Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC157
Excitatory and inhibitory control of L-type Ca2+ current by pH in ventricular myocytes
N. Saegusa2, E. L. Dilworth1, K. W. Spitzer2, R. D. Vaughan-Jones1
1. Burdon Sanderson Cardiac Science Centre, Physiology, Anatomy and Genetics, Oxford University, Oxford, United Kingdom. 2. Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, Utah, United States.
The modulatory effect of H+-ions on the L-type Ca2+-current (ICa,L) in mammalian ventricular myocytes is controversial, with reports suggesting either inhibition (Ref 1) or no effect (Ref 2). Significant modulation would imply that acid-base disturbances control excitation-contraction coupling in heart, at least partly by targeting ICa,L. The sensitivity of ICa,L to experimental changes of pHi and pHo was investigated (whole-cell voltage-clamp), while measuring intracellular Ca2+ (Ca2+i) or pHi (epifluorescence-microscopy), in rabbit & guinea-pig ventricular myocytes (similar results in both species). In rabbit myocytes exposed to 30 μM cariporide (to inhibit Na+/H+ exchange), reducing pHo to 6.50 (Hepes-buffered superfusates) decreased ICa,L, at a test voltage of -10mV, by -52.3 ± 1.9%, and by -13.0 ± 1.7 % at +20mV (n=7). In contrast, reducing pHi from 7.20 to 6.80 (80mM Na+-acetate superfusion) increased ICa,L by +73.8 ± 9.1 % at a test-voltage of -10mV, but more modestly decreased it by -25.1±1.5 % at +20mV (n=14). The effect of pHi on ICa,L was the same when perforated instead of whole-cell patch pipettes were used to control membrane potential (amphotericin B; n=8). When Ca2+i was buffered with 5mM intracellular BAPTA, the excitatory effect of low pHi was even more marked (+221.2 ± 20.0 %) at -10mV, with no significant inhibition at +20mV (n=10). This result suggests that inhibition by low pHi is secondary to a rise of Ca2+i, while stimulation is a direct effect of intracellular H+ ions. We conclude that extracellular H+-ions inhibit while intracellular H+-ions can stimulate ICa,L. In further experiments, low pHi and pHo-effects were additive, tending to cancel when appropriately combined. They persisted after inhibition of CaM-kinase II (with 1.0 μM KN-93; n=8). These effects are consistent with fixed negative charge-screening by H+-ions on both sides of the sarcolemma (altering the voltage-field local to Ca2+ channel proteins), with additional channel-block by H+o and Ca2+i. Action potential duration (APD) was also strongly H+-sensitive, being shortened by low pHo (-17.8 ± 2.7 % at 90% repolarisation, n=9), but lengthened by low pHi (+57.2 ± 6.5 %, n=15). Results of reducing pHo or pHi while measuring ICa,L (nifedipine/Cd2+-sensitive current) under action potential clamp control indicate that that effects on APD are caused mainly by H+-induced changes in late Ca2+-entry through the L-type Ca2+-channel. Kinetic analyses of pH-sensitive channel-gating, when combined with whole-cell modelling, successfully predict the APD-changes, plus many of the accompanying changes in Ca2+-signalling. We conclude that the pHi- versus pHo-control of ICa,L will influence electrical and Ca2+-dependent signalling during acid-base disturbances in the heart.
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