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An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence.

Li M, Farley RA, Lester HA - J. Gen. Physiol. (2000)

Bottom Line: The presence of gamma-aminobutyric acid did not noticeably affect the fluorescence waveforms.Physiol. 114:459-476).Therefore, the study provides verification that conformational changes occur during GAT1 function.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.

ABSTRACT
The rat gamma-aminobutyric acid transporter GAT1 expressed in Xenopus oocytes was labeled at Cys74, and at one or more other sites, by tetramethylrhodamine-5-maleimide, without significantly altering GAT1 function. Voltage-jump relaxation analysis showed that fluorescence increased slightly and monotonically with hyperpolarization; the fluorescence at -140 mV was approximately 0. 8% greater than at +60 mV. The time course of the fluorescence relaxations was mostly described by a single exponential with voltage-dependent but history-independent time constants ranging from approximately 20 ms at +60 mV to approximately 150 ms at -140 mV. The fluorescence did not saturate at the most negative potentials tested, and the midpoint of the fluorescence-voltage relation was at least 50 mV more negative than the midpoint of the charge-voltage relation previously identified with Na(+) binding to GAT1. The presence of gamma-aminobutyric acid did not noticeably affect the fluorescence waveforms. The fluorescence signal depended on Na(+) concentration with a Hill coefficient approaching 2. Increasing Cl(-) concentration modestly increased and accelerated the fluorescence relaxations for hyperpolarizing jumps. The fluorescence change was blocked by the GAT1 inhibitor, NO-711. For the W68L mutant of GAT1, the fluorescence relaxations occurred only during jumps to high positive potentials, in agreement with previous suggestions that this mutant is trapped in one conformational state except at these potentials. These observations suggest that the fluorescence signals monitor a novel state of GAT1, intermediate between the E*(out) and E(out) states of Hilgemann, D.W., and C.-C. Lu (1999. J. Gen. Physiol. 114:459-476). Therefore, the study provides verification that conformational changes occur during GAT1 function.

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The nature of the fluorescent state: a scheme that fits the available data. The scheme is superimposed on the state diagram accompanying Lu and Hilgemann (1999). The fluorescent state is pictured as a novel state, E*out-fluo between the E*out and Eout states, with characteristics intermediate to these states. Hilgemann and Lu 1999 concluded that the transition from the E*out to Eout is voltage dependent, occludes a Na+ onto the transporter, and constitutes the major rate-limiting step in the transport cycle. Because the transition is incomplete in the novel state, with Na+ only partially occluded, the Na+ concentration dependence and voltage dependence for the transition from E*out to E*out-fluo are less than those for the complete transition to Eout. The dashed oval is drawn to include the novel state and the immediate adjacent stable states; any agents that act within the oval would perturb the fluorescent state strongly enough to be detected in our experiments. The W68L mutant is thought to trap transporter in the state now characterized as Eout (Mager et al. 1996), which explains how this mutation eliminates the fluorescence relaxations at most potentials. NO-711 is thought to stabilize the Eout states as well (Mager et al. 1996), explaining how it blocks the relaxations. Agents that act outside the oval would perturb the fluorescent state too weakly to be detected in our experiments. The binding of both GABA and Cl− occur outside the oval, which explains their small effects on the fluorescence relaxations. Therefore, GABA binding and Cl− binding are shown in parentheses.
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Figure 15: The nature of the fluorescent state: a scheme that fits the available data. The scheme is superimposed on the state diagram accompanying Lu and Hilgemann (1999). The fluorescent state is pictured as a novel state, E*out-fluo between the E*out and Eout states, with characteristics intermediate to these states. Hilgemann and Lu 1999 concluded that the transition from the E*out to Eout is voltage dependent, occludes a Na+ onto the transporter, and constitutes the major rate-limiting step in the transport cycle. Because the transition is incomplete in the novel state, with Na+ only partially occluded, the Na+ concentration dependence and voltage dependence for the transition from E*out to E*out-fluo are less than those for the complete transition to Eout. The dashed oval is drawn to include the novel state and the immediate adjacent stable states; any agents that act within the oval would perturb the fluorescent state strongly enough to be detected in our experiments. The W68L mutant is thought to trap transporter in the state now characterized as Eout (Mager et al. 1996), which explains how this mutation eliminates the fluorescence relaxations at most potentials. NO-711 is thought to stabilize the Eout states as well (Mager et al. 1996), explaining how it blocks the relaxations. Agents that act outside the oval would perturb the fluorescent state too weakly to be detected in our experiments. The binding of both GABA and Cl− occur outside the oval, which explains their small effects on the fluorescence relaxations. Therefore, GABA binding and Cl− binding are shown in parentheses.

