Mutation-Induced Pocket Deactivation: How Ser353/Pro245 Alters KCa2.2 vs. KCa3.1 Ligand Selectivity.

The KCa2.2 and KCa3.1 channels are fundamental regulators of membrane potential and calcium signalling and promising targets to treat diseases such as spinocerebellar ataxia and cancer. To fully exploit their therapeutic potential, and to continue studying their pathophysiological role, it is crucial to develop selective modulators for each of these two channels. Here, we present a computational study to identify the molecular determinants behind the selectivity of two recently reported KCa2.2 modulators, namely, N-(2,1,3-benzoxadiazol-4-yl)-3-(4-methoxybenzene-1-sulfonamido)benzamide and N-(2,1,3-benzoxadiazol-4-yl)-4-(trifluoromethyl)benzamide. We leveraged a protocol combining in silico mutagenesis, molecular dynamics simulations, and protein-ligand docking to analyse the pockets targeted by these ligands. We identified the Ser353/Pro245 substitution to be the main driver of the distinct pocket shapes in KCa2.2 and KCa3.1 channels, ultimately defining modulator selectivity. This approach provides novel insights into the structural differences of this binding site across potassium channel subtypes, proposing potential selectivity determinants of the modulators targeting this pocket.