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Address correspondence to Toshinori Hoshi, Dept. of Physiology, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104-6085. Fax: (215) 573-5851; email: hoshi{at}hoshi.org
Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BKCa). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BKCa channels formed by human Slo1 (hSlo1) 
subunits alone. Because native BKCa channel complexes may include the auxiliary subunit ß1, we investigated whether ß1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with ß1 present shifted the half-activation voltage much further in the hyperpolarizing direction (–75 mV) as compared with that with alone (–30 mV). This shift was eliminated in the presence of high [Ca2+]i, but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of ß1. Oxidation of cysteine and methionine residues within ß1 was not involved in these potentiated effects because expression of mutant ß1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type ß1. Unlike the results with alone, oxidation by Ch-T caused a significant acceleration of channel activation only when ß1 was present. The ß1 M177 mutation disrupted normal channel activation and prevented the Ch-T–induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming subunit are greatly amplified by the presence of ß1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within ß1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with ß1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+]i.
Key Words: BKCa • hSlo • chloramine-T • methionine • cysteine
GCa, change in free energy change associated with Ca2+ binding; met-O, methionine sulfoxide; Qapp, apparent equivalent charge movement; ROS/RNS, reactive oxygen/nitrogen species; V0.5, half-activation voltage; z, equivalent charge.
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