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.
Highlights
With regards to the normal physiology of this protein family, the KCNQ2 and KCNQ3 subunits have been shown to form M-type potassium channels whose expression is restricted to neuronal tissue [2]
We found that calmodulin (CaM)1 bound to the C-terminal region of KCNQ channels
19 clones were identified as the product of the gene CALM2, and 13 clones were identified as the product of the gene CALM3. Both genes encode variants of CaM and have an identical amino acid sequence. This interaction was confirmed in a second assay where CaM was used as bait and the C-terminal region of KCNQ2 or KCNQ3 was used as prey, demonstrating that the association of CaM with KCNQ subunits was independent of which protein is fused to the binding or activation domain
Summary
Yeast Two-hybrid Analysis—A cDNA generated by PCR encoding amino acids 310 – 844 of the human KCNQ2 subunit [9] was subcloned in frame with the GAL4 DNA-binding domain of the yeast vector pGBKT7 (CLONTECH) to be used as bait in a yeast two-hybrid screen. The reporter yeast strain Y190 was sequentially transformed with this plasmid and with a human brain cDNA library subcloned in pACT2 (CLONTECH, catalog number HL 4004 AH, lot 5008, mRNA source: normal, whole brain from a 37-year-old Caucasian male, whose probable cause of death was trauma). Constructs containing point mutations and deletions were generated by PCR as described previously [10], sequenced, and subcloned into pGBKT7 (CLONTECH bait vector). The mutated constructs were cotransformed, along with a rat CaM cDNA in pGADT7
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