PI(4,5)P2 regulates the gating of NaV1.4 channels

Abstract

Voltage-gated sodium (NaV) channels are densely expressed in most excitable cells and activate in response to depolarization, causing a rapid influx of Na+ ions that initiates the action potential. The voltage-dependent activation of NaV channels is followed almost instantaneously by fast inactivation, setting the refractory period of excitable tissues. The gating cycle of NaV channels is subject to tight regulation, with perturbations leading to a range of pathophysiological states. The gating properties of most ion channels are regulated by the membrane phospholipid, phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2). However, it is not known whether PI(4,5)P2 modulates the activity of NaV channels. Here, we utilize optogenetics to activate specific, membrane-associated phosphoinositide (PI)-phosphatases that dephosphorylate PI(4,5)P2 while simultaneously recording NaV1.4 channel currents. We show that dephosphorylating PI(4,5)P2 left-shifts the voltage-dependent gating of NaV1.4 to more hyperpolarized membrane potentials, augments the late current that persists after fast inactivation, and speeds the rate at which channels recover from fast inactivation. These effects are opposed by exogenous diC8PI(4,5)P2. We provide evidence that PI(4,5)P2 is a negative regulator that tunes the gating behavior of NaV1.4 channels.

Figure 1.

Dephosphorylation of PI(4,5)P₂ augments Na_V1.4 currents. HEK293T cells were transfected to express CRY2-tagged pseudojanin (CRY2-PJ) and CIBN-CAAX with the PI(4,5)P₂ biosensor, iRFP-PH_PLCδ for TIRFM imaging or rNa_V1.4 and Na_Vβ1 for whole cell patch clamp studies. CRY2-PJ is the 5-phosphatase inositol polyphosphate 5-phosphatase E (INPP5E) fused to the 4-phosphatase Sac1. TIRF data are mean, normalized fluorescence values ± SEM for 30 membrane-delimited regions of interest identified from eight cells. Patch-clamp data are paired before and after at least 3 min of BL photoactivation for 18 cells per group obtained from three independent biological replicates of the experiment. Statistical significance was determined using a Student’s paired t test. (A) Schematic showing that CRY2-PJ is targeted to the membrane by BL, where it dephosphorylates PI(4,5)P₂ to PI(4)P and, subsequently, PI. (B) A montage of TIRF fluorescent micrographs showing decoupling of the PI(4,5)P₂ biosensor iRFP-PH_PLCδ1 from the membrane in response to photoactivation of CRY2-PJ. The images are every 40 s and the scale bar = 10 µm. (C) Left: A representative time course of normalized iRFP-PH_PLCδ fluorescence showing loss of the biosensor from the membrane following BL-activation of CRY2-PJ. Right: Summary data showing that >80% of iRFP-PH_PLCδ is decoupled from the membrane after 200 s by BL-activation of CRY2-PJ. (D) Left: Representative traces of Na_V1.4 currents evoked by a test pulse to −40 mV, before (black) and after (blue) dephosphorylation of PI(4,5)P₂ by CRY2-PJ. Right: Mean I–V relationships for Na_V1.4 channels recorded before (black) and after (blue) dephosphorylation of PI(4,5)P₂ by CRY2-PJ.

Figure 2.

PI(4,5)P₂ tunes the voltage-dependence of Na_V1.4 channels. Whole-cell currents for rNa_V1.4 + Na_Vβ1 were recorded as per Fig. 1 with 200 µM diC₈PI(4,5)P₂ in the patch pipette where indicated. The voltage dependence of activation (G/G_max) and SSI (I/I_max) were determined as described in the Materials and methods using fits with Boltzmann functions for data obtained before and at least 3 min after BL activation of CRY2-PJ. The data are for 18 cells per group obtained from three independent biological replicates of the experiment. Statistical significance was determined using a Student’s paired t test. (A) Left: Mean activation (circle) and SSI (square) curves for Na_V1.4 before (black) and after BL photoactivation of CRY2-PJ (BL, blue). The window current is inset. Right: Summary of the change in the V½_act and V½_SSI following photoactivation of CRY2-PJ (BL). (B) Left: Mean activation (circle) and SSI (square) curves for Na_V1.4 in the presence of 200 µM diC₈PI(4,5)P₂ before (green) and after BL photoactivation of CRY2-PJ (BL, red). The window current is inset. Right: Summary of the change in the V½_act and V½_SSI following photoactivation of CRY2-PJ (BL). (C) Left: The activation and SSI curves from above to show the full excursion in the activation and SSI curves for cells with high levels of diC₈PI(4,5)P₂ (green, from B), and fully dephosphorylated endogenous PI(4,5)P₂ (blue, from A). The control condition is black, from A. The window current is inset. Right: Unpaired data to show the differences in V½_act and V½_SSI described by the Boltzmann functions.

