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 is gated by the second messenger inositol 1,4,5-trisphophate (IP3), To accomplish 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. Two 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 recombinant channels had IP3-dependent open probabilities similar to those observed 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 channels were nonselective for Ca2+ over K+ (PCa2+:PK+ similar to 0.6:1), while the Kf conductance was effectively unchanged (391 +/- 4 pS), These results suggest that amino acid residues Val(2548) and Asp(2550) contribute to the ion conduction pathway. We propose that the pore of IP3R channels has two distinct sites that control monovalent cation permeation (Val(2548)) and Ca2+ selectivity (Asp(2550)).