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Correspondence to Manuel Covarrubias: manuel.covarrubias{at}jefferson.edu
Gating of voltage-dependent K+ channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn2+ coordination. Also, added Zn2+ or a potent Zn2+ chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is 
200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact 
-subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1–T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn2+ binding.
Abbreviations used in this paper: FPLC, fast protein, peptide, and polynucleotide liquid chromatography; MTS, methanethiosulfonate; MTSES, 2-sulfonatoethyl-methanethiosulfonate bromide; MTSET, 2-trimethylammonium-ethyl-methanethiosulfonate bromide; NEM, N-ethyl-maleimide; TPEN, tetrakis-(2-pyridylmethyl) ethylendiamide.
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