NOTE:THE SPECIAL CHARACTERS IN THIS ABSTRACT CANNOT BE DISPLAYED CORRECTLY ON THIS PAGE. PLEASE REFER TO THE ABSTRACT ON THE PUBLISHER'S WEBSITE FOR AN ACCURATE DISPLAY. Structural models suggest that Arg4³6 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 (? ) by substituting Arg 4³6 with Cys and examining the effect of methanethiosulfonate (MTS) reagents on ? . Inclusion of positively charged 2-aminoethyl- MTS (MTSEA) within the electrode solution reduced ? of 5-HT3A(R436C) receptors in outside-out patches from 7.8 ± 0.5 to 5.0 ± 0.5 picosiemens (pS). To increase ? , we substituted Arg4³6 by Cys in the 5-HT3A(R432Q,R440A) mutant, yielding the 5-HT3A(QCA) construct with a ? of 17.7 ± 0.4 pS. Modification of 5-HT3A(QCA) receptors by MTSEA or 2-(trimethylammonium) ethyl-MTS reduced ? 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 (t= 94 s) reduced ?. MTSEA more rapidly (t=15 s) reduced the ? of 5-HT3A(QCA) receptors in inside-out patches, an effect reversed by the reducing agent dithiothreitol. Cys4³6 modification by negatively charged 2-carboxyethyl-MTS and 2-sulfonatoethyl-MTS increased to 23 ± 1.0 and 26 ± 0.7 pS, respectively. MTS reagents did not affect ? values for 5-HT3A(QDA) constructs with Cys substituted for Lys4³¹ 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 ?, consistent with the existence of cytoplasmic portals that impose a rate-limiting barrier to ion conduction in Cys loop receptors.