title: Calcium-dependent stoichiometries of the KCa2.2 (SK) intracellular domain/calmodulin complex in solution.
abstract: Ca(2+) activates SK Ca(2+)-activated K(+) channels through the protein Ca(2+) sensor, calmodulin (CaM). To understand how SK channels operate, it is necessary to determine how Ca(2+) regulates CaM binding to its target on SK. Tagless, recombinant SK peptide (SKp), was purified for binding studies with CaM at low and high Ca(2+) concentrations. Composition gradient multi-angle light scattering accurately measures the molar mass, stoichiometry, and affinity of protein complexes. In 2 mM Ca(2+), SKp and CaM bind with three different stoichiometries that depend on the molar ratio of SKp:CaM in solution. These complexes include 28 kD 1SKp/1CaM, 39 kD 2SKp/1CaM, and 44 kD 1SKp/2CaM. A 2SKp/2CaM complex, observed in prior crystallographic studies, is absent. At <5 nM Ca(2+), 1SKp/1CaM and 2SKp/1CaM were observed; however, 1SKp/2CaM was absent. Analytical ultracentrifugation was used to characterize the physical properties of the three SKp/CaM stoichiometries. In high Ca(2+), the sedimentation coefficient is smaller for a 1SKp:1CaM solution than it is for either 2SKp:1CaM or 1SKp:2CaM. At low Ca(2+) and at >100 µM protein concentrations, a molar excess of SKp over CaM causes aggregation. Aggregation is not observed in Ca(2+) or with CaM in molar excess. In low Ca(2+) both 1SKp:1CaM and 1SKp:2CaM solutions have similar sedimentation coefficients, which is consistent with the absence of a 1SKp/2CaM complex in low Ca(2+). These results suggest that complexes with stoichiometries other than 2SKp/2CaM are important in gating.
authors.affiliation: Department of Neuroscience, 2 Center for Learning and Memory, The University of Texas at Austin, Austin, TX 78712.
authors.forename: D Brent
authors.initials: DB
authors.lastname: Halling
authors.forename: Sophia A
authors.initials: SA
authors.lastname: Kenrick
authors.forename: Austen F
authors.initials: AF
authors.lastname: Riggs
authors.forename: Richard W
authors.initials: RW
authors.lastname: Aldrich
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012996:Solutions
chemical_list: D002147:Calmodulin; C496897:KCNN2 protein, human; C496899:Kcnn2 protein, rat; D011994:Recombinant Proteins; D051640:Small-Conductance Calcium-Activated Potassium Channels; D012996:Solutions; D002118:Calcium