The Gln4863Ala mutation within a putative, pore-lining trans-membrane helix of the cardiac ryanodine receptor channel alters both the kinetics of ryanoid interaction and the subsequent fractional conductance.
The specific, high-affinity interaction of the plant toxin ryanodine with its molecular target the ryanodine receptor channel (RyR) has been instrumental in RyR research. Alanine scanning of putative pore regions of mouse RyR2 has highlighted the amino acid Gln4863, predicted to lie within trans-membrane helix TM10, as an important determinant of ryanodine binding. We have investigated the effects of several ryanodine derivatives, guanidinopropionylryanodine, 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine, 8beta-amino-9alpha-hydroxyryanodine, and 21-amino-9alpha-hydroxyryanodine, with the mouse Q4863A RyR2 mutant at the single-channel level. Our results demonstrate that the rate of dissociation of all ryanoids investigated is increased by the mutation. The modification of channel function after ryanoid binding is qualitatively similar for wild-type and mutant, but in several cases, single-channel conductances were increased with Q4863A. These novel findings have been interpreted within the framework of existing comparative molecular field analysis studies on ryanoids. We suggest that replacement of a glutamine by an alanine residue at position 4863 causes RyR2 to simultaneously alter interactions with both ends of the ryanoid molecule.
Ranatunga
Kishani M
KM
Myocardial Systems Biology Group, National Heart and Lung Institute, Guy Scadding Building, Imperial College London, London SW3 6LY, United Kingdom.
Moreno-King
Tracy M
TM
Tanna
Bhavna
B
Wang
Ruiwu
R
Chen
S R Wayne
SR
Ruest
Luc
L
Welch
William
W
Williams
Alan J
AJ
D000409:Alanine / Q000235:genetics*; D000818:Animals; D005998:Glycine / Q000235:genetics*; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D017354:Point Mutation; D012433:Ryanodine / Q000031:analogs & derivatives / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D000409:Alanine; D005998:Glycine