Lu CC and Hilgemann DW (1999), GAT1 (GABA:Na+:Cl-) cotransport function. Kinet...

XB-ART-12415

J Gen Physiol 1999 Sep 01;1143:445-57.

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GAT1 (GABA:Na+:Cl-) cotransport function. Kinetic studies in giant Xenopus oocyte membrane patches.

Lu CC , Hilgemann DW .

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To explain cotransport function, the "alternating access" model requires that conformational changes of the empty transporter allow substrates to bind alternatively on opposite membrane sides. To test this principle for the GAT1 (GABA:Na+:Cl-) cotransporter, we have analyzed how its charge-moving partial reactions depend on substrates on both membrane sides in giant Xenopus oocyte membrane patches. (a) "Slow" charge movements, which require extracellular Na+ and probably reflect occlusion of Na+ by GAT1, were defined in three ways with similar results: by application of the high-affinity GAT1 blocker (NO-711), by application of a high concentration (120 mM) of cytoplasmic Cl-, and by removal of extracellular Na+ via pipette perfusion. (b) Three results indicate that cytoplasmic Cl- and extracellular Na+ bind to the transporter in a mutually exclusive fashion: first, cytoplasmic Cl- (5-140 mM) shifts the voltage dependence of the slow charge movement to more negative potentials, specifically by slowing its "forward" rate (i.e., extracellular Na+ occlusion); second, rapid application of cytoplasmic Cl- induces an outward current transient that requires extracellular Na+, consistent with extracellular Na+ being forced out of its binding site; third, fast charge-moving reactions, which can be monitored as a capacitance, are "immobilized" both by cytoplasmic Cl- binding and by extracellular Na+ occlusion (i.e., by the slow charge movement). (c) In the absence of extracellular Na+, three fast (submillisecond) charge movements have been identified, but no slow components. The addition of cytoplasmic Cl- suppresses two components (tau < 1 ms and 13 micros) and enables a faster component (tau < 1 micros). (d) We failed to identify charge movements of fully loaded GAT1 transporters (i.e., with all substrates on both sides). (e) Under zero-trans conditions, inward (forward) GAT1 current shows pronounced pre-steady state transients, while outward (reverse) GAT1 current does not. (f) Turnover rates for reverse GAT1 transport (33 degrees C), calculated from the ratio of steady state current magnitude to total charge movement magnitude, can exceed 60 s(-1) at positive potentials.

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Species referenced: Xenopus laevis
Genes referenced: mapt pc.1 slc6a1

