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This Article Full Text Full Text (PDF) All Versions of this Article: 586/9/2307    most recent jphysiol.2008.152777v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Bal, M. Articles by Shapiro, M. S. PubMed PubMed Citation Articles by Bal, M. Articles by Shapiro, M. S. Related Collections Neuroscience

¹ University of Texas Health Science Center at San Antonio, Department of Physiology, MS 7756, San Antonio, TX 78229, USA

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.

(Received 18 February 2008; accepted after revision 12 March 2008; first published online 13 March 2008)
Corresponding author M. S. Shapiro: University of Texas Health Science Center at San Antonio, Department of Physiology, MS 7756, San Antonio, TX 78229, USA. Email: shapirom{at}uthscsa.edu