Calcium dependence of both lobes of calmodulin is involved in binding to a cytoplasmic domain of SK channels.

Abstract

KCa2.1-3 Ca2+-activated K+-channels (SK) require calmodulin to gate in response to cellular Ca2+. A model for SK gating proposes that the N-terminal domain (N-lobe) of calmodulin is required for activation, but an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Although structures support a domain-driven hypothesis of SK gate activation by calmodulin, only a partial understanding is possible without measuring both channel activity and protein binding. We measured SK2 (KCa2.2) activity using inside-out patch recordings. Currents from calmodulin-disrupted SK2 channels can be restored with exogenously applied calmodulin. We find that SK2 activity only approaches full activation with full-length calmodulin with both an N- and a C-lobe. We measured calmodulin binding to a C-terminal SK peptide (SKp) using both composition-gradient multi-angle light-scattering and tryptophan emission spectra. Isolated lobes bind to SKp with high affinity, but isolated lobes do not rescue SK2 activity. Consistent with earlier models, N-lobe binding to SKp is stronger in Ca2+, and C-lobe-binding affinity is strong independent of Ca2+. However, a native tryptophan in SKp is sensitive to Ca2+ binding to both the N- and C-lobes of calmodulin at Ca2+ concentrations that activate SK2, demonstrating that the C-lobe interaction with SKp changes with Ca2+. Our peptide-binding data and electrophysiology show that SK gating models need deeper scrutiny. We suggest that the Ca2+-dependent associations of both lobes of calmodulin to SKp are crucial events during gating. Additional investigations are necessary to complete a mechanistic gating model consistent with binding, physiology, and structure.