Evidence for a Deep Pore Activation Gate in Small Conductance Ca²⁺-activated K⁺ Channels

Small conductance calcium-gated potassium (SK) channels share an overall topology with voltage-gated potassium (K_v) channels, but are distinct in that they are gated solely by calcium (Ca²⁺), not voltage. For K_v channels there is strong evidence for an activation gate at the intracellular end of the pore, which was not revealed by substituted cysteine accessibility of the homologous region in SK2 channels. In this study, the divalent ions cadmium (Cd²⁺) and barium (Ba²⁺), and 2-aminoethyl methanethiosulfonate (MTSEA) were used to probe three sites in the SK2 channel pore, each intracellular to (on the selectivity filter side of) the region that forms the intracellular activation gate of voltage-gated ion channels. We report that Cd²⁺ applied to the intracellular side of the membrane can modify a cysteine introduced to a site (V391C) just intracellular to the putative activation gate whether channels are open or closed. Similarly, MTSEA applied to the intracellular side of the membrane can access a cysteine residue (A384C) that, based on homology to potassium (K) channel crystal structures (i.e., the KcsA/MthK model), resides one amino acid intracellular to the glycine gating hinge. Cd²⁺ and MTSEA modify with similar rates whether the channels are open or closed. In contrast, Ba²⁺ applied to the intracellular side of the membrane, which is believed to block at the intracellular end of the selectivity filter, blocks open but not closed channels when applied to the cytoplasmic face of rSK2 channels. Moreover, Ba²⁺ is trapped in SK2 channels when applied to open channels that are subsequently closed. Ba²⁺ pre-block slows MTSEA modification of A384C in open but not in closed (Ba²⁺-trapped) channels. The findings suggest that the SK channel activation gate resides deep in the vestibule of the channel, perhaps in the selectivity filter itself.