HpTx2 gating modification involves distinct amino acids in S3b region of Kv4

Kv4 channels are responsible for many of the fast-inactivating potassium currents found in cardiovascular, neural, and smooth muscle tissues. Kv4 channels are selectively inhibited by HpTx2, an inhibitor cysteine knot (ICK) gating modifier toxin. It is the goal of this study to understand how HpTx2 modifies gating of Kv4 channels by finding the binding and specificity determinants, and the basis for the difference in voltage-dependent gating modification among Kv4 subtypes. The binding site for HpTx2 on Kv4.3 was found by alanine scanning mutagenesis of the S3b-S4 linker region. L275A and V276A mutants decreased HpTx2 inhibition of Kv4.3; the double mutant eliminated inhibition, showing that the binding site for HpTx2 is LV275. The specificity determinants for HpTx2 with Kv4.3 were also examined. HaTx modifies gating of Kv4 and Kv2 channels; whereas HpTx2 specifically modifies gating of Kv4 channels. To find the specificity determinants, the S3b region of Kv4.3 was mutated to amino acids in S3b of Kv2.1. The LV275 binding site of Kv4.3 was mutated to amino acids of the homologous site IF275 in Kv2.1, where HaTx binds. HpTx2 modified gating of Kv4.3 LV275IF similar to the wild-type channel. These results suggest that binding determinants of HpTx2 are shared with other ICK toxins. The size of the S3-S4 linker in Kv2.1 is 7 amino acids larger than Kv4.3. Swapping the linker region of Kv2.1 into Kv4.3 eliminated the ability of HpTx2 to modify gating of Kv4.3. This showed that linker size determines specificity of HpTx2 in modifying gating of Kv4.3. HpTx2 modifies gating of Kv4.3 in a strongly voltage-dependent manner compared to Kv4.1. The molecular basis of the difference in gating modification is due to four amino acids in S3b. Activation models including toxin-bound states were developed. The models closely follow the experimental data and predict a difference in closed-state occupancy as being responsible for the differences in voltage-dependent gating modification.