Molecular determinants of ion permeation and selectivity in inositol 1,4,5-trisphosphate receptor Ca2+ channels

We tested the hypothesis that key residues in a putative intraluminal loop contribute to determination of ion permeation through the intracellular Ca2 + release channel (inositol 1,4,5-trisphosphate receptors (IP(3)Rs)) that i s gated by the second messenger inositol 1,4,5-trisphophate (IP3), To accom plish this, we mutated residues within the putative pore forming region of the channel and analyzed the functional properties of mutant channels using a Ca-45(2+) flux assay and single channel electrophysiological analyses. T wo IP3R mutations, V2548I and D2550E, retained the ability to release Ca-45 (2+) in response to IP3. When analyzed at the single channel level; both re combinant channels had IP3-dependent open probabilities similar to those ob served in wild-type channels. The mutation V2548I resulted in channels that exhibited a larger K+ conductance (489 +/- 13 picosiemens (pS) for V2548I versus 364 +/- 5 pS for wildtype), but retained a Ca2+ selectivity similar to wild-type channels (PCa2+:PK+ similar to 4:1). Conversely, D2550E channe ls were nonselective for Ca2+ over K+ (PCa2+:PK+ similar to 0.6:1), while t he Kf conductance was effectively unchanged (391 +/- 4 pS), These results s uggest that amino acid residues Val(2548) and Asp(2550) contribute to the i on conduction pathway. We propose that the pore of IP3R channels has two di stinct sites that control monovalent cation permeation (Val(2548)) and Ca2 selectivity (Asp(2550)).