Mentions: A conformational change produces the fluorescence signal. Conformational changes form the basis of kinetic-state models for GAT1 function studied by Hilgemann and Lu 1999. Their favored model is shown in Fig. 15. Dr. Hilgemann kindly spent some effort at investigating states in this model that might parallel our signals, but was unable to correlate the fluorescence signals shown here with a kinetic state in their model. We suggest that the fluorescence relaxations observed with TMRM-labeled GAT1 accompany transitions to and from a novel intermediate state that was not detectable in the experiments of Hilgemann and Lu (Fig. 15). Hilgemann concluded that the transition from E*out to Eout is voltage dependent, occludes a Na+ onto the transporter, and constitutes the major rate-limiting step in the transport cycle (Hilgemann and Lu 1999). It is therefore not surprising that an additional detection technique—fluorescence labeling in this case—has produced evidence for the existence of an additional step during this complex E*out to Eout transition. Because the novel state lies between the two states, E*out and Eout, we term the new state E*out-fluo. Our measurements do not accurately reflect the steady state fluorescence, but do track changes from this baseline level; therefore, the measurements are expected to reflect primarily the relative levels of E*out-fluo and the two adjacent states, E*out and Eout. Fig. 15 explains how the scheme accounts for the data in the present study: membrane potential, Na+, NO-711, and the W68L mutation all affect the fluorescence relaxations strongly, while Cl− and GABA affect the relaxations more weakly, if at all.


An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence.

Li M, Farley RA, Lester HA - J. Gen. Physiol. (2000)

The nature of the fluorescent state: a scheme that fits the available data. The scheme is superimposed on the state diagram accompanying Lu and Hilgemann (1999). The fluorescent state is pictured as a novel state, E*out-fluo between the E*out and Eout states, with characteristics intermediate to these states. Hilgemann and Lu 1999 concluded that the transition from the E*out to Eout is voltage dependent, occludes a Na+ onto the transporter, and constitutes the major rate-limiting step in the transport cycle. Because the transition is incomplete in the novel state, with Na+ only partially occluded, the Na+ concentration dependence and voltage dependence for the transition from E*out to E*out-fluo are less than those for the complete transition to Eout. The dashed oval is drawn to include the novel state and the immediate adjacent stable states; any agents that act within the oval would perturb the fluorescent state strongly enough to be detected in our experiments. The W68L mutant is thought to trap transporter in the state now characterized as Eout (Mager et al. 1996), which explains how this mutation eliminates the fluorescence relaxations at most potentials. NO-711 is thought to stabilize the Eout states as well (Mager et al. 1996), explaining how it blocks the relaxations. Agents that act outside the oval would perturb the fluorescent state too weakly to be detected in our experiments. The binding of both GABA and Cl− occur outside the oval, which explains their small effects on the fluorescence relaxations. Therefore, GABA binding and Cl− binding are shown in parentheses.
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Related In: Results  -  Collection

