Effects of membrane potential and tension on prestin, the outer hair cell lateral membrane motor protein

Abstract

1. Under whole-cell voltage clamp, the effects of initial voltage conditions and membrane tension on gating charge and voltage-dependent capacitance were studied in human embryonic kidney cells (TSA201 cell line) transiently transfected with the gene encoding the gerbil protein prestin. Conformational changes in this membrane-bound protein probably provide the molecular basis of the outer hair cell (OHC) voltage-driven mechanical activity, which spans the audio spectrum. 2. Boltzmann characteristics of the charge movement in transfected cells were similar to those reported for OHCs (Q(max) = 0.99 +/- 0.16 pC, z = 0.88 +/- 0.02; n = 5, means +/- S.E.M.). Unlike that of the adult OHC, the voltage at peak capacitance (V(pkcm)) was very negative (-74.7 +/- 3.8 mV). Linear capacitance in transfected cells was 43.7 +/- 13.8 pF and membrane resistance was 458 +/- 123 Mohms. 3. Voltage steps from the holding potential preceding the measurement of capacitance-voltage functions caused a time- and voltage-dependent shift in V(pkcm). For a prepulse to -150 mV, from a holding potential of 0 mV, V(pkcm) shifted 6.4 mV, and was fitted by a single exponential time constant of 45 ms. A higher resolution analysis of this time course was made by measuring the change in capacitance during a fixed voltage step and indicated a double exponential shift (tau(0) = 51.6 ms, tau(1) = 8.5 s) similar to that of the native gerbil OHC. 4. Membrane tension, delivered by increasing pipette pressure, caused a positive shift in V(pkcm). A maximal shift of 7.5 mV was obtained with 2 kPa of pressure. The effect was reversible. 5. Our results show that the sensitivity of prestin to initial voltage and membrane tension, though present, is less than that observed in adult OHCs. It remains possible that some other interacting molecular species within the lateral plasma membrane of the native OHC amplifies the effect of tension and prior voltage on prestin's activity.

Figure 1. Prestin's charge movement is sensitive to initial voltage

A, gating current (a) in a prestin-transfected TSA201 cell induced by a voltage step (c) to -100 mV from a holding potential of 0 mV. Non-transfected cells did not produce gating currents (b). P/-4 protocol was used with a subtraction holding potential of +50 mV. B, whole-cell C_m determined with dual sinusoidal technique (top panel). The bottom panel shows the voltage protocol. Sinusoids have been removed for visual clarity. Data were obtained from 4 episodes; voltage was stepped to -150 mV with incrementing durations from a holding potential of 0 mV. Five-second intervals at the holding potential were allowed for recovery between episodes. C, capacitance data from B are plotted vs. ramp voltage. Traces are shifted downward (1 pF decrements) for visual clarity. Fits indicate a shift in V_pkcm denoted by • (V_pkcm, Q_max, z and C_lin were, respectively: -87.15 mV, 0.89 pC, 0.66 and 14.7 pF), ▾ (-84.2 mV, 0.91 pC, 0.64 and 15.0 pF), ▪ (-81.5 mV, 0.97 pC, 0.61 and 15.0 pF) and ♦ (-79.8 mV, 0.96 pC, 0.63 and 14.9 pF). The same symbols are used in the inset plot to show the time course of the shift. A single exponential fit gave τ= 116 ms.

Figure 2. Average V_pkcm vs. prepulse duration

Data were obtained from experimental protocols as in Fig. 1. Plotted are the means ±s.e.m. for five transfected cells. The single exponential fit gave τ= 45 ms.

Figure 3. Time course of capacitance change following voltage step

A, transfected cell capacitance decreased with τ₀= 51.6 ms and τ₁= 8.5 s indicative of the time-dependent shift in V_pkcm. B, OHC capacitance decreased with τ₀= 69.2 ms and τ₁= 2.79 s. These results are predicted by a visco-elastic model of motor interactions (Santos-Sacchi et al. 1998).

Figure 4. Effects of membrane tension on V_pkcm

A, C-V functions were obtained with transient analysis under conditions where pipette pressure was modified. Capacitance functions are offset by -4 pF for visual clarity. •, fitted V_pkcm. A straight line is drawn through the top three values, indicating the depolarizing shift in V_pkcm that accompanied pipette pressure increases. Pipette pressure was less than 0 kPa (–), ∼0.6 kPa (+), ∼1.3 kPa (++) and ∼2 kPa (+++). When pressure was made negative, V_pkcm shifted back in the hyperpolarizing direction. Fits (from top trace to bottom trace) for V_pkcm, Q_max, z and C_lin were, respectively: -68.0 mV, 0.81 pC, 0.66 and 50.6 pF; -66.2 mV, 0.91 pC, 0.60 and 50.6 pF; -60.5 mV, 0.91 pC, 0.58 and 50.5 pF; -65.4 mV, 0.96 pC, 0.55 and 50.8 pF. B, V_pkcm was tracked during changes in pipette pressure. Photographs of patch-clamped transfected cells correspond to points (arrows) before and after a pressure increase that changed cell diameter by 8 %. A slight depolarizing shift in V_pkcm was observed during the pressure increase.