Figure 3.

Na_V1.4 currents are augmented by CRY2-5P_OCRL dephosphorylation of PI(4,5)P₂. HEK293T cells were transfected to express CRY2-tagged 5-phosphatase OCRL (CRY2-5P_OCRL), and CIBN-CAAX with the PI(4,5)P₂ biosensor, iRFP-PH_PLCδ for TIRFM imaging or rNa_V1.4 and Na_Vβ1 for whole cell patch clamp studies. TIRF data are from 30 membrane-delimited regions from eight cells. Patch-clamp data are paired before and after at least 3 min of BL photoactivation for 12 cells per group obtained from three independent biological replicates of the experiment. Statistical significance was determined using a Student’s paired t test. (A) Schematic showing that CRY2-5P_OCRL is targeted to the membrane by BL where it dephosphorylates PI(4,5)P₂ to PI(4)P. (B) A montage of TIRF fluorescent micrographs showing decoupling of the PI(4,5)P₂ biosensor iRFP-PH_PLCδ1 from the membrane following photoactivation of CRY2-5P_OCRL. The images are every 40 s; scale bar = 10 µm. (C) Left: A representative time course of normalized iRFP-PH_PLCδ fluorescence showing the decrease in membrane association of the biosensor following BL activation of CRY2-5P_OCRL. Right: Summary data showing that >80% of iRFP-PH_PLCδ is decoupled from the membrane 200 s after BL activation of CRY2-5P_OCRL. (D) Left: Representative traces of Na_V1.4 currents evoked by a test pulse to −40 mV, before (black) and after (blue) dephosphorylation of PI(4,5)P₂ by CRY2-5P_OCRL. Right: Mean I–V relationships for Na_V1.4 channels recorded before (black) and after (blue) dephosphorylation of PI(4,5)P₂ by CRY2-5P_OCRL.

Figure 4.

5-dephosphorylation of PI(4,5)P₂ tunes the voltage-dependence of Na_V1.4 channels. Whole-cell currents for rNa_V1.4 + Na_Vβ1 were recorded as per Fig. 3 with 200 µM diC₈PI(4,5)P₂ in the patch pipette where indicated. The voltage-dependence of activation (G/G_max) and SSI (I/I_max) were determined as described in the Materials and methods using fits with Boltzmann functions for data obtained before and at least 3 min after activation of CRY2-5P_OCRL by BL. The data are from 12 cells per group obtained from three independent biological replicates of the experiment. Statistical significance was determined using a Student’s paired t test. (A) Left: Mean activation (circle) and SSI (square) curves for Na_V1.4 before (black) and after BL photoactivation of CRY2-5P_OCRL (BL, blue). The window current is inset. Right: Summary of the change in the V½_act and V½_SSI following photoactivation of CRY2-5P_OCRL (BL). (B) Left: Mean activation (circle) and SSI (square) curves for Na_V1.4 in the presence of 200 µM diC₈PI(4,5)P₂ before (green), and after BL photoactivation of CRY2-5P_OCRL (BL, red). The window current is inset. Right: Summary of the change in the V½_act and V½_SSI following photoactivation of CRY2-5P_OCRL (BL). (C) Left: The activation and SSI curves from above to show the full excursion in the activation and SSI curves for cells with high levels of diC₈PI(4,5)P₂ (green, from B), and fully dephosphorylated endogenous PI(4,5)P₂ (blue, from A). The control condition is black, from A. The window current is inset. Right: Unpaired data to show the differences in V½_act and V½_SSI described by the Boltzmann functions.