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Figure 15: The nature of the fluorescent state: a scheme that fits the available data. The scheme is superimposed on the state diagram accompanying Lu and Hilgemann (1999). The fluorescent state is pictured as a novel state, E*out-fluo between the E*out and Eout states, with characteristics intermediate to these states. Hilgemann and Lu 1999 concluded that the transition from the E*out to Eout is voltage dependent, occludes a Na+ onto the transporter, and constitutes the major rate-limiting step in the transport cycle. Because the transition is incomplete in the novel state, with Na+ only partially occluded, the Na+ concentration dependence and voltage dependence for the transition from E*out to E*out-fluo are less than those for the complete transition to Eout. The dashed oval is drawn to include the novel state and the immediate adjacent stable states; any agents that act within the oval would perturb the fluorescent state strongly enough to be detected in our experiments. The W68L mutant is thought to trap transporter in the state now characterized as Eout (Mager et al. 1996), which explains how this mutation eliminates the fluorescence relaxations at most potentials. NO-711 is thought to stabilize the Eout states as well (Mager et al. 1996), explaining how it blocks the relaxations. Agents that act outside the oval would perturb the fluorescent state too weakly to be detected in our experiments. The binding of both GABA and Cl− occur outside the oval, which explains their small effects on the fluorescence relaxations. Therefore, GABA binding and Cl− binding are shown in parentheses.
Mentions: A conformational change produces the fluorescence signal. Conformational changes form the basis of kinetic-state models for GAT1 function studied by Hilgemann and Lu 1999. Their favored model is shown in Fig. 15. Dr. Hilgemann kindly spent some effort at investigating states in this model that might parallel our signals, but was unable to correlate the fluorescence signals shown here with a kinetic state in their model. We suggest that the fluorescence relaxations observed with TMRM-labeled GAT1 accompany transitions to and from a novel intermediate state that was not detectable in the experiments of Hilgemann and Lu (Fig. 15). Hilgemann concluded that the transition from E*out to Eout is voltage dependent, occludes a Na+ onto the transporter, and constitutes the major rate-limiting step in the transport cycle (Hilgemann and Lu 1999). It is therefore not surprising that an additional detection technique—fluorescence labeling in this case—has produced evidence for the existence of an additional step during this complex E*out to Eout transition. Because the novel state lies between the two states, E*out and Eout, we term the new state E*out-fluo. Our measurements do not accurately reflect the steady state fluorescence, but do track changes from this baseline level; therefore, the measurements are expected to reflect primarily the relative levels of E*out-fluo and the two adjacent states, E*out and Eout. Fig. 15 explains how the scheme accounts for the data in the present study: membrane potential, Na+, NO-711, and the W68L mutation all affect the fluorescence relaxations strongly, while Cl− and GABA affect the relaxations more weakly, if at all.

Bottom Line: The presence of gamma-aminobutyric acid did not noticeably affect the fluorescence waveforms.Physiol. 114:459-476).Therefore, the study provides verification that conformational changes occur during GAT1 function.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.

ABSTRACT
The rat gamma-aminobutyric acid transporter GAT1 expressed in Xenopus oocytes was labeled at Cys74, and at one or more other sites, by tetramethylrhodamine-5-maleimide, without significantly altering GAT1 function. Voltage-jump relaxation analysis showed that fluorescence increased slightly and monotonically with hyperpolarization; the fluorescence at -140 mV was approximately 0. 8% greater than at +60 mV. The time course of the fluorescence relaxations was mostly described by a single exponential with voltage-dependent but history-independent time constants ranging from approximately 20 ms at +60 mV to approximately 150 ms at -140 mV. The fluorescence did not saturate at the most negative potentials tested, and the midpoint of the fluorescence-voltage relation was at least 50 mV more negative than the midpoint of the charge-voltage relation previously identified with Na(+) binding to GAT1. The presence of gamma-aminobutyric acid did not noticeably affect the fluorescence waveforms. The fluorescence signal depended on Na(+) concentration with a Hill coefficient approaching 2. Increasing Cl(-) concentration modestly increased and accelerated the fluorescence relaxations for hyperpolarizing jumps. The fluorescence change was blocked by the GAT1 inhibitor, NO-711. For the W68L mutant of GAT1, the fluorescence relaxations occurred only during jumps to high positive potentials, in agreement with previous suggestions that this mutant is trapped in one conformational state except at these potentials. These observations suggest that the fluorescence signals monitor a novel state of GAT1, intermediate between the E*(out) and E(out) states of Hilgemann, D.W., and C.-C. Lu (1999. J. Gen. Physiol. 114:459-476). Therefore, the study provides verification that conformational changes occur during GAT1 function.

Show MeSH
Related in: MedlinePlus