The Architecture of Selectivity: Molecular Basis of BlTx2 Discrimination Among Kv Channels and Kv4 Isoforms

Kv4 channels generate transient A-type potassium currents, but the contribution of Kv4.1 remains difficult to define because few pharmacological tools discriminate among Kv4 isoforms. We investigated the molecular basis of Kv4.1-selective inhibition by BlTx2, an α-KTx15 scorpion toxin from Buthacus leptochelys. Using electrophysiology, mutagenesis, tandem-channel analysis, structural modeling, and evolutionary analysis, we mapped the determinants governing BlTx2 activity and selectivity. Substitution of conserved α-KTx residues Lys27 and Tyr36 abolished detectable inhibition, whereas N30A reduced potency, supporting a canonical pore-blocking mechanism. A Kv4.1/Kv4.3 tandem chimera reduced BlTx2 potency, consistent with a contribution of adjacent Kv4.1 subunits to the high-affinity toxin-binding surface. Channel mutagenesis identified the Kv4.2/Kv4.3 lysine corresponding to Kv4.1 Asn355 as a key barrier to BlTx2 activity, since introducing this lysine into Kv4.1 abolished detectable inhibition. Mutational mapping of BlTx2 revealed that pore-facing residues required for inhibition are separated from peripheral residues that tune potency and cross-reactivity. In particular, Q9K and I18K conferred Kv1.2 activity, partly through interaction with Kv1.2 Glu355. Evolutionary analysis linked conserved pore-facing residues to purifying selection, and peripheral determinants to diversification. These findings identify distinct conserved and peripheral determinants that together shape BlTx2 activity and selectivity, thereby establishing BlTx2 as a useful probe for Kv4.1 function.