directly associate with Cav1.2 subunit and regulate the LTCC function by enhancing the surface expression, density and biophysical properties of the ICa,L.