HpTx2 Interaction With Kv4.3 and Kv4.1 Reveals Differences in Gating Modification

Abstract

HpTx2 is an ICK gating modifier toxin specific for Kv4 channels. The Kd of HpTx2 for two similar channels, Kv4.3 and Kv4.1, was 2.4 μM and 7.1 μM, respectively. HpTx2 inhibition of Kv4.3 is highly voltage dependent, shifting the current-voltage relationship to more depolarized potentials; consistent with the classic behavior of gating modifier toxins. However, modification of Kv4.1 gating is much less voltage dependent. Site directed mutagenesis of the S3b interaction site of Kv4.3 and Kv4.1 shows that the same two conserved bulky hydrophobic amino acids are required for HpTx2 interaction with each channel. While the interior of the binding site is conserved between Kv4.3 and Kv4.1, three amino acids adjacent to the binding site are not conserved. Swapping these amino acids between Kv4.3 and Kv4.1 swaps the phenotypic response to toxin, while having minimal effect on gating properties of the channels. We modeled the activation gating of Kv4.3 and Kv4.1 and incorporated the effects of HpTx2 into the kinetic parameters of activation. The model is similar for both channels; it has four voltage-dependent transitions between the closed states followed by voltage-independent transition to an open state. Voltage-dependent transitions in Kv4.3 are more strongly affected by toxin. In Kv4.1, the voltage-independent transition from the closed pre-open to open state is most affected by HpTx2. Therefore, a higher proportion of toxin-bound Kv4.1 channels are in the closed pre-open state, compared to toxin-bound Kv4.3 channels. This decreases the voltage dependence of toxin-bound Kv4.1 opening. The model closely recapitulates our experimental data. These data show that amino acids near the HpTx2 binding site play a role in the kinetics of Kv4 channel activation gating.