G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to cytosolic and luminal Ca2.

ABSTRACT

The ryanodine receptor ion channel RyR1 is present in skeletal muscle and has a large cytoplasmic N-terminal domain and smaller C-terminal pore-forming domain comprising six transmembrane helices, a pore helix, and a selectivity filter. The RyR1 S6 pore-lining helix has two conserved glycines, Gly-4934 and Gly-4941, that facilitate RyR1 channel gating by providing S6 flexibility and minimizing amino acid clashes. Here, we report that substitution of Gly-4941 with Asp or Lys results in functional channels as indicated by caffeine-induced Ca2+ release response in HEK293 cells, whereas a low response of the corresponding Gly-4934 variants suggested loss of function. Following purification, the RyR1 mutants G4934D, G4934K, and G4941D did not noticeably conduct Ca2+ in single-channel measurements. Gly-4941 replacement with Lys resulted in channels having reduced K+ conductance and reduced selectivity for Ca2+ compared with wildtype. RyR1-G4941K did not fully close at nanomolar cytosolic Ca2+ concentrations and nearly fully opened at 2 µm cytosolic or sarcoplasmic reticulum luminal Ca2+, and Ca2+- and voltage-dependent regulation of RyR1-G4941K mutant channels was demonstrated. Computational methods and single-channel recordings indicated that the open G4941K variant results in the formation of a salt bridge to Asp-4938. In contrast, wildtype RyR1 was closed and not activated by luminal Ca2+ at low cytosolic Ca2+ levels. A model suggested that luminal Ca2+ activates RyR1 by accessing a recently identified cytosolic Ca2+-binding site in the open channel as the Ca2+ ions pass through the pore.