Structural basis for the subtype-selectivity of KCa2.2 channel activators

Small-conductance (K_Ca2.2) and intermediate-conductance (K_Ca3.1) Ca²⁺-activated K⁺ channels are gated by a Ca²⁺-calmodulin dependent mechanism. NS309 potentiates the activity of both K_Ca2.2 and K_Ca3.1, while rimtuzalcap selectively activates K_Ca2.2. Rimtuzalcap has been used in clinical trials for the treatment of spinocerebellar ataxia and essential tremor. We report cryo-electron microscopy structures of NS309-bound K_Ca2.2 and K_Ca3.1, in addition to structures of rimtuzalcap-bound K_Ca2.2 and mutant K_Ca3.1_R355K. The different conformations of calmodulin and the cytoplasmic HC helices in the two channels underlie the subtype-selectivity of rimtuzalcap for K_Ca2.2. NS309 binds to pre-existing pockets in both channels, while the bulkier rimtuzalcap binds in an induced-fit pocket in K_Ca2.2 requiring conformational changes. In K_Ca2.2, calmodulin's N-lobes are sufficiently far apart to enable conformational changes to accommodate either NS309 or rimtuzalcap. In K_Ca3.1, calmodulin's N-lobes are closer to each other and constrained by K_Ca3.1's HC helices, which allows binding of NS309 but not rimtuzalcap. Replacement of arginine-355 in K_Ca3.1's HB helix with lysine (K_Ca3.1_R355K) allows the binding of rimtuzalcap and renders the mutant channel sensitive to rimtuzalcap. These structures provide a framework for structure-based drug design targeting K_Ca2.2 channels.