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	Scheme S1.
	Figure 1. Slow GAT1 charge movements, Qslow, in an excised oocyte patch. (A and B) Charge transfer, the time integral of membrane current, was recorded directly from an integrating patch-clamp amplifier. The signals presented are records with no cytoplasmic substrates, from which records with 0.13 mM cytoplasmic NO-711 (A) or with 120 mM cytoplasmic Cl− (B) were subtracted. Extracellular [Na+] and [Cl−] were both 40 mM. Holding potential was −40 mV; 60-ms voltage pulses to voltages between −160 and +120 mV were applied in 40-mV steps. The record at the holding potential was fitted by a straight line, representing a background current of ∼1 pA, and this baseline was subtracted from all records in the same pulse series. Dotted lines, barely visible outside of signal noise, plot the best fit of each record by a single exponential function during pulse to various potentials (Qon) and 0.6 ms after returning to −40 mV (Qoff). (C and D) The charge magnitudes (Qon and Qoff) and rate constants (kslow) from the exponential fits are plotted against membrane potential: ▪, Qon; □, Qoff; •, kslow. The Q–V and kslow–V relations in C correspond to the inhibitor-defined charge movements (A), and those in D correspond to the Cl−i-defined records (B). Values at −40 mV are the mean for all the relaxation curves; standard error was within the symbol size. The voltage dependencies of Qon and Qoff were fitted by the Boltzmann relation, ΔQV=ΔQmax1+eV12−V·q·FR·T,where ΔQmax = Qmax − Qmin, V1/2 is the potential where ΔQ(V) = ΔQmax/2, q is the equivalent charge moved; F, R, and T have their usual meanings. At 33°C, the fitted parameters are (C) V1/2 = −45 mV, q = 0.94, Qmin = −2.47, Qmax = 1.89; and (D) V1/2 = −35 mV, q = 0.76, Qmin = −1.41, Qmax = 1.52. The kslow–V relations were fitted by the sum of two single exponentials kslowV=A1·exp−V·q1·FR·T+A2·expV·q2·FR·T: (C) q1 = 0.49 and q2 = 0.30, and (D) q1 = 0.39 and q2 = 0.36.
	Figure 3. Linkage of cytoplasmic Cl− binding to slow GAT1 charge movements. In the presence of 40 mM extracellular Na+, current transients are elicited by stepping [Cl−]i from 0 to 120 mM and back to 0 mM. (A, top) Current traces before (a) and after (b) adding 0.13 mM cytoplasmic NO-711 (no cytoplasmic GABA or Na+). (Bottom) The inhibitor-defined current transient (a–b). (B) Current transients, as in A, were monitored at various membrane potentials. The magnitude of Cl−i-linked charge movement was calculated as the area bounded by the zero-current line and the outward-current transient. Note that charge movement is absent at the extreme potentials. (C, top) NO-711i-defined membrane current in response to combined Cl−i concentration jumps and voltage jumps (no cytoplasmic GABA or Na+). (Bottom) In the same patch, GAT1 transport current was turned on and off by applying and removing 120 mM cytoplasmic Cl− in the presence of 20 mM cytoplasmic GABA and 120 mM cytoplasmic Na+. Dashed lines in A and C represent zero current.
	Figure 2. Negative shift and slowing of Qslow by cytoplasmic Cl−. (A) GAT1 current transients elicited by an 80-ms test pulse to +80 mV after 360-ms prepulses applied in 40-mV increments from −160 to +40 mV (no cytoplasmic GABA or Na+; 100 mM extracellular NaCl). The top traces are without and the bottom traces are with 120 mM cytoplasmic Cl−. (B) Charge movement magnitudes, determined from the current traces at +80 mV, are plotted in the absence (▪) and presence (□) of 120 mM cytoplasmic Cl− (100 mM extracellular NaCl). The solid curves are fits of Boltzmann functions to the data (see Fig. 1, legend), whereby the midpoints are −36 and −96 mV in the absence and presence of 120 mM cytoplasmic Cl−, respectively. See text for details. (C) GAT1 current transients in 0, 15, and 30 mM cytoplasmic Cl− were elicited by a 180-ms pulse to −150 mV from the holding potential of 0 mV, followed by a 60-ms pulse to +50 mV (no cytoplasmic Na+ or GABA; 120 mM extracellular NaCl). The current transients at −150 mV were fitted by single exponentials (dashed lines and inset). The time constants are 12.1, 18.9, and 36.6 ms with 0, 15, and 30 mM cytoplasmic Cl−, respectively.
