Molecular Determinants of Selectivity for Kv1.3 K+ Channels

Abstract

The voltage-gated Kv1.3 potassium channel plays a key role in the activation of T lymphocytes by maintaining a negative membrane potential. By blocking these channels the proliferation of T cells can be inhibited. Anuroctoxin (AnTX), a 35-amino-acid peptide isolated and characterized previously from the venom of the scorpion Anuroctonus phaiodactylus, blocks Kv1.3 with high affinity (Kd = 0.7 nM). Although with lower affinity, the toxin blocks another K+channel, Kv1.2 (Kd = 6 nM). The aim of the current experiments was to improve the selectivity of AnTx for Kv1.3 by point mutations strategically designed based on sequence and affinity analysis of other K+ channel blocker toxins. Wild-type and mutant AnTX variants were produced by solid-phase synthesis. Whole-cell patch-clamp was used to measure hKv1.3 hKv1.1, hKv1.2 and hIKCa1 currents. The effect of synthetic wild-type anuroctoxin was similar to that of the natural toxin (Kv1.3: Kd = 0.3 nM and Kv1.2: Kd = 5.3 nM). AnTx F32T practically lost its affinity for Kv1.2 but also showed a slight decrease in affinity for Kv1.3 (Kd = 7.5 nM). The affinity of AnTx N17A for Kv1.3 did not change significantly (Kd = 0.9 nM) and a slight improvement in selectivity could be observed (Kv1.2: Kd = 18.9 nM). Combining the two mutations in one toxin we constructed the mutant N17A,F32T where the advantageous effect of both mutations could be detected. The N17A,F32T mutant is highly selective (no effect on Kv1.2 at 100 nM) and a high affinity blocker of Kv1.3 (Kd = 0.6 nM). In summary, with targeted mutations we designed and produced a selective and high affinity blocker of Kv1.3. Our results provide the foundation for the possibility of the production and future therapeutic application of additional, even more selective toxins.