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Originally published In Press as doi:10.1074/jbc.M102751200 on June 26, 2001

J. Biol. Chem., Vol. 276, Issue 34, 31760-31771, August 24, 2001
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Functional Characterization of Mutants in the Predicted Pore Region of the Rabbit Cardiac Muscle Ca2+ Release Channel (Ryanodine Receptor Isoform 2)*

Guo Guang DuDagger , Xinghua Guo, Vijay K. Khanna, and David H. MacLennan§

From the Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada

A highly conserved amino acid sequence, GVRAGGGIGD4831, which may form part of the Ca2+ release channel pore in RyR2, was subjected to Ala scanning or Ala to Val mutagenesis; function was then measured by expression in HEK-293 cells, followed by Ca2+ photometry, high affinity [3H]ryanodine binding, and single-channel recording. All mutants except I4829A and I4829T (corresponding to the I4897T central core disease mutant in RyR1) displayed caffeine-induced Ca2+ release in HEK-293 cells; only mutants G4826A, I4829V, and G4830A retained high affinity [3H]ryanodine binding; and single-channel function was found for all mutants tested, except for G4822A and A4825V. EC50 values for caffeine-induced Ca2+ release were increased for G4822A, R4824A, G4826A, G4828A, and D4831A; decreased for V4823A; and unchanged for A4825V, G4827A, I4829V, and G4830A. Ryanodine (10 µM), which did not stimulate Ca2+ release in wild type (wt), did so in Ala mutants in amino acids 4823-4827. It inhibited the caffeine response in wt and most mutants, but enhanced the amplitude of caffeine-induced Ca2+ release in mutant G4828A. It also restored caffeine-induced Ca2+ release in mutants I4829A and I4829T. In single-channel recordings, mutants I4829V and G4830A retained normal conductance, whereas all others had decreased unitary channel conductances ranging from 27 to 540 picosiemens. Single-channel modu-lation was retained in G4826A, I4829V, and G4830A, but was lost in other mutants. In contrast to wt and G4826A, I4829V, and G4830A, in which divalent metals were preferentially conducted, mutants with loss of modulation had no selectivity of divalent cations over a monovalent cation. Analysis of Gly4822 to Asp4831 mutants in RyR2 supports the view that this highly conserved sequence constitutes part of the ion-conducting pore of the Ca2+ release channel and plays a key role in ryanodine and caffeine binding and activation.

* This work was supported in part by Grant MT-3399 (to D. H. M.) from the Canadian Institutes for Health Research.

Dagger Postdoctoral fellow of the Heart and Stroke Foundation of Canada.

§ To whom correspondence should be addressed: Banting and Best Dept. of Medical Research, University of Toronto, Charles H. Best Inst., 112 College St., Toronto, Ontario M5G 1L6, Canada. Tel.: 416-978-5008; Fax: 416-978-8528; E-mail: david.maclennan@utoronto.ca.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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