Small conductance Ca 2ϩ -activated K ϩ channels (SK channels) are complexes of four ␣ pore-forming subunits each bound by calmodulin (CaM) that mediate Ca 2ϩ gating. Proteomic analysis indicated that SK2 channels also bind protein kinase CK2 (CK2) and protein phosphatase 2A (PP2A). Coexpression of SK2 with the CaM phosphorylation surrogate CaM(T80D) suggested that the apparent Ca 2ϩ sensitivity of SK2 channels is reduced by CK2 phosphorylation of SK2-bound CaM. By using 4,5,6,7-tetrabromo-2-azabenzimidazole, a CK2-specific inhibitor, we confirmed that SK2 channels coassemble with CK2. PP2A also binds to SK2 channels and counterbalances the effects of CK2, as shown by coexpression of a dominant-negative mutant PP2A as well as a mutant SK2 channel no longer able to bind PP2A. In vitro binding studies have revealed interactions between the N and C termini of the channel subunits as well as interactions among CK2 ␣ and ␤ subunits, PP2A, and distinct domains of the channel. In the channel complex, lysine residue 121 within the N-terminal domain of the channel activates SK2-bound CK2, and phosphorylation of CaM is state dependent, occurring only when the channels are closed. The effects of CK2 and PP2A indicate that native SK2 channels are multiprotein complexes that contain constitutively associated CaM, both subunits of CK2, and at least two different subunits of PP2A. The results also show that the Ca 2ϩ sensitivity of SK2 channels is regulated in a dynamic manner, directly through CK2 and PP2A, and indirectly by Ca 2ϩ itself via the state dependence of CaM phosphorylation by CK2.

In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca2+-activated K+ channels type 2 (SK2 channels) provides a negative feedback on N-methyl-d-aspartate receptors (NMDARs), reestablishing Mg2+ block that reduces Ca2+ influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia.

Summary Small-conductance Ca2+-activated K+ (SK) channels are widely expressed in neuronal tissues where they underlie post-spike hyperpolarizations, regulate spike-frequency adaptation and shape synaptic responses. SK channels constitutively interact with calmodulin (CaM), which serves as Ca2+ sensor, and with protein kinase CK2 and protein phosphatase 2A, which modulate their Ca2+ gating. By recording coupled activities of Ca2+ and SK2 channels, we showed that SK2 channels can be inhibited by neurotransmitters independently of changes in the activity of the priming Ca2+ channels. This inhibition involves SK2-associated CK2 and results from a 3-fold reduction in the steady-state Ca2+ sensitivity of channel gating. CK2 phosphorylated SK2-bound CaM but not KCNQ2-bound CaM, thereby selectively regulating Ca2+ gating of SK2 channels. We extended these observations to sensory neurons by showing that noradrenaline inhibits SK current and enhances signaling of primary afferent neurons in a CK2- dependent fashion. Hence, neurotransmitter-initiated signaling cascades can dynamically regulate Ca2+ sensitivity of SK channels and directly influence somatic excitability.