Opening TRPP2 (PKD2L1) requires the transfer of gating charges

Abstract

The opening of voltage-gated ion channels is initiated by transfer of gating charges that sense the electric field across the membrane. Although transient receptor potential ion channels (TRP) are members of this family, their opening is not intrinsically linked to membrane potential, and they are generally not considered voltage gated. Here we demonstrate that TRPP2, a member of the polycystin subfamily of TRP channels encoded by the PKD2L1 gene, is an exception to this rule. TRPP2 borrows a biophysical riff from canonical voltage-gated ion channels, using 2 gating charges found in its fourth transmembrane segment (S4) to control its conductive state. Rosetta structural prediction demonstrates that the S4 undergoes ∼3- to 5-Å transitional and lateral movements during depolarization, which are coupled to opening of the channel pore. Here both gating charges form state-dependent cation-π interactions within the voltage sensor domain (VSD) during membrane depolarization. Our data demonstrate that the transfer of a single gating charge per channel subunit is requisite for voltage, temperature, and osmotic swell polymodal gating of TRPP2. Taken together, we find that irrespective of stimuli, TRPP2 channel opening is dependent on activation of its VSDs.

Keywords: TRP channels; biophysics; gating mechanisms; ion channels; polycystins.

Fig. 1.

Gating currents generated by gating charges in TRPP2’s voltage sensor. (A) A structural alignment of TRPP1, TRPP2, and the prokaryotic voltage-gated sodium channel NavMs (PDB accession codes 5T4D, 5Z1W, and 5HVX, respectively) (7, 20, 26). (Right) Expanded view of the VSD helices. Note the conserved gating charge positions R3/K3 and R4/K4 from TRPP2 and NavMs. (B) Steady-state voltage-dependent Cs⁺ currents (blue traces, Top) recorded from SF9 insect cells and blocked Gd³⁺ (gray traces, Bottom). (C) HA-TRPP2-GFP gating currents (black traces) activated by voltage steps shown above. The integrated gating currents (red) convert from current to charge (femtocoulombs [fC]). (D) The voltage dependence of the central pore tail current (black) and the estimated charges per channel (red), fit to the Boltzmann equation. (E) A comparison of the rates of central pore current opening (τ_open) and gating charge activation (τ_act.). Number replicates are indicated in the parentheses, and error bars are SEM.

Fig. 2.

Mutant cycle analysis reveals coupling energy between gating charges and Y366F. (A) (Top) Steady-state voltage-dependent currents from single- and double-mutant TRPP2 channels recorded from HEK cells, with the same protocol shown in Fig. 1A. (Bottom) Resulting normalized tail current shown as current–voltage relationship from proposed interacting pairs. (B) Structural locations of the proposed interacting (red dotted line) and noninteracting residues within the TRPP2 voltage sensor. (C) The amount of nonadditive or coupled free energy (ΔΔG°) from 4 pairs cycle analyzed (SI Appendix, Methods).

Fig. 3.

MTSES modification of H404C is state dependent. (A) Transmembrane views of the TRPP2 channel structure, showing H404 (red) side chain is exposed and accessible in the up state. (B) (Top) Reaction scheme of H404C thiol modification by 30 μM MTSES. (Bottom Left) Currents from Wt and H404C channels activated by a 0.2-Hz train of 100-mV depolarizations. (Bottom Right) The time course of normalized tail current magnitudes before and after 30 μM MTSES modification (blue shaded regions). The decay in tail current is only observed with the cysteine substitution and if MTSES is applied when the VSD is activated. (C) (Left) H404C TRPP2 gating currents before (black traces) and after cystine modification (gray traces) with MTSES. Gating currents were activated by depolarizing the membrane potential to 60 mV from a holding potential of −100 mV. (Right) Inward and outward gating charge transfer measured before and after MTSES modification. Four cells expressing the H404C channel were tested, and the error bars are equal to SD. (D) Extracellular view of the Rosetta structural model of the TRPP2 VSD. Note the displacement of the TOP domain by the S3 during transition from the deactivated (Top) to activated (Bottom) states and the accessibly of H404 (red) from the extracellular side in the activated state.

Fig. 4.

Rosetta model of TRPP2 in the open and closed states. (A) A transmembrane view of the open (yellow) and closed (dark gray) TRPP2 channel models. The arrows depict the conformational changes expected to occur in 1 TRPP2 protomer during channel opening. The S4 segment translates upward during voltage sensor activation, shifting the gating charges K3 (green) and K4 (blue). The de novo predicted S4–S5 linker in the open state also shifts compared with the closed state as a result. Finally, the S6 moves laterally away from the central pore, allowing the opening of the lower gate in D. (B) The gating charges K3 and K4 form sequential cation–π interactions with the gating charge transfer center Y366 when the voltage sensor is activated. S3 residue H404 (red) does not change significantly between the predicted states. (C) An extracellular view of the channel pore in the open and closed states. Residue G522 forms the putative upper gate and dilates by 2.7 Å when the channel opens. (D) An intracellular view of the channel pore and the lower gate. The narrowest point in the lower gate, N561, widens by 7.6 Å when TRPP2 opens.

Fig. 5.

Osmotic swell potentiation of TRPP2 is dependent on gating charge transfer. (A) The onset of the TRPP2 current potentiation and swelling of the cell membrane. (Left) Images of a patch clamped SF9 insect cell in the on-cell configuration, the whole-cell configuration, and after swelling the membrane by decreasing the external osmolarity (240 mOs). The pipette was filled with standard internal saline and 30 nM Alexa Fluor 588 (red color; Invitrogen) to visualize the continuity between electrode and the volume of the intracellular compartment while the cell swelled in response to lowering the external osmolarity. (Scale bar, 15 μm.) (Middle) TRPP2 currents activated by a 0.2-Hz train of voltage ramps before and after membrane swell. Note that the outward (I_peak) and inward (I_tail) currents are antagonized by the TRPP2 channel antagonist 30 μM Gd³⁺. (Right) The time course of the increase in cell volume (red) and the onset of TRPP2 current potentiation. (B) (Left) Exemplar steady TRPP2 whole-cell currents activated before and after membrane swelling. (Middle) Rate of channel opening is estimated by the onset of current rise time. (Inset) Exemplar currents from the normal and swell conditions. (Right) Resulting normalized tail currents demonstrating the shift in the voltage dependence of activation. (C) (Left) Exemplar gating current (green) and integrated current (red) traces measured after blocking the central pore current with Gd³⁺ and osmotically swelling the cell membrane. (Right) Corresponding estimate of gating charges per channel and membrane voltage relationship. Note the −28-mV shift in QV_1/2 after 240 mOs membrane swelling (green circles, QV_1/2 = −6 mV) compared with the 300 mOs isotonic condition (white circles, QV_1/2 = 22 mV).