期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 282, 期 9, 页码 6172-6182出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M607698200
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资金
- Wellcome Trust [073388] Funding Source: Medline
Structural models suggest that Are 36 lies within five cytoplasmic portals of the 5-HT3A receptor. We tested both the accessibility of residue 436 and the influence of its charge on single channel conductance (gamma) by substituting Arg(436) with Cys and examining the effect of methanethiosulfonate (MTS) reagents on gamma. Inclusion of positively charged 2-aminoethyl-MTS (MTSEA) within the electrode solution reduced gamma of 5-HT3A(R436C) receptors in outside-out patches from 7.8 +/- 0.5 to 5.0 +/- 0.5 picosiemens (pS). To increase gamma, we substituted Arg(436) by Cys in the 5-HT3A(R432Q,R440A) mutant, yielding the 5-HT3A(QCA) construct with a gamma of 17.7 +/- 0.4 pS. Modification of 5-HT3A(QCA) receptors by MTSEA or 2-(trimethylammonium) ethyl-MTS reduced gamma to 8.7 +/- 0.5 and 6.7 +/- 0.4 pS, respectively, both significantly below that of channels exposed to nonpolar propyl-MTS. Extracellular MTSEA, but not 2-(trimethylammonium) ethyl-MTS, crossed the membrane and in so doing slowly (tau=94s) reduced gamma. MTSEA more rapidly (tau=15s) reduced the gamma of 5-HT3A(QCA) receptors in inside-out patches, an effect reversed by the reducing agent dithiothreitol. Cys(436) modification by negatively charged 2-carboxyethyl-MTS and 2-sulfconatoethyl-MTS increased gamma to 23 +/- 1.0 and 26 +/- 0.7 pS, respectively. MTS reagents did not affect gamma values for 5-HT3A(QDA) constructs with Cys substituted for Lys(431) predicted to be outside the entrance to the portals. Collectively, the data demonstrate that the dynamic modification of the charge of a cytoplasmic residue regulates gamma, consistent with the existence of cytoplasmic portals that impose a rate-limiting barrier to ion conduction in Cys loop receptors.
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