Pivoting between calmodulin lobes triggered by calcium in the Kv7.2/calmodulin complex.
Kv7.2 (KCNQ2) is the principal molecular component of the slow voltage gated M-channel, which strongly influences neuronal excitability. Calmodulin (CaM) binds to two intracellular C-terminal segments of Kv7.2 channels, helices A and B, and it is required for exit from the endoplasmic reticulum. However, the molecular mechanisms by which CaM controls channel trafficking are currently unknown. Here we used two complementary approaches to explore the molecular events underlying the association between CaM and Kv7.2 and their regulation by Ca(2+). First, we performed a fluorometric assay using dansylated calmodulin (D-CaM) to characterize the interaction of its individual lobes to the Kv7.2 CaM binding site (Q2AB). Second, we explored the association of Q2AB with CaM by NMR spectroscopy, using (15)N-labeled CaM as a reporter. The combined data highlight the interdependency of the N- and C-lobes of CaM in the interaction with Q2AB, suggesting that when CaM binds Ca(2+) the binding interface pivots between the N-lobe whose interactions are dominated by helix B and the C-lobe where the predominant interaction is with helix A. In addition, Ca(2+) makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca(2+).
Unidad de Biofísica, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
Alessandro
A
Alaimo
Unidad de Biofísica, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
Araitz
A
Alberdi
Unidad de Biofísica, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
Carolina
C
Gomis-Perez
Unidad de Biofísica, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
Juncal
J
Fernández-Orth
Unidad de Biofísica, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
Ganeko
G
Bernardo-Seisdedos
Unidad de Biofísica, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
Covadonga
C
Malo
Structural Biology Unit, CICbioGUNE, Bizkaia Technology Park, Derio, Spain.
Oscar
O
Millet
Departamento de Farmacología, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
Pilar
P
Areso
Unidad de Biofísica, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
Alvaro
A
Villarroel
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007477:Ions; D051658:KCNQ2 Potassium Channel / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D007477:Ions; D051658:KCNQ2 Potassium Channel; D050505:Mutant Proteins; D002118:Calcium