title: Cooperativity between calmodulin-binding sites in Kv7.2 channels.
abstract: Among the multiple roles assigned to calmodulin (CaM), controlling the surface expression of Kv7.2 channels by binding to two discontinuous sites is a unique property of this Ca(2+) binding protein. Mutations that interfere with CaM binding or the sequestering of CaM prevent this M-channel component from exiting the endoplasmic reticulum (ER), which reduces M-current density in hippocampal neurons, enhancing excitability and offering a rational mechanism to explain some forms of benign familial neonatal convulsions (BFNC). Previously, we identified a mutation (S511D) that impedes CaM binding while allowing the channel to exit the ER, hinting that CaM binding may not be strictly required for Kv7.2 channel trafficking to the plasma membrane. Alternatively, this interaction with CaM might escape detection and, indeed, we now show that the S511D mutant contains functional CaM-binding sites that are not detected by classical biochemical techniques. Surface expression and function is rescued by CaM, suggesting that free CaM in HEK293 cells is limiting and reinforcing the hypothesis that CaM binding is required for ER exit. Within the CaM-binding domain formed by two sites (helix A and helix B), we show that CaM binds to helix B with higher apparent affinity than helix A, both in the presence and absence of Ca(2+), and that the two sites cooperate. Hence, CaM can bridge two binding domains, anchoring helix A of one subunit to helix B of another subunit, in this way influencing the function of Kv7.2 channels.
authors.affiliation: Unidad de Biofísica, CSIC-UPV/EHU, Universidad del País Vasco, Barrio Sarriena s/n, 48940 Leioa, Spain.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Alaimo
authors.forename: Araitz
authors.initials: A
authors.lastname: Alberdi
authors.forename: Carolina
authors.initials: C
authors.lastname: Gomis-Perez
authors.forename: Juncal
authors.initials: J
authors.lastname: Fernández-Orth
authors.forename: Juan Camilo
authors.initials: JC
authors.lastname: Gómez-Posada
authors.forename: Pilar
authors.initials: P
authors.lastname: Areso
authors.forename: Alvaro
authors.initials: A
authors.lastname: Villarroel
mesh_terms: D001665:Binding Sites; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D004594:Electrophysiology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D051658:KCNQ2 Potassium Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D002147:Calmodulin; D051658:KCNQ2 Potassium Channel; D021122:Protein Subunits