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J. Gen. Physiol.,
Volume 113, Number 2, February 1, 1999 177-186
From the * Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153; Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1-5 kHz and filtered at 0.2-1.0 kHz. Channel incorporations were obtained
in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca2+ currents were monitored when 2-30
mM Ca2+ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca2+
current (at 0 mV holding potential) and lumenal [Ca2+] was hyperbolic and saturated at ~4 pA. This relationship
was then defined in the presence of different symmetrical CsCH3SO3 concentrations (5, 50, and 150 mM). Under
these conditions, unitary current amplitude was 1.2 ± 0.1, 0.65 ± 0.1, and 0.35 ± 0.1 pA in 2 mM lumenal Ca2+;
and 3.3 ± 0.4, 2.4 ± 0.2, and 1.63 ± 0.2 pA in 10 mM lumenal Ca2+ (n > 6). Unitary Ca2+ current was also defined
in the presence of symmetrical [Mg2+] (1 mM) and low [Cs+] (5 mM). Under these conditions, unitary Ca2+ current in 2 and 10 mM lumenal Ca2+ was 0.66 ± 0.1 and 1.52 ± 0.06 pA, respectively. In the presence of higher symmetrical [Cs+] (50 mM), Mg2+ (1 mM), and lumenal [Ca2+] (10 mM), unitary Ca2+ current exhibited an amplitude of 0.9 ± 0.2 pA (n = 3). This result indicates that the actions of Cs+ and Mg2+ on unitary Ca2+ current were
additive. These data demonstrate that physiological levels of monovalent cation and Mg2+ effectively compete with
Ca2+ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca2+ is 2 mM, then our results indicate that unitary Ca2+ current under physiological conditions should be <0.6 pA.
Department of
Molecular Biophysics and Physiology, Rush University School of Medicine, Chicago, Illinois 60612; and § Laboratory of Cardiovascular
Science, Gerontology, Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21214
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