The episodic ataxia type 1 mutation I262T alters voltage-dependent gating and disrupts protein biosynthesis of human Kv1.1 potassium channels

Abstract

Voltage-gated potassium (Kv) channels are essential for setting neuronal membrane excitability. Mutations in human Kv1.1 channels are linked to episodic ataxia type 1 (EA1). The EA1-associated mutation I262T was identified from a patient with atypical phenotypes. Although a previous report has characterized its suppression effect, several key questions regarding the impact of the I262T mutation on Kv1.1 as well as other members of the Kv1 subfamily remain unanswered. Herein we show that the dominant-negative effect of I262T on Kv1.1 current expression is not reversed by co-expression with Kvβ1.1 or Kvβ2 subunits. Biochemical examinations indicate that I262T displays enhanced protein degradation and impedes membrane trafficking of Kv1.1 wild-type subunits. I262T appears to be the first EA1 mutation directly associated with impaired protein stability. Further functional analyses demonstrate that I262T changes the voltage-dependent activation and Kvβ1.1-mediated inactivation, uncouples inactivation from activation gating, and decelerates the kinetics of cumulative inactivation of Kv1.1 channels. I262T also exerts similar dominant effects on the gating of Kv1.2 and Kv1.4 channels. Together our data suggest that I262T confers altered channel gating and reduced functional expression of Kv1 channels, which may account for some of the phenotypes of the EA1 patient.

Figure 1. I262T exerts dominant effects on functional expression and voltage-dependent gating of Kv1.1 channels.

Functional characterization of the I262T mutant in Xenopus oocytes. (A) Dominant-negative effects of I262T. (Left) Representative current traces of Kv1.1 WT, I262T, and equal-molar co-expression of WT and I262T [WT + I262T (1:1)]. In all oocyte injection conditions hereafter, the cRNA concentration is 0.5 μg/μl for each construct. The external bath solution contains 3 mM KCl. The holding potential is −90 mV. The voltage protocol comprises a 370-ms test potential (ranging from −80 mV to + 60 mV in + 10 mV steps) followed by −90-mV tail potential. (Right) Normalized peak current amplitudes at the + 60-mV test potential. In the co-expression group (WT + I262T), the molar ratio of the mutant was increased from 0.5 up to 10. Data were normalized with respect to the average values of Kv1.1 WT recorded from the same batch of oocytes on the same day. Asterisks denote a significant difference from the WT control (*, t-test: p < 0.05). (B) I262T shifts the voltage-dependence of Kv1.1. (Left) Representative current traces in the external solution containing 60 mM KCl. (Right) Steady-state activation (P_o–V) curves derived from isochronal tail currents at −90 mV in response to various test pulse potentials. (C) Representative current traces in 3-mM KCl bath solution (left) and normalized peak current amplitudes (right) of Kv1.1 WT-WT dimer and WT-I262T dimer. Asterisks denote a significant difference from the WT-WT dimer control (*, t-test: p < 0.05). (D) Representative current traces in 60-mM KCl bath solution (left) and P_o–V curves (right) of WT-WT and WT-I262T dimers. (E,F) I262T slows activation kinetics but speeds deactivation kinetics of Kv1.1 channels. Activation and deactivation time constants were derived from one-exponential fits of the rising phase of test currents (3 mM KCl) and tail currents (60 mM KCl), respectively. See Supplementary Table S1 for more details on P_o–V parameters.

Figure 2. Co-expression with Kvβ subunits does not reverse the current suppression effect of I262T.

(A) Normalized peak current amplitudes (at + 60 mV) in Xenopus oocytes. Despite the presence of Kvβ1.1 or Kvβ2 subunits, 262T displays significant dominant-negative effects. (B,C) Normalized peak current densities (at + 60 mV) in HEK293T cells. I262T shows comparable current suppression effects in the absence or presence of Kvβ subunits. (D) Normalized peak current densities (at + 60 mV) of Kv1.1 WT-WT dimer and WT-I262T dimer in HEK293T cells. The voltage protocol is the same as that in Fig. 1A. Asterisks denote a significant difference from the corresponding WT control (*, t-test: p < 0.05). Kv1.1α and Kvβ subunits were co-expressed in the molar ratio 1:5. See Supplementary Figs S1 and S2 for representative current traces.

Figure 3. Biochemical analyses of the mechanism underlying the dominant-negative effect of I262T.

(A) Surface biotinylation analyses of Myc-tagged Kv1.1 (Myc-Kv1.1) WT and I262T in HEK293T cells. (Left) Representative immunoblots. The molecular weight markers (in kilodaltons) are labeled to the left, and the immunoblotting antibodies (α-Myc and α-GAPDH) are specified below the immunoblots. Cell lysates from biotinylated intact cells were either directly employed for immunoblotting analyses (total) or subject to streptavidin pull-down before being used for immunoblotting analyses (surface). (Right) Quantification of total protein level (total signal), surface protein level (surface signal), and surface expression efficiency (surface/total signal). I262T shows reduced protein level. The total protein density was standardized as the ratio of total Myc signal to the signal of the loading control GAPDH. The surface protein density was standardized as the ratio of surface Myc signal to the cognate total GAPDH signal. The efficiency of surface presentation is expressed as surface protein density divided by the corresponding standardized total protein density. (B) The kinetics of Myc-Kv1.1-WT and Myc-Kv1.1-I262T protein degradation in the presence of 100 μg/ml cycloheximide (CHX) treatments of different durations. (Left) Representative immunoblots. (Right) Quantification of Kv1.1 protein degradation time course. Protein densities were standardized as the ratio of Kv1.1 signals to the cognate GAPDH signals, followed by normalization to those of the corresponding control at 0 hr. See Supplementary Fig. S3 for details on semi-logarithmic linear-regression analyses of the degradation time course. (C) Surface biotinylation analyses of Myc-Kv1.1-WT co-expressed with untagged WT or I262T (1:1 molar ratio). (D) Surface biotinylation analyses of Kv1.1 WT-WT dimer and WT-I262T dimer. Kv1.1 dimers were detected with the anti-Kv1.1 (αKv1.1) antibody. Asterisks denote significant difference from the WT control (*, t-test: p < 0.05; n = 3–6). The gels were run under the same experimental conditions. Uncropped images of immunoblots are shown in Supplementary Fig. S5.

