To investigate the binding site of verapamil which blocks currents through hKv1.3 channels from the intracellular side of the channel, we used wt as well as a mutant channel (H399T). The position H399 in hKv1.3 is located at the extracellular loop of the channel between the pore and the S6 helix and the mutation H399T resulted in a strong reduction of the C-type inactivated state of the channel. Using the whole-cell recording mode of the patch clamp technique we performed electrophysiological experiments evaluating the different abilities of verapamil to block currents through hKv1.3 channels when the intracellular pipette solution contained 135 mM TMA+ and 10 mM of either K+ or Rb+ . In the wt channel the Kd for verapamil with 10 mM K+i was 9 mM and with 10 mM Rb + i was 34 mM. Therefore the affinity for verapamil was reduced by a factor of ~4 with Rb+i compared to K+i. The H399T mutant channel did not show a change of verapamil affinity in K+i compared to Rb+i (Kd ~6 mM). Interestingly, in the absence of internal and external K + with 10 mM NH4+i the H399T mutant showed a marked NH4+ conductance in contrast to the wt channel indicating that the H399T mutation not only resulted in a change in C-type inactivation but also in a change in selectivity.

Supported by grants from 4SC AG (Martinsried), the Wilhelm- Sander Stiftung (2004.046.1) and the Land Baden-Württemberg (1423/74)