Carpaneto A et al. (2010), Sucrose- and H-dependent charge movements assoc...

XB-ART-42063

PLoS One 2010 Sep 07;59:e12605. doi: 10.1371/journal.pone.0012605.

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Sucrose- and H-dependent charge movements associated with the gating of sucrose transporter ZmSUT1.

Carpaneto A , Koepsell H , Bamberg E , Hedrich R , Geiger D .

Abstract
In contrast to man the majority of higher plants use sucrose as mobile carbohydrate. Accordingly proton-driven sucrose transporters are crucial for cell-to-cell and long-distance distribution within the plant body. Generally very negative plant membrane potentials and the ability to accumulate sucrose quantities of more than 1 M document that plants must have evolved transporters with unique structural and functional features.To unravel the functional properties of one specific high capacity plasma membrane sucrose transporter in detail, we expressed the sucrose/H(+) co-transporter from maize ZmSUT1 in Xenopus oocytes. Application of sucrose in an acidic pH environment elicited inward proton currents. Interestingly the sucrose-dependent H(+) transport was associated with a decrease in membrane capacitance (C(m)). In addition to sucrose C(m) was modulated by the membrane potential and external protons. In order to explore the molecular mechanism underlying these C(m) changes, presteady-state currents (I(pre)) of ZmSUT1 transport were analyzed. Decay of I(pre) could be best fitted by double exponentials. When plotted against the voltage the charge Q, associated to I(pre), was dependent on sucrose and protons. The mathematical derivative of the charge Q versus voltage was well in line with the observed C(m) changes. Based on these parameters a turnover rate of 500 molecules sucrose/s was calculated. In contrast to gating currents of voltage dependent-potassium channels the analysis of ZmSUT1-derived presteady-state currents in the absence of sucrose (I = Q/τ) was sufficient to predict ZmSUT1 transport-associated currents.Taken together our results indicate that in the absence of sucrose, 'trapped' protons move back and forth between an outer and an inner site within the transmembrane domains of ZmSUT1. This movement of protons in the electric field of the membrane gives rise to the presteady-state currents and in turn to C(m) changes. Upon application of external sucrose, protons can pass the membrane turning presteady-state into transport currents.

