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

Abstract

The IP3R (inositol 1,4,5-trisphosphate receptor) Ca 2+-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 approx. 70% of the 60 cysteine residues in the type-I IP3R are maintained 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 IP 3R. 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 domain 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 C206/214/326A blocked MPEG reactivity in fragment I. We conclude that a set of highly reactive cysteine residues in fragment I are differentially accessible in the SI(+) and SI(-) splice variants of the type-I IP3R. © 2006 Biochemical Society.