Inhibition by nickel of the L-type Ca channel in guinea pig ventricular myocytes and effect of internal cAMP.
The characteristics of nickel (Ni) block of L-type Ca current (I(Ca, L)) were studied in whole cell patch-clamped guinea pig cardiac myocytes at 37 degrees C in the absence and presence of 100 microM cAMP in the pipette solution. Ni block of peak I(Ca,L) had a dissociation constant (K(d)) of 0.33 +/- 0.03 mM in the absence of cAMP, whereas in the presence of cAMP, the K(d) was 0.53 +/- 0.05 mM (P = 0.006). Ni blocked Ca entry via Ca channels (measured as I(Ca, L) integral over 50 ms) with similar kinetics (K(d) of 0.35 +/- 0.03 mM in cAMP-free solution and 0.30 +/- 0.02 mM in solution with cAMP, P = not significant). Under both conditions, 5 mM Ni produced a maximal block that was complete for the first pulse after application. Ni block of I(Ca,L) was largely use independent. Ni (0. 5 mM) induced a positive shift (4 to 6 mV) in the activation curve of I(Ca,L). The block of I(Ca,L) by 0.5 mM Ni was independent of prepulse membrane potential (over the range of -120 to -40 mV). Ni (0.5 mM) also induced a significant shift in I(Ca,L) inactivation: by 6 mV negative in cAMP-free solution and by 4 mV positive in cells dialyzed with 100 microM cAMP. These data suggest that, in addition to blocking channel conductance by binding to a site in the channel pore, Ni may bind to a second site that influences the voltage-dependent gating of the L-type Ca channel. They also suggest that Ca channel phosphorylation causes a conformational change that alters some effects of Ni. The results may be relevant to excitation-contraction coupling studies, which have employed internal cAMP dialysis, and where Ni has been used to block I(Ca,L) and Ca entry into cardiac cells.
Hobai
I A
IA
Cardiovascular Research Laboratories, Bristol Heart Institute, and Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom. ionhobai@welchlink.welch.jhu.edu
Hancox
J C
JC
Levi
A J
AJ
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology / Q000502:physiology*; D006168:Guinea Pigs; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D006352:Heart Ventricles; D007700:Kinetics; D009206:Myocardium / Q000166:cytology; D009532:Nickel / Q000494:pharmacology*
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D009532:Nickel; D000242:Cyclic AMP