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, 284 (31), 20668-75

Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit From the Endoplasmic Reticulum

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Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit From the Endoplasmic Reticulum

Alessandro Alaimo et al. J Biol Chem.

Abstract

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K+ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an "active" conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca2+ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca2+ sensor, conferring Ca2+ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.

Figures

FIGURE 1.
FIGURE 1.
Topological representation of a KCNQ subunit. The consensus IQ residues are shown in bold. Circles and squares correspond to the residues mutated here (the squares indicate the mutations causing BFNC). The boxes indicate the regions with a high probability of adopting an α helix configuration, and the thick lines delineate the region fused to GST.
FIGURE 2.
FIGURE 2.
Mutations in helix A of the KCNQ2 binding site affect the maximal D-CaM fluorescence emission. A, emission spectra of 12.5 nm D-CaM in the presence of 1.6 μm free Ca2+ (dotted lines) and in the absence of Ca2+ (solid lines; 10 mm EGTA added), as well as in the presence (bold traces) and absence (light traces) of the GST-Q2AB fusion protein (200 nm). B, relative concentration-dependent enhancement of 12.5 nm D-CaM fluorescence by the indicated GST-Q2AB proteins in the presence and absence (filled circles) of 1.6 μm Ca2+ (open circles). The parameters used to fit a Hill equation to the data (continuous and dashed lines) can be found in Table 1. The data represent the means ± S.E. from three or more independent experiments. The error bars are smaller than the symbols.
FIGURE 3.
FIGURE 3.
The A343D mutant displaces D-CaM from the NR1a receptor-binding site (fused to the C0C1C2 C-terminal region of the NR1 NMDA receptor, amino acids 818–922) and from the neurogranin-binding site (Nrg, amino acids 1–78). The relative effect of 50 nm GST and GST-Q2 A343D on the relative fluorescence emission from 12.5 nm D-CaM complexed with GST-Nrg (taken as 100%) in the absence of Ca2+ (filled bars) and GST-NR1a in the presence of 1.6 μm free Ca2+ (empty bars, n = 3) is shown. To make the assay more sensitive, the concentrations of GST-Nrg and GST-NR1a that caused 50% of the maximal increase in D-CaM fluorescence emission were employed (30 and 8.75 nm in the absence -Nrg- and presence -NR1a- of Ca2+, respectively). The small effect observed after the addition of 50 nm GST was due to the dilution of the sample.
FIGURE 4.
FIGURE 4.
Characterization of the BFNC causing L339R mutant. A, top panel, representative current trace from Xenopus oocytes injected with a 1:1 ratio of cRNAs for KCNQ3 and KCNQ2-L339R. The dotted line is a reference current trace from oocytes expressing wild type channels. The voltage protocol is depicted in the inset. Bottom panel, averaged maximal current (gray box, n ≥ 5) and the relative surface expression of KCNQ3-HA (hatched boxes, n ≥ 11). The values given are the means (±S.E.) normalized to values obtained from WT-KCNQ2/KCNQ3 or WT-KCNQ2/KCNQ3-HA channels from the same batch. B, protein complexes from HEK293T cells expressing YFP-tagged CaM and Myc-tagged KCNQ2 subunits were immunoprecipitated (IP) with an anti-Myc antibody. The YFP-CaM bands revealed with an anti-CaM antibody are indicated by arrows, and the heavy chain of the anti-Myc antibody is labeled with an asterisk. Bottom panel, densitometric quantification of CaM (CaM band density/KCNQ2 band density; n = 2). Similar results were obtained when endogenous CaM was co-immunoprecipitated (see supplemental Fig. S1 and Ref. 7). C, co-immunoprecipitation of HA-tagged KCNQ3 and YFP-tagged CaM assembled with Myc-tagged KCNQ2. The anti-Myc antibody was used to immunoprecipitate proteins from HEK293T cells expressing the constructs indicated, and the proteins were detected with anti-Myc, anti-HA, or anti-CaM antibodies. The asterisk represents the heavy chain of the primary antibody. Right lane, molecular weight marker. Bottom panel, densitometric quantification of YFP-CaM (n = 3). D, the confocal images of HEK293T cells co-transfected with the mCFP-tagged KCNQ2 L339R and with an ER marker were processed as described under “Experimental Procedures” (n ≥ 10). The KCNQ2 subunits and the ER marker were pseudo-colored in green and red, respectively, producing a yellow color upon co-localization. Middle panel, effect of elevated CaM expression. Right panel, effect of elevated expression of a Ca2+ binding incompetent CaM mutant (CaM1234). The bar represents 10 μm. E, Western blot probed with an anti-GFP antibody of HEK293T cell extracts expressing the indicated Tac chimeras (n = 3). The upper bands represent immature (*) and mature (**) Tac chimeras. The mature band indicates a complex glycosylation state acquired after processing in the Golgi system.
FIGURE 5.
FIGURE 5.
Relationship of the in vitro maximal D-CaM fluorescence emission with trafficking parameters. A, plot of the Manders' index of ER co-localization from cells overexpressing CaM (open circles) or CaM1234 (filled circles) and different KCNQ2 mutants (from left to right, WT, R353G, L339R, and I340E). The co-localization indices for WT, R353, and I340E were taken from Ref. . The continuous lines are the results of a linear regression fit to the data. For CaM the parameters of the fit were: intercept, 296.9 ± 38.9; slope, −325.5 ± 49.5 (r2 = 0.96). For CaM1234 the parameters of the fit were: intercept, 653.6 ± 24.2; slope, −686.6 ± 17.8 (r2 = 0.88). B, plot of the glycosylation index of the Tac-AB proteins (from left to right: I340E, L339R, R353G, and WT) versus D-CaM fluorescence. The glycosylation index used was the average ratio of the optical density values (mature OD/[immature OD + mature OD]; see Fig. 3E). For CaM the parameters of the fit were: intercept, −80.5 ± 9.6; slope, 4.3 ± 0.3 (r2 = 0.99). For CaM1234 the parameters of the fit were: intercept, −135.5 ± 20.3; slope, 6.8 ± 0.8 (r2 = 0.97). C, the relationship between D-CaM emission and surface expression (filled circles), and the current (empty circles) of heteromeric KCNQ2/KCNQ3 channels expressed in Xenopus oocytes. The lines are the result of the best fit of a transfer function to the data (continuous line, surface expression; dotted line, current). This function has no mechanistic implications, and its purpose is to facilitate the comparison with other sets of data. The function is y = ax/(b + x), where y is fluorescence emission, and x is current or surface expression. The parameters for surface expression were: a = 142.0 ± 22.7, b = 0.42 ± 0.15 (r2 = 0.96), current: a = 133.5 ± 15.5, b = 0.34 ± 0.10 (r2 = 0.98). Small changes in D-CaM emission are reflected by large changes in the number of functional channels at the plasma membrane.
FIGURE 6.
FIGURE 6.
Model of Ca2+-CaM regulation of KCNQ2 trafficking. At resting intracellular Ca2+ concentrations, CaM binds to helices A and B of KCNQ2, and subsequently, it suffers a conformational change that stabilizes the complex and conceals the retention/retrieval signals within the binding site, thereby facilitating the exit of the channel from the ER. Subsequent Ca2+ binding triggers an additional conformational change that allows CaM to occlude other retention/retrieval signals located at the binding site or elsewhere, further facilitating the translocation of the channel to the plasma membrane.

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