Probing the Role of Negatively Charged Amino Acid Residues in Ion Permeation of Skeletal Muscle Ryanodine Receptor

¹ University of North Carolina
² Rush University

^* To whom correspondence should be addressed. E-mail: meissner{at}med.unc.edu.

Submitted on November 9, 2004
Revised on February 10, 2005
Accepted on 27 April 2005

	Abstract

Sequence comparison suggests that the RyRs have pore architecture similar to that of the bacterial K+ channel KcsA. The lumenal loop linking the two most C-terminal transmembrane spanning segments in the RyRs has a predicted pore helix and an amino acid motif (GGGIG) homologous similar to the selectivity filter (TVGYG) of KcsA identified by x-ray analysis (Doyle et al., 1998). The RyRs have many negatively charged amino acid residues in the two regions linking the GGGIG motif and predicted pore helix with the two most C-terminal transmembrane spanning segments. We tested the role of these residues by generating single-site mutants, focusing on amino acid residues conserved among the mammalian RyRs. Replacement of two acidic residues immediately following the GGGIG motif in skeletal muscle ryanodine receptor (RyR1-D4899 and -E4900) with asparagine and glutamine adjacent to the GGGIG motif profoundly affected ion permeation and selectivity. By comparison, mutagenesis of aspartate and glutamate residues in the putative linker regions showed a K+ conductance and selectivity for Ca2+ compared to K+ (PCa/PK) close to wild type. The results show that the negatively charged carboxyl oxygens of D4899 and E4900 side chains are major determinants of RyR ion conductance and selectivity. A model emphasizing the role of the two amino acid residues in determining ion permeation of RyR is presented in (Gillespie et al., (submitted).

Key Words: Ca2+ release channel, ion permeation, ion selectivity, ryanodine receptor, skeletal muscle

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