Slc7a5 regulates Kv1.2 channels and modifies functional outcomes of epilepsy-linked channel mutations

Figures

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  Fig. 1. Mass spectrometry and screening of Kv1.2 channel complexes. Kv1.2-1D4 was expressed in HEK cells, and EGS-cross-linked complexes were immunoprecipitated and analyzed by LC–MS/MS. Thirty proteins were chosen for further screening. a Abundance of proteins relative to untransfected cells, with cross-linking performed in ambient redox conditions (gray) or 1 mM DTT (black). Proteins containing labile extracellular disulfides are indicated by the black bar. b V1/2 (mean ± s.d.) was determined for Kv1.2 when co-expressed with each candidate gene (mEGFP-tagged) in ltk-mouse fibroblasts. Conductance-voltage relationships were measured by stepping between −120 and +60 mV (100 ms in 10 mV steps, −80 mV holding potential) followed by a tail current voltage of −30 mV. In addition, a 100 ms depolarization to +60 mV was delivered before each sweep to relieve use-dependent activation (n = 5–15, n = 31 for WT Kv1.2). c Kv1.2 current densities at +60 mV (pulsed from a holding potential of −80 mV) after co-expression with each candidate gene. The data from individual cells are superimposed on bars that represent mean ± s.d. (n = 5–15, n = 31 for WT Kv1.2)

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  Fig. 2. Slc7a5 alters Kv1.2 expression and current density. a Combinations of Kv1.2, mCherry-Slc7a5, and mEGFP-Slc3a2 were expressed in ltk-mouse fibroblasts. Current density was measured at +60 mV (pulsed from a holding potential of −80 mV). Transfection ratios were (with Kv1.2 maintained constant): Kv1.2:Slc7a5 (2:1); Kv1.2:Slc3a2 (1:1); Kv1.2:Slc7a5:Slc3a2 (2:1:2), n = 14–19 cells. A non-parametric Kruskal–Wallis ANOVA on ranks test, followed by Dunn’s post hoc was used to compare treatments to WT Kv1.2, (* indicates p < 0.05 relative to WT Kv1.2 current density). b Exemplar anti-Kv1.2 western blot of ltk-mouse fibroblasts transfected as in a except Kv1.2 + Slc7a5+Slc3a2 (1:1:1) for 72 h. c Densitometry measurements of the cell surface:total Kv1.2 protein. No significant differences were detected between groups (n = 3–5). d Densitometry of total Kv1.2 protein, normalized to WT Kv1.2 alone. ANOVA followed by Dunnett’s post hoc test was used to compare treatments to WT Kv1.2 (* indicates p < 0.05 relative to WT Kv1.2 transfection). The data from individual experiments are superimposed on bars that depict mean ± S.D. (n = 3–5)

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  Fig. 3. Hyperpolarization disinhibits Kv1.2 co-expressed with Slc7a5. a ltk-mouse fibroblast cells transfected with Kv1.2 + Slc7a5 (1:1) were held at a range of voltages: −80 mV (n = 7), −100 mV (n = 12) and −120 mV (n = 25). Currents were measured at +10 mV (50 ms pulses, every 300 ms), and normalized to the peak current after 30 s at −120 mV (mean ± s.d.). b Cell-by-cell current density (at +10 mV) of the first (gray) and final (green) pulses of a train of depolarizations with a −120 mV holding voltage. c Fold change between the initial and final test pulses of a 30 s pulse train with the indicated holding voltages (mean ± s.d.: 1.4 ± 0.1 at −80 mV; 1.6 ± 0.3 at −100 mV; 5 ± 2-fold at −120 mV, ANOVA followed by Dunnett’s post hoc test was used to compare groups)

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  Fig. 4. Slc7a5 shifts the voltage-dependence of Kv1.2 activation. a Indicated combinations of Kv1.2, Slc7a5, and Slc3a2 were transfected in ltk- mouse fibroblasts. Cells were hyperpolarized to −120 mV for 30 s prior to recording, leading to disinhibition of Kv1.2 currents. Conductance-voltage relationships were measured as described in Fig. 1. Conductance-voltage relationships were generated from the tail current amplitudes and fit with a Boltzmann function (Kv1.2 V1/2 = −11 ± 3, k = 11 ± 3 mV, n = 5; Kv1.2 + Slc7a5 V1/2 = −58 ± 3, k = 10 ± 2 mV, n = 16; Kv1.2 + Slc3a2 V1/2 = −11 ± 10, k = 10 ± 3 mV, n = 10; and Kv1.2 + Slc7a5 + Slc3a2 V1/2 = −16 ± 3 mV, k = 11 ± 2 mV, n = 9). b Representative traces of the activation curves measured in a, with the pulse to −20 mV highlighted. c HEK cells were transfected with equal amounts of the indicated combinations of Kv1.2, Slc7a5, and Kvβ1.3. Conductance-voltage relationships were generated as in a (Kv1.2 V1/2 = −16 ± 3, k = 9 ± 4 mV; Kv1.2 + Kvβ V1/2 = −22 ± 4, k = 9 ± 2 mV; Kv1.2 + Slc7a5 V1/2 = −60 ± 1, k = 9 ± 1 mV; and Kv1.2 + Slc7a5 + Kvβ V1/2 = −56 ± 3 mV, k = 9 ± 3 mV). d Current density at +10 mV was measured at the beginning and of a…