PubMed ID: 20838661
PMC ID: PMC2935479
Article link: PLoS One

Article Images: [+] show captions

	Figure 1. Sucrose dependent membrane capacitance changes in ZmSUT1 expressing oocytes.A) While continuously recording the membrane capacitance (Cm) at −20 mV and pH 4, various external solutions (indicated in the figure) were applied. Upon perfusion with sucrose containing external media the Cm decreased in a dose dependent manner. B) When plotting Cm against the membrane potential it became apparent that at all tested voltages, Cm decreased in the presence of sucrose in a dose dependent manner (standard solution pH 4, sucrose concentrations are indicated in the figure, n≥4, ±SD).
	Figure 2. Capacitance changes in response to varying proton concentrations.A) Continuous recording of Cm at −20 mV and indicated pH values. When exchanging the bath solution by stepwise decreasing proton concentrations from pH 4 to 7 in the absence of sucrose, Cm decreased reversibly. Increasing the proton concentration from pH 4 to 3 at −20 mV led to a decrease of Cm as well. Subsequent application of saturating sucrose concentrations decreased Cm to similar levels as in the presence of neutral buffers. B) Cm/voltage plot at various pH values without sucrose and at pH 4 in the presence of sucrose. Decreasing the proton concentration resulted in a shift of the Cm peak towards more negative membrane potentials. At pH 4 and in the presence of saturating sucrose concentrations Cm was depressed at all voltages tested (n≥5, ±SD). Cm values were normalized to values at pH 4 in the absence of sucrose.
	Figure 3. Presteady-state currents of ZmSUT1.A) Inset: currents recorded in the absence (No suc) and in the presence of saturating sucrose concentration (+suc). Same currents presented in the inset after forcing their stationary level to zero. Holding and pulse voltages were –20 and –80 mV respectively. B) Presteady-state currents versus time obtained after subtracting the stationary currents in the absence and in the presence of saturating sucrose. Holding voltage –20 mV, voltage pulses from +40 mV to –120 mV (step –40 mV). Inset: presteady-state current elicited by a voltage of –80 mV. C) Charge associated to presteady-state currents plotted versus the voltage. The charge was obtained from integration of the presteady-state currents (see Materials and Methods). The continuous line is the fit of Q with the Boltzmann function shown in Material and Methods with, in this oocyte, Qmax = −9 nC, Vh = −80 mV and s = 103 mV. Inset of panel C: Capacitance (empty symbols) measured using the method of Adrian and Almers (1976). The continuous line is the capacitance derived from the Q-V plot of panel C (see materials and methods). D) Qon and Qoff, the charge movements during the on and off response of the voltage steps obtained from the integrals of the presteady-state currents in 20 different oocytes, are plotted for various membrane voltages from –120 and +60 mV in 10 mV steps. The dotted line has a slope of 1. The linear fits (not shown) of the experimental points for each single oocyte give regression coefficients with a minimum value of −0.993, indicating very strong correlation. E) Time constants of the decay of the on and off presteady-state currents versus the applied voltage. The final decay of the presteady-state currents can be fitted by a two-exponential function with a fast and a slow τ (see Materials and Methods). The value of the fast time constants (▪ for the on and for the off presteady-state current) is under 1 ms and is limited by the speed of the voltage-clamp. Symbols • and + represent the slow time constants of the on and off presteady-state currents respectively. F) Transported (measured, empty symbol) and predicted (filled symbol) currents plotted against voltage for a single oocyte.
	Figure 4. Properties of charge movement.A) Relation between I-120 and Q-120 for 20 individual oocytes; the linear regression (r = 0.92) gives a slope of 520±53 nA nC−1 or s−1. C) Transported (measured, empty symbols) and predicted (filled symbols) currents plotted against voltage for two oocytes with different level of ZmSUT1 expression.
	Figure 5. Sucrose dependence of ZmSUT1 presteady-state currents.A) Presteady-state currents of a single oocyte at varying external sucrose concentrations. For the sake of clarity only currents recorded from +40 to –120 mV, in 40 mV step, are shown; the holding voltage was –20 mV. B) Charge associated to presteady-state currents versus voltage at different sucrose concentrations. The continuous lines are fit of Q using a Boltzmann function (see Materials and Methods). Data for each oocyte were normalized to the maximum charge obtained in the absence of external sucrose, Qmax(No suc). The fits gave the following parameters (mean±SEM, n = 4): no external sucrose: V1/2 = −65±8 mV, s = 96±5 mV; 0.5 mM sucrose: V1/2 = −123±17 mV, Qmax(suc = 0.5 mM)/Qmax(No suc) = 0.69±0.02; 1 mM sucrose V1/2 = −159±20 mV, Qmax(suc = 1 mM)/Qmax(No suc) = 0.50±0.02. At 0.5 and 1 mM sucrose the slope s of the fit was fixed at the value obtained at 0 external sucrose. C) Isat – IX against the applied membrane potential. Isat is the current at saturating external sucrose concentration; Ix are the currents in the presence of 0, 0.5 mM and 1 mM external sucrose. Empty and filled symbols refer to the measured and predicted currents respectively. The following relationship was used in order to evaluate the predicted currents: Isat – IX = QX/τ where X represents the external sucrose concentration and τ is the slow time constant at zero external sucrose.
	Figure 6. pH dependence of ZmSUT1 presteady-state currents.A) Presteady-state currents of a single oocyte at varying external pH. Holding voltage –20 mV, voltage pulses from +40 mV to –120 mV (step –40 mV). B) Normalized charge associated to presteady-state currents at different external pH versus voltage. For the details of the pH analysis see Materials and Methods. Qmax(pH = 3)/Qmax(pH = 4) = 0.85±0.03. Data were obtained by four different oocytes. C) Half activation voltages (V1/2) obtained by data in panel B versus external pH. The slope of the linear fit from pH 7 to 4 is equal to 83±5 mV. D) Slow time constant τ recorded at pH 4 and 5 plotted against applied voltage. E) Predicted (filled symbols) and measured (empty symbols) transported currents versus voltage (see Materials and Methods for details).

References [+] :

Adrian, The voltage dependence of membrane capacity. 1976, Pubmed