Figure 4. I262T alters Kvβ1.1-mediated voltage-dependent inactivation of Kv1.1 channels.

(A) (Left) Representative Kv1.1 current traces of WT, WT-I262T dimer, or I262T co-expressed with Kvβ1.1 (molar ratio 1:5) in Xenopus oocytes. The voltage protocol is the same as that in Fig. 1A. The external solution contains 3 mM KCl. (Right) I262T decelerates the fast inactivation kinetics of Kv1.1 channels. Inactivation time constants were derived from two-exponential fits of the falling phase of test currents. (B) I262T shifts the voltage-dependent inactivation of Kv1.1. (Left) Representative current traces in response to the voltage protocol comprising a 350-ms depolarizing prepulse potential (ranging from −80 mV to + 20 mV in + 5 mV steps), followed by a test pulse (fixed at + 60 mV) for 100 ms. (Right) Steady-state inactivation curves derived from the normalization of peak current amplitudes (at the + 60-mV test pulse; I/I_max) in response to different prepulse potentials. See Supplementary Table S1 for more details on the parameters of the inactivation curves. (C) Lack of effect of I262T on the inactivation recovery of Kv1.1. (Left) Representative current traces in response to a double-pulse voltage protocol comprising two consecutive 100-ms + 40-mV test pulses that were separated by −90- mV interpulses of increasing durations (from 0.05 to 9 sec). The dotted line denotes the recovery time course of the K⁺ current in response to the second test pulse. (Right) Normalized peak current amplitudes (at the second + 40-mV pulse) as a function of interpulse duration. (D) I262T changes the cumulative inactivation of Kv1.1. (Left) Representative current traces in response to a 40-Hz train of 3-ms + 40-mV test pulses with 25-ms interpulse intervals (holding at −90 mV). No –P/4 leak subtraction was performed. The dotted line denotes the time course of K⁺ current reduction in response to 15 consecutive test pulses. (Right) Normalized peak current amplitudes (at + 40 mV) as a function of test pulse number. See Supplementary Table S2 for more details on the kinetics of inactivation recovery and cumulative inactivation.

Figure 5. I262T exhibits dominant effects on voltage-dependent gating of Kv1.4 channels.

Co-expression of Kv1.4 and Kv1.1 I262T in Xenopus oocytes. (A) Kv1.4 fails to affect the defective expression of I262T. (Left) Representative current traces of Kv1.4 WT alone (Kv1.4), co-expression of Kv1.4 WT and Kv1.1 WT (Kv1.4 + Kv1.1 WT), and co-expression of Kv1.4 WT and Kv1.1 I262T (Kv1.4 + Kv1.1 I262T). The inherent fast inactivation kinetics of Kv1.4 decelerates in the presence of Kv1.1. The voltage protocol is the same as that in Fig. 1A. The external solution contains 3 mM KCl. (Right) Normalized current amplitudes (at + 60 mV) for the two Kv1.4 + Kv1.1 co-expression conditions. (B) Representative current traces (left) and normalized current amplitudes (right) for Kv1.4-Kv1.1 WT dimer and Kv1.4-Kv1.1 I262T dimer. (C) Representative current traces (left) and normalized current amplitudes (right) for the two Kv1.4-Kv1.1 dimers in the presence of Kvβ1.1 (molar ratio 1:5). Asterisks denote a significant difference from the WT control (*, t-test: p < 0.05). (D) I262T shifts the voltage-dependent activation (left) and inactivation (center, right) of Kv1.4. Steady-state activation curves of the two Kv1.4-Kv1.1 dimers were expressed as normalized peak current amplitudes in response to different test pulse potentials. Steady-state inactivation curves in the absence or presence of Kvβ1.1 were obtained by employing the same voltage protocol as described in Fig. 4B. See Supplementary Table S3 for more details on the parameters of the activation and inactivation curves. (E,F) I262T does not prominently affect the inactivation recovery but significantly slowed the cumulative inactivation of Kv1.4. Normalized peak current amplitudes (with or without Kvβ1.1) of the two Kv1.4-Kv1.1 dimers were plotted against interpulse duration (E) and test pulse number (F) as described in Fig. 4C,D, respectively. See Supplementary Table S4 for more details on the kinetics of inactivation recovery and cumulative inactivation.

Figure 6. I262T modifies the window currents of Kv1.1 and Kv1.4 channels.

Steady-state activation and inactivation curves of “Kv1.1 vs. I262T” (A), “Kv1.1 WT-WT dimer vs. WT-I262T dimer” (B), and “Kv1.4-Kv1.1 WT dimer vs. Kv1.4-Kv1.1 I262T dimer” (C) were assembled from those shown in Figs 1,4 and 5. Kv1.1 inactivation curves are based on Kvβ1.1-mediated fast inactivation, whereas Kv1.4-Kv1.1 inactivation curves concern the inherent fast inactivation of Kv1.4. Solid lines refer to constructs comprising the mutant, whereas dashed lines denote the corresponding WT control. The window current of each indicated construct is defined by the triangular area underneath the overlap of activation and inactivation curves. See Supplementary Table S5 for more details on the parameters of window currents.