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  Fig. 5. Slc7a5 influences inactivation of Kv1.2. a Kv1.2[V381T] channels were expressed alone or with Slc7a5 (1:1 transfection ratio). Conductance-voltage relationships were generated with the same protocol as Fig. 4, after disinhibition by a 30 s hyperpolarization to −120 mV (Kv1.2[V381T] V1/2 = −16 ± 4 mV, k = 8.5 ± 0.7 mV, n = 6; + Slc7a5 V1/2 = −55 ± 11 mV, k = 13.1 ± 3.2 mV, n = 8). b Current density at +60 mV was measured at the beginning and of a pulse train with −120 mV holding potential (identical to Fig. 3; Kv1.2[V381T] n = 8; +Slc7a5 n = 16). c Exemplar traces of Kv1.2[V381T] ± Slc7a5 (green) currents elicited by a 1 s depolarization to +60 mV (after current disinhibition by holding at −120 mV). d Steady-state inactivation was measured by pulsing to test voltages for 6 s and allowing channels to recover for 7 s at −100 mV. Curves were fit with a Boltzmann function (Kv1.2[V381T] V1/2 = −31 ± 3 mV, k = 11.1 ± 2.1 mV, n = 5; Kv1.2[V381T] + Slc7a5 V1/2 = −69 ± 6 mV, k = 6.7 ± 1.8 mV, n = 5). e, f Identical experiments as in a, b were performed with Kv1.2[LT] channels (Kv1.2[LT] V1/2 = +80 ± 10 mV, k = 18 ± 2 mV, n = 7; +Slc7a5 V1/2 = +40 ± 11 mV, k = 17 ± 2 mV, n = 7). g Current…

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  Fig. 6. Measurement of the proximity of Slc7a5 and Kv1.2 with bioluminescence resonance energy transfer (BRET). a Emission spectra were collected from HEK293 cells transfected with Kv1.2-nanoluc + EGFP-Slc7a5 (green line normalized to peak). The Kv1.2-nanoluc (donor) spectrum was subtracted to yield the mEGFP component. Weighted components of the nanoluc (1.0) and mEGFP (0.1) spectra were used to fit the experimental spectrum (black dashed line). b mEGFP fluorescence (nanoluc-subtracted) was measured for Kv1.2-nanoluc co-expressed with various acceptor constructs, as indicated. c The area under the curve (AUC) for each BRET acceptor in b was normalized to the positive control AUC (Kv1.2-EGFP). Data are shown as mean ± SD (n = 4–5). ANOVA and a Tukey post hoc test were used to compare the BRET signals from EGFP-Slc7a5 (±Slc3a2) and EGFP-Slc1a5. d Western blot of dissociated cortical and hippocampal neurons from P2 rat pups at 14 days in vitro blotted with anti-Kv1.2 (left), anti-Slc7a5 (middle) and anti-β actin (right). e RT-PCR of Slc7a5 and Slc3a2 from dissociated cortical neurons from P1 rat pups after 4 days in vitro. f Cortical and hippocampal neurons from P2 rat pups at 7 days in…

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  Fig. 7. Using split YFP constructs to measure competition for Slc7a5 between Kv1.2 and Slc3a2. YFP fragments (YFPN residues 1–157, YN; and YFPC residues 158–239, YC) were fused to Kv1.2, Slc3a2, and Slc7a5 as indicated. a Fluorescence-activated cell sorting was performed on split-YFP tagged versions of Kv1.2, Slc7a5, and Slc3a2, with the third protein tagged with LSS-mOrange. All DNA transfections were done at a 1:1:1 ratio. Bars represent the distribution of transfected cells with prominent mOrange fluorescence (upper left quadrant in b), prominent YFP (lower right quadrant), or prominent signals from both fluorophores (upper right quadrant). b Representative data from FACS sorting of cells in the Kv1.2-YFPC + Slc7a5-YFPN + mOrange or +mOrange-Slc3a2 conditions. c Kv1.2-YFPC was expressed with combinations of Slc7a5-YFPN and Slc3a2 as indicated. The data from individual cells are plotted along with the mean ± s.d., n = 7–20. d Conductance-voltage relationships were generated for each condition after current disinhibition with a 30 s hyperpolarization to −120 mV (Kv1.2-YFPC V1/2 = −8.4 ± 7, n = 10; Kv1.2-YFPC + Slc7a5-YFPN V1/2 = −49 ± 2, n = 5; Kv1.2-YFPC + YFPN-Slc3a2 V1/2 = −11 ± 4, n…

