Reactivity of free thiol groups in type-I inositol trisphosphate receptors

Affordable Access

Reactivity of free thiol groups in type-I inositol trisphosphate receptors

Publisher
Portland Press Ltd.
Publication Date
Dec 23, 2005
Source
PMC
Keywords
License
Unknown

Abstract

bic281.dvi Biochem. J. (2006) 393, 575–582 (Printed in Great Britain) doi:10.1042/BJ20050889 575 Reactivity of free thiol groups in type-I inositol trisphosphate receptors Suresh K. JOSEPH1, Steven K. NAKAO and Siam SUKUMVANICH Department of Pathology, Thomas Jefferson University, Room 230A JAH, 1020 Locust Street, Philadelphia, PA 19107, U.S.A. The IP3R (inositol 1,4,5-trisphosphate receptor) Ca2+-release channel is known to be sensitive to thiol redox state. The present study was undertaken to characterize the number and location of reactive thiol groups in the type-I IP3R. Using the fluorescent thiol-reactive compound monobromobimane we found that ap- prox. 70% of the 60 cysteine residues in the type-I IP3R are main- tained in the reduced state. The accessibility of these residues was assessed by covalently tagging the IP3R in membranes with a 5 kDa or 20 kDa MPEG [methoxypoly(ethylene glycol) maleimide]. MPEG reaction caused a shift in the mobility of IP3R on SDS/PAGE that was blocked by pretreatment of the membranes with dithiothreitol, N-ethylmaleimide, mersalyl or thimerosal, indicating that MPEG reactivity was specific to thiol groups on the IP3R. Trypsin cleavage of the type-I IP3R generates five defined domains. In cerebellum membranes, MPEG reacted over a 5 min interval with tryptic fragment I and fragment III, but not fragments II, IV or V. Fragment I appears as a doublet in cerebellum membranes, corresponding to the presence and absence of the SI splice site in this region (SI is a spliced do- main corresponding to amino acids 318–332). Only the fragment I band corresponding to the SI(+) splice form shifted after reaction with MPEG. Expression of SI(+) and SI(−) spliced forms in COS cell microsomes confirmed this result. The MPEG-induced shift was not prevented when the cysteine residue present in the SI splice domain (C326A) or the remaining seven cysteine residues in fragment I were individually mutated. Of the combination mutations screened, only the mutation of C206/214/3

Report this publication

Statistics

Seen <100 times