Functional Characterization of Mutants in the Predicted Pore Region of the Rabbit Cardiac Muscle Ca²⁺ Release Channel (Ryanodine Receptor Isoform 2)*

Abstract

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.