A Push–Pull Mechanism Between PRRT2 and β4-subunit Differentially Regulates Membrane Exposure and Biophysical Properties of NaV1.2 Sodium Channels

Figures

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  Fig. 1. PRRT2 and the NaV β4-subunit do not interact or compete for binding to NaV1.2. A Left: representative immunoblot of co-immunoprecipitation of β1/β4-subunits by PRRT2. HA-tagged PRRT2 (PRRT2), bacterial alkaline phosphatase (BAP), and FLAG-tagged β1/β4-subunits were expressed in wild-type HEK293 cells. Cell lysates (INPUT, 10 µg protein) and samples immunoprecipitated by anti-HA beads (HA-pellet) were analyzed by western blotting with anti-FLAG and anti-HA antibodies. The representative blots were cut from the same gel. Right: quantification of the FLAG immunoreactive signal in PRRT2 immunoprecipitates normalized to the control BAP values. Means ± sem of n = 3 independent experiments. B Left: representative immunoblot of co-immunoprecipitation of PRRT2 and NaV1.2 from extracts of NaV1.2-expressing stable HEK293 clones transiently transfected with either HA-tagged PRRT2 alone or with HA-PRRT2 + β4 subunit. BAP was used as a control. Cell lysates (INPUT, 10 µg protein) and samples immunoprecipitated by anti-HA beads (HA-pellet) were analyzed by western blotting with anti-panNaV and anti-HA antibodies. Right: quantification of the NaV immunoreactivity in PRRT2 immunoprecipitates…

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  Fig. 2. PRRT2 and the NaV β4-subunit have opposite effects on membrane targeting and exposure of NaV1.2 channels. A Schematics of the biotinylation experiment. B Representative immunoblots of cell surface biotinylation performed in HEK293 cells expressing NaV1.2 and transfected with empty vector (MOCK), PRRT2-HA, β4-FLAG, or both. Total lysates (input; left) and biotinylated (cell surface; right) fractions were analyzed by western blotting. Membranes were probed with antibodies to panNaV, HA, FLAG, and Na/K-ATPase (Na/K), with the latter used as marker of cell surface fractions. C The cell surface NaV immunoreactivity is expressed in percent of the control MOCK value after normalization to Na/K-ATPase immunoreactivity. Means ± sem of n = 4 independent experiments. Two-way ANOVA revealed no significant interaction between PRRT2 and β4-subunit (F1,12 = 3.238; p = 0.1). *p < 0.05, **p < 0.01; one-way ANOVA/Fisher’s least significant difference tests versus MOCK

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  Fig. 3. PRRT2 and the β4-subunit have opposite effects on the expression of the NaV1.2 transient current. A Representative whole-cell transient Na+ currents recorded in HEK293 cells stably expressing NaV1.2 and transiently transfected with empty vector (MOCK, black), PRRT2 (gray), β4-subunit (orange), or PRRT2 + β4 subunit (dark red). Currents were elicited by a protocol (inset) consisting of 5-mV depolarization steps from − 80 to 65 mV from a holding potential of − 120 mV. For clarity, the first 6 ms of the 100-ms steps for eight representative traces per condition are plotted. B, C Current density (J) versus voltage (V) relationship (B) for all the studied experimental conditions. The statistical analysis of J values at three representative voltages (− 40/− 20/− 10 mV) is reported (C). Data are expressed as means ± sem (MOCK, n = 36; PRRT2, n = 22; β4, n = 18; PRRT2 + β4, n = 17). Two-way ANOVA revealed no significant interaction between PRRT2 and β4-subunit on the amplitude of the macroscopic Na+ current (− 40 mV: F1,88 = 0.021; p = 0.88; − 20 mV: F1,90 = 1.2; p = 0.276; − 10 mV: F1,89 = 0,804; p = 0.37). *p < 0.05, **p < 0.01, ***p < 0.001 versus MOCK, Kruskal–Wallis/Dunn’s tests

