Lack of correlation between surface expression and currents in epileptogenic AB-calmodulin binding domain Kv7.2 potassium channel mutants

Figures

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  Figure 1.. Kv7.2 Helices A-B architecture. (a), Cartoon representation of a Kv7.2 subunit. The amino acids corresponding to helices A and B are shadowed in green [6,26]. The CaM binding domains are indicated in blue, while putative residues of Helix A [28,29] and Helix B [30] implicated in the interaction with PIP2 are boxed in brown. The residues mutated R333 and K526 are in red. (b), Structure of the CaM/Kv7.2-hAB complex. Ribbon diagram of the complex between Kv7.2 AB helices and CaM [26]. The N-lobe and C-lobe of CaM are colored in blue and the helices A (hA) and B (hB) are in green. The Kv7.2 residues R333 and K526 and the putative interacting CaM aminoacids L112 and D50 are indicated. The figure was rendered using Pymol.

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  Figure 2.. Characterization of BFNE-causing mutations in oocytes. (a), Representative current recordings from Xenopus oocytes injected in a 1:1 ratio with cRNAs for Kv7.3 and Kv7.2, or either the K526N or R333Q mutated Kv7.2 subunits. Inset: imposed voltage protocol. Each trace corresponds to the currents recorded in response to membrane depolarizations in 20 mV increments from −90 mV to + 50 mV (2 s duration), followed by a pulse to a constant voltage of −20 mV (1 s). (b), Normalized average maximal conductance of the indicated Kv7.2 subunits co-expressed with Kv7.3 (n ≥ 10 from 2 or more batches of oocytes). The following parameters were obtained after fitting a Boltzmann distribution to I-V relationships from tail currents measured at −20 mV: Kv7.2/Kv7.3: V1/2 = −41.7 ± 0.4, S = 9.4 ± 0.4; Kv7.2R333Q/Kv7.3: V1/2 = −39.1 ± 0.4, S = 8.8 ± 0.8; Kv7.2K526N/Kv7.3: V1/2 = −40.1 ± 0.26, S = 9.4 ± 0.5. Asterisks indicate values significantly different (*** = p < 0.001) versus WT-Kv7.2/Kv7.3. (c), Normalized conductance–voltage relationship of tail currents measured in 800 msec current traces at −20 mV for Kv7.3 channels co-expressed with WT Kv7.2 (black circles) or K526N (white circles).…

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  Figure 3.. Surface expression is reduced in K526N and R333Q mutants. (a), Relative surface expression levels of Kv7.3-HA co-expressed with Kv7.2 subunits (n ≥ 11) in Xenopus oocytes. Kv7.3/Kv7.3-HA represents the negative control. The number of Kv7.3-HA containing channels in the oocyte membrane was quantified using a whole-oocyte chemiluminescence assay. The background of uninjected oocytes was subtracted, and the values given are the means ± SE normalized to values obtained from WT-Kv7.2/Kv7.3-HA channels from the same batch. Asterisks indicate values significantly different (* = p < 0.05; *** = p < 0.001) versus WT-Kv7.2/Kv7.3-HA. B Left, Representative flow cytometry histogram distribution of surface stained HEK293T cells expressing Tac-AB-CFP reporter construct carrying the indicated constructs. (b) Right, Plot of the surface expression index obtained from the normalized sum of the product of the number of events and fluorescence intensity from the flow cytometry histogram distribution (n = 4; ** significance at p < 0.01).

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  Figure 4.. The K526N mutation impairs CaM binding. (a), Anti-CaM immunoblot after GST pull-down of GST-Kv7.2 (AB) with added CaM (10 μg) in the presence of Ca2+ (100 μM) or EGTA (5 mM). GST-neurogranin (Nrg, aa 1–78) and GST-NR1 (C0C1C2 C-terminal region of the NR1 receptor, aa 818–922) were used as positive controls to demonstrate interactions with apo-CaM and Ca2+-CaM, respectively. (b), Relative immunoblot signal of CaM. The data were obtained by WB from the following (n = 3; percent ± SEM): WT, 100.0 ± 1.0; R333Q, 85.2 ± 14.5; K526N, 41.4 ± 8.5, in the presence of Ca2+ and WT, 100.0 ± 0.8; R333Q, 91.1 ± 8.7; K526N, 52.9 ± 12.4, in the absence of Ca2+. Asterisks indicate values significantly different (* = p < 0.05; ** = p < 0.01) versus WT. (c), D-CaM (12.5 nM) fluorescence enhancement in the presence of Ca2+ (3.9 μM free Ca2+, left) or in the absence of Ca2+ (10 mM EGTA added, right) for the indicated recombinant GST-fusion protein concentrations. The lines are the result of fitting a Hill equation to the data. Asterisks indicate values of maximal fluorescence significantly different (** = p < 0.01; *** = p < 0.001). The data represent the means ± SEM from 3 or more independent…

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  Figure 5.. Co-immunoprecipitation experiments confirm altered interaction of CaM with the K526N channel. HEK293T cells were transfected with Myc-tagged Kv7.2 subunits, immunoprecipitated with an anti-Myc antibody, separated by SDS-PAGE, and analyzed by WB. The arrows indicate Kv7.2 subunits and immunoprecipitated endogenous CaM detected with anti-Myc (top panel) and anti-CaM antibodies (middle panel), respectively. The heavy chain of the anti-Myc antibody are also indicated with arrows. Cells were transfected with YFP-tagged CaM (YFP-CaM) to increase the amount of CaM in HEK293T, immunoprecipitated with an anti-Myc antibody, separated by SDS-PAGE, analyzed by WB and detected with an anti-CaM antibody (bottom panel). The densitometric quantification of CaM associated with the K526N mutant gave a value of 0.47, expressed as the ratio of the optical density of immunoprecipitated CaM/Kv7.2-K526N normalized to the CaM/WT-Kv7.2 value (n = 3) .

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  Figure 6.. The K526N channel presents a reduced PIP2 affinity. (a), Averaged time course of the current decline during 2.560 ms of Dr-VSP activation at + 100 mV in cells transfected with WT + Dr-VSP (red, n = 6) or K526N + Dr-VSP (green, n = 6) subunits. The shadows represent the mean ± SEM. (b), Normalized current at 20 mV (after/before step to + 100 mV, see arrows in the inset) for different durations at + 100 mV. Each point represents the mean ± SEM for WT (n = 6) and K526N (n = 6) .