Complex modulation of L-type Ca2+ current inactivation by sorcin in isolated rabbit cardiomyocytes
Modulation of the L-type Ca2+ channel (LTCC) by sorcin was investigated by measuring the L-type Ca2+ current (I Ca,L) in isolated rabbit ventricular myocytes using ruptured patch, single electrode voltage clamp in the absence of extracellular Na+. Fifty millimolars EGTA (170 nM Ca2+) in the pipette solution buffered bulk cytoplasmic [Ca2+], but retained rapid Ca2+-dependant inactivation of I Ca,L,. Recombinant sorcin (3 μM) in the pipette significantly slowed time-dependant inactivation (τ fast: 8.8 ± 0.9 vs. 15.1 ± 1.7 ms). Sorcin had no significant effect on I Ca,L, after inhibition of the sarcoplasmic reticulum (SR). Using 10 mM 1,2-bis(o-N,N,N′,N′-tetraacetic acid (170 nM Ca2+), I Ca,L inactivation was then determined by a Ca2+ -independent, voltage-dependant process. Under these conditions, 3 μM sorcin speeded up inactivation. A similar effect was observed by substitution of Ca2+ with Ba2+. Down-regulation of endogenous sorcin to 27 ± 7% using an RNAi adenoviral vector slowed inactivation of I Ca,L by ∼42%. The effects of sorcin on voltage-dependant inactivation were mimicked by a truncated form of the protein containing only the Ca2+-binding domain. This data is consistent with two independent actions of sorcin on the LTCC: (1) slowing Ca2+-dependant inactivation and (2) stimulating voltage-dependant inactivation. The net effect of sorcin on the time-dependent inactivation of I Ca,L was a balance between these two effects. Under normal conditions, sorcin slows I Ca,L inactivation because the effects of Ca2+-dependant inactivation out-weigh the effects on voltage-dependant inactivation.
KeywordsCardiac myocytes Excitation–contraction coupling Intracellular calcium Ca2+ current Inactivation Membrane current
The authors would like to thank Aileen Rankin and June Irvine for preparation of cardiomyocytes. This work was funded by the British Heart Foundation (Mark Fowler and Godfrey Smith) and the Deutsche Forschungsgemeinschaft (DFG, grant 1233/7-3 (Gerd Hasenfuß and Tim Seidler); the authors would also like to thank Carlotta Zamparelli and Daniela Verzili for help in generation and purification of recombinant proteins.
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