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Biophys J, April 2000, p. 1835-1851, Vol. 78, No. 4

Effects of Cytoplasmic and Luminal pH on Ca2+ Release Channels from Rabbit Skeletal Muscle

D. R. Laver,* K. R. Eager,dagger L. Taoube,Dagger and G. D. Lambdagger

 *Division of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, ACT 2601;  dagger School of Zoology, La Trobe University, Bundoora, Victoria 3083; and  Dagger John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia

Ryanodine receptor (RyR)-Ca2+ release channels from rabbit skeletal muscle were incorporated into lipid bilayers. The effects of cytoplasmic and luminal pH were studied separately over the pH range 5-8, using half-unit intervals. RyR activity (at constant luminal pH of 7.5) was inhibited at acidic cytoplasmic pH, with a half-inhibitory pH (pHI) ~6.5, irrespective of bilayer potential and of whether the RyRs were activated by cytoplasmic Ca2+ (50 µM), ATP (2 or 5 mM), or both. Inhibition occurred within ~1 s and could be fully reversed within ~1 s after brief inhibition or within ~30-60 s after longer exposure to acidic cytosolic pH. There was no evidence of any hysteresis in the cytoplasmic pH effect. Ryanodine-modified channels were less sensitive to pH inhibition, with pHI at ~5.5, but the inhibition was similarly reversible. Steady-state open and closed dwell times of RyRs during cytoplasmic pH inhibition suggest a mechanism where the binding of one proton inhibits the channel and the binding of two to three additional protons promotes further inhibited states. RyR activity was unaffected by luminal pH in the pH range 7.5 to 6.0. At lower luminal pH (5-5.5) most RyRs were completely inhibited, and raising the pH again produced partial to full recovery in only ~50% of cases, with the extent of recovery not detectably different between pH 7.5 and pH 9. The results indicate that isolated skeletal muscle RyRs are not inhibited as strongly by low cytoplasmic and luminal pH, as suggested by previous single-channel studies.

Biophys J, April 2000, p. 1835-1851, Vol. 78, No. 4
© 2000 by the Biophysical Society   0006-3495/00/04/1835/17  $2.00


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