Molecular Characterization of the Inositol 1,4,5-Trisphosphate Receptor Pore-forming Segment

Zachary T Schug,Paula C.A Da Fonseca,Cunnigaiper D Bhanumathy,Larry Wagner,Xianchao Zhang,Bradley Bailey,Edward P Morris,David I Yule,Suresh K Joseph

doi:10.1074/jbc.m706645200

Abstract

Specific residues in the putative pore helix, selectivity filter, and S6 transmembrane helix of the inositol 1,4,5-trisphosphate receptor were mutated in order to examine their effects on channel function. Mutation of 5 of 8 highly conserved residues in the pore helix/selectivity filter region inactivated the channel (C2533A, G2541A, G2545A, G2546A, and G2547A). Of the remaining three mutants, C2527A and R2543A were partially active and G2549A behaved like wild type receptor. Mutation of a putative glycine hinge residue in the S6 helix (G2586A) or a putative gating residue at the cytosolic end of S6 helix (F2592A) had minimal effects on function, although channel function was inactivated by G2586P and F2592D mutations. The mutagenesis data are interpreted in the context of a structural homology model of the inositol 1,4,5-trisphosphate receptor.

Highlights

In common with many voltage-gated ion channels, the IP3R and its close relative the ryanodine receptor (RyR) contain a short luminal pore helix and a selectivity filter [1]
A model for the gating mechanism of the IP3R channel has been proposed in which the N-terminal region of the receptor interacts with the channel domain at sites located in the cytosol-exposed S4-S5 linker [9]
It has been proposed that conformational changes in the ligand-binding domain resulting from IP3 binding cause a mechanical movement of the S4-S5 linker that releases the constriction of the S6 helix bundle and allows Ca2ϩ ions to exit the channel [9]

Summary

Introduction

In common with many voltage-gated ion channels, the IP3R and its close relative the ryanodine receptor (RyR) contain a short luminal pore helix and a selectivity filter [1]. To test if mutations of the conserved residues to alanine affect function, we employed a 45Ca2ϩ flux assay using microsomal vesicles prepared from COS-7 cells co-transfected with various mutant IP3R constructs along with the Ca2ϩ pump, SERCA2b.

Results

Conclusion