Figure 5.

Dephosphorylating PI(4,5)P₂ speeds the recovery of Na_V1.4 channels from fast inactivation. (A–D) Recovery from fast inactivation at −30 mV in HEK293T cells transfected with rNa_V1.4, Na_Vβ1, CIBN-CAAX, and CRY2-PJ (A and B) or CRY2-5P_OCRL (C and D) were tested in whole-cell patch clamp experiments according to the protocol described in the Material and methods. Normalized data are fit with an exponential function to get the time-constant, τ. The data are paired before and after at least 3 min of BL photoactivation for eight cells per group obtained from two independent biological replicates of the experiment. Statistical significance was determined using a Student’s paired t test. (A) Left: Time course of recovery from fast inactivation in response to a paired-pulse protocol before (black) and after activation of CRY2-PJ with BL. Right: Paired τ values. (B) Left: Photoactivation of CRY2-PJ (red) did not speed τ when 200 µM diC₈PI(4,5)P₂ was included in the patch-pipette (green). (C) Left: τ was also speeded following activation of CRY2-5P_OCRL with BL. Right: Paired τ values. (D) Left: Photoactivation of CRY2-5P_OCRL (red) did not speed τ when 200 µM diC₈PI(4,5)P₂ was included in the patch-pipette (green).

Figure 6.

Dephosphorylating PI(4,5)P₂ augments the late sodium current. (A–E) HEK293T cells transfected with rNa_V1.4, Na_Vβ1, CIBN-CAAX, and CRY2-PJ (A–C) or CRY2-5P_OCRL (D and E) were studied by a 400 ms pulse to −20 mV before and at least 3 min after illumination with BL. I_LATE is expressed as a percent of I_PEAK. The time course for fast inactivation is determined by an exponential fit and is expressed as τ. The data are for 10–15 cells per group obtained from three independent biological replicates of the experiment. Statistical significance was determined using a Student’s paired t test. (A) Representative late current traces before (black) and after BL activation of CRY2-PJ (blue). The scale bars are 50 pA (vertical) and 50 ms (horizontal). Inset: A zoomed-in view of the late current. (B) I_LATE is increased eightfold by BL activation of CRY2-PJ (blue); the effect is precluded when 200 μM diC₈PI(4,5)P₂ is included in the patch pipette. (C) τ is slowed by activation of CRY2-PJ (blue) in control cells but does not change with 200 μM diC₈PI(4,5)P₂ in the patch pipette. (D) I_LATE increases eightfold following BL activation of CRY2-5P_OCRL (blue) in control cells but not when 200 μM diC₈PI(4,5)P₂ (green) is included in the patch pipette. (E) τ is slowed by activation of CRY2-5P_OCRL (blue) in control cells but does not change with 200 μM diC₈PI(4,5)P₂ in the patch pipette.

Figure 7.

PI(4,5)P₂-dependent tuning of V½-activation requires the fast inactivation process. Xenopus oocytes were injected with cRNA for Na_V1.4-WCW, Na_Vβ1, CIBN-CAAX, and the CRY2-fused phosphatases indicated and studied 48 h later by two-electrode voltage clamp experiments. Currents were evoked by a step protocol before (black), and 3 min after sustained photoactivation with BL (blue). The voltage-dependence of activation was determined as described in the Materials and methods using fits with Boltzmann functions. Data are mean whole-oocyte currents ± SEM for 15–18 cells per condition obtained from three independent biological replicates of the experiment. (A) Left: Representative traces evoked by a step to −10 mV from oocytes expressing CRY2-5P_OCRL and CRY2-Sac2 before (black) and after (blue) photoactivation. Right: A normalized I–V relationship for the same experiment. (B) Normalized G–V relationships for data in A show no change V½_act before (−23 ± 4 mV) and after (−23 ± 3 mV) BL. (C) Normalized G–V relationships for oocytes expressing CRY2-5P_OCRL show no change V½_act before (−22 ± 5 mV) and after (−21 ± 5 mV) BL.