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  Fig. 4. PRRT2 and β4-subunit differentially affect the kinetics of activation and inactivation of NaV1.2 channels. A, B Voltage dependence of activation (A) and steady-state inactivation (B) curves fit with a Boltzmann function for all conditions tested. Activation was studied using recordings obtained with the protocol depicted in Fig. 3A. Steady-state inactivation was obtained with a protocol in which the cell under study was held at a series of voltages ranging from − 130 mV to 30 mV for 500 ms followed by a 20-ms step pulse to − 10 mV to measure channel availability using a holding potential of − 120 mV. C, D Mean parameters of activation (C) and steady-state inactivation (D) curves fit to data obtained from all condition tested. All data are expressed as means ± sem. Activation: MOCK, n = 36; PRRT2, n = 22; β4, n = 18; PRRT2 + β4, n = 17. Steady-state inactivation: MOCK, n = 44; PRRT2, n = 16; β4, n = 28; PRRT2 + β4, n = 24. No significant differences were found in the activation kinetics between β4-subunit alone and PRRT2 + β4-subunit groups, as well as in the inactivation kinetics between PRRT2 alone and PRRT2 + β4-subunit groups. Two-way ANOVA revealed no significant interaction…

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  Fig. 5. PRRT2, but not the β4-subunit, modulates the NaV1.2 recovery kinetics from fast inactivation. A Representative channel recovery from inactivation current traces recorded for all the experimental conditions. Recordings were obtained pre-pulsing cells to − 20 mV for 20 ms to inactivate Na+ currents and then coming back to a recovery potential of − 100 mV for increasing durations before the repetition of test pulse to − 20 mV. For clarity, 6 of the nine time intervals are shown. The dotted line represents the first pulse peak amplitude. B Time courses of the recovery from inactivation of peak currents at − 100 mV for all condition studied are plotted on a semi-logarithmic scale. C Mean (± sem) values of plateau and τ of recovery estimated from one-phase decay fit to the data. D Relationship between the 10th and the 1st test pulse evoked by protocol displayed in A. All data are expressed as means ± sem (MOCK, n = 46; PRRT2, n = 25; PRRT2 + β4, n = 28; β4, n = 29). Two-way ANOVA revealed no significant differences were found between PRRT2 alone and PRRT2 + β4-subunit groups (plateau of recovery: F1,124 = 3.286, p = 0.072; τ of recovery: F1,124 = 0.09, p = 0.764; 10th/1st response:…

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  Fig. 6. PRRT2 and β4-subunit independently modulate the NaV1.2 persistent and resurgent currents. A Representative MOCK persistent NaV1.2 currents evoked by depolarizing steps from − 60 to 50 mV with 5-mV increments, lasting 600 ms (inset) in MOCK-transfected NaV1.2-expressing HEK293. For clarity, only currents evoked at − 60, − 40, − 20, 0, 20, and 40 mV are reported. The highlighted box at the end of stimulation indicates the region of the trace in which the persistent current was measured. The insets show zoomed views of the maximal persistent current for all conditions tested. B Persistent current, measured as the mean current in the last 60 ms of each 600 ms step and normalized to the transient current peak, is plotted versus voltage in each cell. C Bar plots of the mean (± sem) values of the normalized persistent current amplitude recorded at three distinct voltages (− 40, 0, and 40 mV) for all tested conditions. Data are expressed as means ± sem (MOCK, n = 24; PRRT2, n = 10; PRRT2 + β4, n = 20; β4, n = 21). D Representative peak resurgent current traces generated by NaV1.2-expressing HEK293 cells either mock-transfected or transfected with PRRT2 recorded in the presence…

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  Fig. 7. Summary of the differential modulation of NaV1.2 channels by PRRT2 and the β4-subunit. Radar plot showing the effects of the single or combined expression of PRRT2 and NaV β4-subunit on NaV1.2 properties. The plot was built with each spoke arranged in such a way that points lying outside the control condition (MOCK plot; black) are indicative of a gain-of-function, while points lying within the MOCK plot indicate a loss-of-function. All parameters are expressed in percent of the MOCK group. Values of transient peak current density were recorded at − 20 mV. The percentage values of persistent current were obtained from recordings performed at 0 mV