Subtype-selective regulation of IP3 receptors by thimerosal via cysteine residues within the IP3-binding core and suppressor domain

Samir A. Khan, Ana M. Rossi, Andrew M. Riley, Barry V.L. Potter, Colin W. Taylor

Research output: Contribution to journalArticlepeer-review

19 Citations (Scopus)
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Abstract

IPR (IP [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IPR, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP -evoked Ca release via IPR1 and IP R2, but inhibited IPR3. Activation of IPR is initiated by IP binding to the IBC (IP-binding core; residues 224-604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1-223). Thimerosal (100 μM) stimulated IP binding to the isolated NT (N-terminal; residues 1-604) of IPR1 and IPR2, but not to that of IP R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric- IP) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IPR activation. IP binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IPR was repla ced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP binding to the chimaeric NT and IP -evoked Ca release from the chimaeric IPR. This is the first systematic analysis of the effects of a thiol reagent on each IPR subtype. We conclude that thimerosal selectively sensitizes IPR1 and IPR2 to IP by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.
Original languageEnglish
Pages (from-to)177-184
JournalBiochemical Journal
Volume451
Issue number2
DOIs
Publication statusPublished - 15 Apr 2013

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