Selective open-channel block of KV1 potassium channels by S-nitrosodithiothreitol (SNDTT)

Blockade of voltage-gated K+ (Kv) channels is a feature of many large quaternary and tertiary amines. These compounds bind with a 1:1 stoichiometry in an aqueous cavity along the channel pore that is exposed to the cytoplasm only when channels are open. This thesis addresses the action of S-nitrosodithiothreitol (SNDTT; ONSCH₂CH(OH)CH(OH)CH₂SNO), which produces qualitatively similar “open-channel block” in Kv channels despite its unconventional (small, electrically neutral, and polar) structure. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of delayed-rectifier K + channels, but not Na+ or Ca2+ channels. The inactivation-removed ShakerB (ShBΔ) Kv1 channel expressed in HEK 293 cells is blocked in a similar manner and was used to further characterize the action of SNDTT. Dose-response data for ShBΔ indicate that two molecules of SNDTT can bind to each open channel, but binding of a single molecule is sufficient for block. The dissociation constant for the second molecule bound (0.14 mM) is lower than for the first (0.67 mM), indicating cooperativity. Surprisingly, the steady-state level of block by this electrically neutral compound has a voltage-dependence (∼−0.25 e₀) similar in magnitude but opposite in directionality to that reported for amines. Both nitrosyl (-NO) groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site. Through targeted mutagenesis, we have identified two contiguous residues (Thr469 and Ile470) in the channel cavity that are strong determinants of SNDTT sensitivity. At position 469, a side chain -OH group is required for high affinity block, and may form a hydrogen bond with an -NO group on SNDTT. Finally, SNDTT is remarkably selective for Kv1 subfamily channels. When individually expressed in HEK 293 cells, rat Kv1.1–1.6 display profound time-dependent block by SNDTT, an effect not seen for rat Kv2.1, 3.1, or 4.2. SNDTT may therefore be useful as a pharmacological probe of Kv subtype, and may represent the prototype for a new class of pharmaceuticals selectively targeting Kv1 channels.