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  Fig. 8. Disease-linked Slc7a5 mutations have attenuated effects on Kv1.2. a Kv1.2 channels were co-expressed with Slc7a5[A246V] or Slc7a5[P375L] (1:1) in ltk-mouse fibroblast cells. Conductance-voltage relationships were gathered as described in Fig. 1 (+Slc7a5[A246V] V1/2 = −10 ± 6 mV, k = 11 ± 3 mV, n = 6; +Slc7a5[P375L] V1/2 = −32 ± 8 mV, k = 16 ± 8 mV, n = 10). Dashed lines illustrate conductance-voltage relationships for WT Kv1.2 (black) and Slc7a5 (green) as a reference. b Representative BRET signals were calculated for mEGFP-tagged Slc7a5 mutants co-expressed with Kv1.2-nanoluc, as described in Fig. 6 (representative data from three experiments). c Current density at +60 mV was measured from Kv1.2 co-expressed with various Slc7a5 mutants, before and after a 30 s train of −120 mV hyperpolarizations to −120 mV. d Cell lysates were probed for Slc7a5 expression using western blot for cells transfected with Kv1.2 plus each of the Slc7a5 mutations. No significant changes were detected for expression of the Slc7a5 mutants (n = 3, p > 0.05). e–g Cortical and hippocampal neurons from P2 rat pups transiently transfected at 7 days in vitro with either mCherry-Slc7a5 WT or…

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  Fig. 9. Kv1.2 disease-linked mutations are strongly suppressed by Slc7a5. a, b Current density at +60 mV was measured before and after a 30 s hyperpolarizing train to −120 mV for Kv1.2[R297Q] alone (n = 6) or with Slc7a5 (n = 13), and Kv1.2[L298F] alone (n = 12) or with Slc7a5 (n = 14), as indicated. c, d Exemplar currents recorded from Kv1.2[R297Q] or Kv1.2[L298F], expressed in ltk- mouse fibroblasts. Highlighted sweeps are to −120 mV with a tail current voltage of −30 mV. e, f Conductance-voltage relationships for Kv1.2[R297Q] (V1/2 = −57 ± 4 mV, k = 8 ± 1 mV, n = 6) and Kv1.2[R297Q] + Slc7a5 (2:1 transfection, V1/2 = −143 ± 6 mV, k = 17 ± 2 mV, n = 9), Kv1.2[L298F] (V1/2 = −56 ± 1 mV, 7 ± 1 mV, n = 4) and Kv1.2[L298F] + Slc7a5 (4:1 transfection, V1/2 < −200 mV, estimated to be −210 ± 5 mV, k = 35 ± 6 mV, n = 9). g Exemplar western blot of cell lysates from ltk- mouse fibroblasts transfected with indicated combinations of Kv1.2 mutants and Slc7a5 as indicated, each at a 1:1 transfection ratio. h Densitometric quantification of Slc7a5 effects on total protein levels of various Kv1.2 mutants (data shown as mean ± s.d., n = 3 per condition); Student’s t-test was used to compare expression…

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  Fig. 10. Inactivation trap gating effect of Slc7a5 on Kv1.2. a Generic gating scheme of Kv1.2 cycling between closed, activated, and inactivated states. b Slc7a5 accelerates channel activation and inactivation, and slows recovery from inactivation, leading to accumulation of channels in an inactivated state. c Activation curves from Fig. 4 and inactivation curves from Fig. 5 are shown, illustrating the greater propensity for inactivation of Kv1.2 when assembled with Slc7a5. d Disease-linked gain-of-function mutants (GoF Kv1.2) exhibit greatly exaggerated sensitivity to Slc7a5 gating effects, such that Kv1.2 channels in complex with Slc7a5 are predominantly inactivated at resting voltages. Activation curves are reproduced from Fig. 9e. Inactivation curves are hypothesized, as they could not be measured due to the extreme voltages required