title: Calmodulin binding to M-type K+ channels assayed by TIRF/FRET in living cells.
abstract: Calmodulin (CaM) binds to KCNQ2-4 channels within their carboxy termini, where it regulates channel function. The existing data have not resolved the Ca2+ dependence of the interaction between the channels and CaM. We performed glutathione S-transferase (GST)-pull-down assays between purified KCNQ2-4 carboxy termini and CaM proteins to determine the Ca2+ dependence of the interaction in vitro. The assays showed substantial Ca2+ dependence of the interaction of the channels with wild-type (WT) CaM, but not with dominant-negative (DN) CaM. To demonstrate CaM-channel interactions in individual living cells, we performed fluorescence resonance energy transfer (FRET) between ECFP-tagged KCNQ2-4 channels and EYFP-tagged CaM expressed in CHO cells, performed under total internal reflection fluorescence (TIRF) microscopy, in which excitation light only penetrates several hundred nanometres into the cell, thus isolating membrane events. FRET was assayed between the channels and either WT or DN CaM, performed under conditions of normal [Ca2+]i, low [Ca2+]i or high [Ca2+]i induced by empirically optimized bathing solutions. The FRET data suggest a strong Ca2+ dependence for the interaction between WT CaM and KCNQ2, but less so for KCNQ3 and KCNQ4. FRET between all KCNQ2-4 channels and DN CaM was robust, and not significantly Ca2+ dependent. These data show interactions between CaM and KCNQ channels in living cells, and suggest that the interactions between KCNQ2-4 channels and CaM are likely to have Ca2+-dependent and Ca2+-independent components.
authors.affiliation: University of Texas Health Science Center at San Antonio, Department of Physiology, MS 7756, San Antonio, TX 78229, USA.
authors.forename: Manjot
authors.initials: M
authors.lastname: Bal
authors.forename: Oleg
authors.initials: O
authors.lastname: Zaika
authors.forename: Pamela
authors.initials: P
authors.lastname: Martin
authors.forename: Mark S
authors.initials: MS
authors.lastname: Shapiro
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D015640:Ion Channel Gating / Q000502:physiology; D051656:KCNQ Potassium Channels / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D002147:Calmodulin; D051656:KCNQ Potassium Channels; D002118:Calcium