Effect of verapamil on the action of methanethiosulfonate reagents on human voltage-gated K(v)1.3 channels: implications for the C-type inactivated state.

BACKGROUND AND
PURPOSE: Voltage-gated K(v)1.3 channels appear on T-lymphocytes and are characterized by their typical C-type inactivation. In order to develop drugs stabilizing the C-type inactivated state and thus potentially useful in treatment of autoimmune diseases, it is important to know more about the three-dimensional structure of this inactivated state of the channel. EXPERIMENTAL APPROACH: The patch-clamp technique was used to study effects of methanethiosulphonate (MTS) compounds on currents through wild-type human K(v)1.3 (hK(v)1.3) and two mutant channels, hK(v)1.3 V417C and hK(v) 1.3 H399T-V417C, in the closed, open and inactivated states. KEY
RESULTS: Extracellular application of 2-aminoethyl methanethiosulphonate (MTSEA) irreversibly reduced currents through hK(v) 1.3 V417C channels in the open and inactivated, but not in the closed state, indicating that a modification was possible. Co-application of verapamil prevented this reduction. Intracellular application of MTSEA and [2-(trimethylammonium)ethyl] methanethiosulphonate (MTSET) also modified the mutant channels, whereas extra- and intracellular application of sodium (2-sulfonatoethyl)methanethiosulphonate (MTSES) and intracellular application of MTSET did not. CONCLUSIONS AND IMPLICATIONS: Our experiments showed that the binding site for MTS compounds was intracellular in the mutant channels and that the V417C mutant channels were modified in the open and the inactivated states, and this modification was prevented by verapamil. Therefore, the activation gate on the intracellular side of the selectivity filter must be open during inactivation. Furthermore, although the S6 segment is moving further apart during inactivation, this change does not include a movement of the side chain of the amino acid at position 417, away from lining the channel pore.