Calmodulin Is an Auxiliary Subunit of KCNQ2/3 Potassium Channels

Abstract

Calmodulin (CaM) was identified as a KCNQ2 and KCNQ3 potassium channel-binding protein, using a yeast two-hybrid screen. CaM is tethered constitutively to the channel, in the absence or presence of Ca2+, in transfected cells and also coimmunoprecipitates with KCNQ2/3 from mouse brain. The structural elements critical for CaM binding to KCNQ2 lie in two conserved motifs in the proximal half of the channel C-terminal domain. Truncations and point mutations in these two motifs disrupt the interaction. The first CaM-binding motif has a sequence that conforms partially to the consensus IQ motif, but both wild-type CaM and a Ca2+-insensitive CaM mutant bind to KCNQ2. The voltage-dependent activation of the KCNQ2/3 channel also shows no Ca2+ sensitivity, nor is it affected by overexpression of the Ca2+-insensitive CaM mutant. On the other hand, KCNQ2 mutants deficient in CaM binding are unable to generate detectable currents when coexpressed with KCNQ3 in CHO cells, although they are expressed and targeted to the cell membrane and retain the ability to assemble with KCNQ3. A fusion protein containing both of the KCNQ2 CaM-binding motifs competes with the full-length KCNQ2 channel for CaM binding and decreases KCNQ2/3 current density in CHO cells. The correlation of CaM binding with channel function suggests that CaM is an auxiliary subunit of the KCNQ2/3 channel.

Dual Phosphorylations Underlie Modulation of Unitary KCNQ K+ Channels by Src Tyrosine Kinase

“…For co-expression of KCNQ2/Q3-Y349F, the mean current amplitude in control cells was 294 Ϯ 68 pA, and in cells co-expressed with Src, it was 248 Ϯ 59 pA (n ϭ 8, 9). Interestingly Tyr-349 is in the domain suggested to be a site for binding of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to the channels (22) and for calmodulin-mediated assembly (31). For the other mutants, Y88F, Y243F, and Y513F, overexpression of Src inhibited the heteromeric current in a way similar to the inhibition of currents from wt KCNQ2/3 channels (Fig.…”

“…Thus, regulation by phosphoinositides and Ca 2ϩ has been suggested to be mediated by PIP 2 and Ca 2ϩ / calmodulin binding to regions in the carboxyl terminus (22,25), and tetrameric assembly has likewise been shown to be mediated by carboxyl-terminal domains (31,38,39). Our laboratory has shown KCNQ3 in particular to have a much greater maximal P o than KCNQ2, KCNQ4, or KCNQ5 and that this difference also localizes to the carboxyl terminus of the channel (29).…”

Expression of a calmodulin-binding KCNQ2 potassium channel fragment modulates neuronal M-current and membrane excitability

“…The ubiquitous calcium sensor calmodulin (CaM) has been shown to associate tightly with the extended C-terminal tail domains of both KCNQ2 and KCNQ3 (13,14) and to regulate the M-like current produced by these subunits when they are coexpressed in heterologous cells (13,15). In the present study, we extend this analysis to the M-current in cultured hippocampal neurons.…”

“…The pEGFP-C2 vector with no inserted channel sequence was used to express GFP alone. Previously, we used GST fusion protein constructs to introduce these WT and mutant channel sequences into heterologous cells (13).…”

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

“…Ca 2þ -binding to VGCC-associated calmodulin can have a range of effects on channel function, including mediating Ca 2þ -dependent facilitation or Ca 2þ -inactivation (Lee et al 2000;DeMaria et al 2001;Catterall and Few 2008;Liu et al 2010). Calmodulin is also constitutively associated with and regulates opening of Ca 2þ -activated potassium channels (Xia 1998;Schumacher et al 2001) and other types of potassium channels (Wen and Levitan 2002). Two other major modes of action of calmodulin are exerted indirectly through its target proteins Ca 2þ /calmodulin-dependent kinases (CaMKs) and calcineurin.…”