Selective open-channel block of Shaker (Kv1) potassium channels by S-nitrosodithiothreitol (SNDTT)

Large quaternary ammonium (QA) ions block voltage-gated K+ (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to t he cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; O NSCH2CH(OH)CH(OH)CH2SNO) produces qualitatively similar "open-channel block " in Kv channels despite a radically different structure. SNDTT is small, e lectrically neutral, and not very hydrophobic. In whole-cell voltage-clampe d squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time -dependent block of Kv channels, but not Na+ or Ca2+ channels. Inactivation -removed ShakerB (ShB Delta) Kv1 channels expressed in HEK 293 cells are si milarly blocked and were used to study further the action of SNDTT. Dose-re sponse data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficien t to produce block. The dissociation constant for the binding of the second SNDTT molecule (K-d2 = 0.14 mM) is lower than that of the first molecule ( K-d1 = 0.67 mM), indicating cooperativity. The half-blocking concentration (K-1/2) is similar to0.2 mM. Steady-state block b this electrically neutral compound has a voltage dependence (about -0.3 e(0)) similar in magnitude b ut opposite in directionality to that reported for QA ions. Both nitrosyl g roups on SNDTT (one oil each sulfur atom) are required for block, but trans fer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (tau approximate to 770 s) i n which these NO groups are liberated, leading to spontaneous reversal of t he SNDTT effect. Competition with internal tetraethylammonium indicates tha t bath-applied SNDTT crosses the cell membrane to act at an internal site, most likek, within the channel cavity. Finally, SNDTT is remarkably selecti ve for Kv1 channels. When individually, expressed in HEK 293 cells, rat Kv1 .1-1.6 display profound time-dependent block by SNDTT, an effect not seen f or Kv2.1, 3.1b, or 4.2.