Abstract 20669: LRRC10 and Dilated Cardiomyopathy-Associated I195T LRRC10 Mutation Differentially Regulate Cav1.2 L-Type Ca2+ Channels
Leucine-rich repeat containing protein 10 (LRRC10) is a cardiac-specific protein and loss of LRRC10 in LRRC10-/- mice results in dilated cardiomyopathy (DCM). Here we describe a novel mutation in human LRRC10, I195T, associated with early onset DCM. However, the mechanism by which LRRC10 and mutations in LRRC10 contribute to DCM remains unknown. Because LRRC10-/- hearts exhibit defective contractility and LRRC10 is localized to the dyad, we tested the hypothesis that LRRC10 interacts with and regulates Cav1.2 channels. We performed whole-cell patch clamp electrophysiology on isolated ventricular myocytes from wild-type (WT) and LRRC10-/- mice. The L-type Ca2+ currents (ICa,L) was significantly decreased in LRRC10-/- myocytes (-4.9 ± 0.22 pA/pF, N=10; 7 mice) compared to wild-type (-7.7 ± 0.72 pA/pF, N=5; 4 mice). The voltage dependence of current activation and inactivation, however, were not significantly different between LRRC10-/- and WT. Western blot experiments using heart lysates from LRRC10-/- and WT showed no significant difference in the expression of the Cav1.2 L-type Ca2+ channel protein. Co-immunoprecipitation experiments using WT heart lysates revealed an association between LRRC10 and the Cav1.2 channel subunit. To determine whether LRRC10 or I195T mutation modulates Cav1.2 L-type Ca2+ channels, additional whole-cell patch clamp electrophysiology and biochemistry experiments were performed using HEK293 cells transiently transfected with the L-type Ca2+ channel complex (Cav1.2, β2CN2, α2δ subunits) alone or with WT or I195T LRRC10. Electrophysiology studies showed an increase in ICa,L with WT LRRC10 coexpression (-81 ± 5.3 pA/pF, N=12), but a decrease in ICa,Lwith I195T LRRC10 coexpression (-18.2 ± 3.3 pA/pF, N=9) compared to control (-34.1 ± 2.2 pA/pF, N=17) at 0mV. Surface biotinylation experiments using lysates prepared from transiently transfected HEK293 cells revealed that neither the WT nor I195T LRRC10 altered the expression of L-type Ca2+ channels on the plasma membrane. Taken together, these findings identify a role for LRRC10 in the regulation of cardiac L-type Ca2+ channels and that mutations in the LRR motif of LRRC10 can directly impact L-type Ca2+ channel function possibly contributing to the development of DCM.
Author Disclosures: M.T. Woon: None. P. Long: None. L. Reilly: None. J.M. Evans: None. A.M. Keefe: None. M.R. Lea: None. R.C. Balijepalli: None. T.M. Olson: None. Y. Lee: None. T.J. Kamp: None.
- © 2016 by American Heart Association, Inc.