title: The identification and characterization of a noncontinuous calmodulin-binding site in noninactivating voltage-dependent KCNQ potassium channels.
abstract: We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+. Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two alpha-helices (A and B). These two helices encompass approximately 85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of approximately 130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1-5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the [Ca2+] is raised. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels.
authors.affiliation: Instituto Cajal, Consejo Superior de Investigaciones, Avenida, Dr. Arce 37, 28002 Madrid, Spain.
authors.forename: Eva
authors.initials: E
authors.lastname: Yus-Najera
authors.forename: Irene
authors.initials: I
authors.lastname: Santana-Castro
authors.forename: Alvaro
authors.initials: A
authors.lastname: Villarroel
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002147:Calmodulin / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051656:KCNQ Potassium Channels; D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; D008297:Male; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020798:Two-Hybrid System Techniques
chemical_list: D002147:Calmodulin; D051656:KCNQ Potassium Channels; D051658:KCNQ2 Potassium Channel; C496914:KCNQ2 protein, human; D051659:KCNQ3 Potassium Channel; C496916:KCNQ3 protein, human; C412231:KCNQ5 protein, human; C496915:Kcnq2 protein, rat; C496918:Kcnq3 protein, rat; C501028:Kcnq5 protein, rat; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase