Functional characterization of mutants in the predicted pore region of the rabbit cardiac muscle Ca2+ release channel (Ryanodine receptor isoform 2)

A highly conserved amino acid sequence, GVRAGG-GIGD(4831), 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 [H-3]ryanodine binding, and single-channel recording. All mutants except I4829A and 14829T (corresponding to the I4S97T 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 muM), 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 I4929T. In single-channel recordings, mutants 14829V 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, I4S29V, and G4830A, but was lost in other mutants. In contrast to wt and G4826A, I4S29V, 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 Gly(4822) to Asp(4831) 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.