	Figure 5. Identification of multiple fast GAT1 charge movement components with NO-711i. GAT1 capacitance (A) and charge movements (B) were measured simultaneously. After a 100-ms prepulse to +120 mV from 0 mV, 60-ms pulses to voltages between −160 and +120 mV were applied in 40-mV decrements. The extracellular solution contained 40 mM Na+ and 120 mM Cl−, cytoplasmic Na+ and GABA were absent. (A) Capacitance signals are shown for four different cytoplasmic solutions: (a) with 0 Cl−i and 0 NO-711i, (b) 120 mM Cl−i and 0 NO-711i, (c) 0 Cl−i and 0.13 mM NO-711i, and (d) 120 mM Cl−i and 0.13 mM NO-711i. (B) Cl−i- (left) and NO-711i–defined (right) slow charge movements. Charge records accompanying the capacitance signals in A were subtracted as indicated.
	Figure 4. Linkage of slow and fast charge movements revealed by capacitance measurements. (A) GAT1 capacitance was measured with 0, 60, and 120 mM cytoplasmic Cl− and 100 mM extracellular NaCl (no cytoplasmic Na+ or GABA). The patch was prepulsed from the holding potential of 0 to −150 mV for 1 s (not shown) before applying the voltage pulse shown at bottom. The capacitance records at −150 mV were fitted by single exponentials (dotted lines), and the time constant at each Cl−i concentration is given to the right of each trace. (B) Superimposed GAT1 capacitance records with 120 mM cytoplasmic Cl− and 100 mM extracellular NaCl. Same prepulse and holding potential as in A. In both A and B, the capacitance was measured with a 1-mV, 20-kHz sinusoidal perturbation.
	Figure 6. Multiple components of fast Cl−i-dependent GAT1 charge movements (no cytoplasmic Na+ or GABA). (A) Superimposed records of GAT1 charge movements elicited by a series of 2.7-ms pulses in 40-mV increments. The holding potential was −40 mV. Records in the presence of 120 mM cytoplasmic Cl− are subtracted from those in the absence (left) and presence (right) of 12 mM cytoplasmic Cl−. The extracellular solution contained 20 mM Cl− and no Na+. (B) The charge magnitude at the end of each voltage pulse in A is plotted against membrane voltage. (C, top) Fast Cl−i-defined charge movements resolved during 0.27-ms voltage pulses. Records shown are subtractions of records with and without 120 mM cytoplasmic Cl−. (Bottom) Traces from the top panel to +160 mV (solid line) and −200 mV (dotted line).
	Figure 7. Current transients during forward and reverse GAT1 transport. (A) Outward GAT1 transport currents with 120 mM cytoplasmic NaCl, 120 mM extracellular Cl−, and no extracellular Na+. The solid line shows current activated by 20 mM cytoplasmic GABA, and the dotted line shows NO-711i–defined current. (B) Inward GAT1 transport current with 0.4 mM extracellular GABA and 120 mM extracellular NaCl. Current records in the presence of 120 mM cytoplasmic Cl−(solid line) or 0.13 mM cytoplasmic NO-711 (dotted line) were subtracted from those with zero cytoplasmic substrates and zero inhibitor. Dashed lines represent zero current. Holding potential was 0 mV.
	Figure 8. Estimation of GAT1 turnover rates from the slow charge movements and steady state current magnitudes. (A) The NO-711i–sensitive GAT1 transient (top) and steady state outward transport current (bottom) were recorded in the same patch in the presence of 120 mM extracellular NaCl. The current transients are a subtraction of records with no cytoplasmic substrates (no inhibitor record minus 0.13 mM cytoplasmic NO-711 record). Steady state GAT1 transport current (bottom) is with 20 mM cytoplasmic GABA and 120 mM cytoplasmic NaCl (no inhibitor record minus 0.13 mM NO-711 record). Dashed lines under the current trace represents zero current. Holding potential was 0 mV. (B) Voltage dependence of charge movement (○ and •), rate constants of the slow charge (▪), and turnover rates (□). The magnitude and rate of charge movement in A were estimated from extrapolation of the exponential current decay for pulses to (•) and from (○) various voltages. The fit of the Q–V relation by a Boltzmann function (smooth line) gives a midpoint voltage of −33.1 mV, a slope of 0.99, and a total charge of 2.6 pC. The turnover rates were calculated as the quotient of steady state transport current and total charge moved, assuming that one elementary charge moves per transport cycle.

References [+] :

Chen, Fast voltage clamp discloses a new component of presteady-state currents from the Na(+)-glucose cotransporter. 1996, Pubmed, Xenbase