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PLoS One
2010 Jan 21;51:e8752. doi: 10.1371/journal.pone.0008752.
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New mechanism for voltage induced charge movement revealed in GPCRs--theory and experiments.
Zohar A
,
Dekel N
,
Rubinsky B
,
Parnas H
.
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Depolarization induced charge movement associated currents, analogous to gating currents in channels, were recently demonstrated in G-protein coupled receptors (GPCRs), and were found to affect the receptor's Agonist binding Affinity, hence denoted AA-currents. Here we study, employing a combined theoretical-experimental approach, the properties of the AA-currents using the m2-muscarinic receptor (m2R) as a case study. We found that the AA-currents are characterized by a "bump", a distinct rise followed by a slow decline, which appears both in the On and the Off responses. The cumulative features implied a directional behavior of the AA-currents. This forced us to abandon the classical chemical reaction type of models and develop instead a model that includes anisotropic processes, thus producing directionality. This model fitted well the experimental data. Our main findings are that the AA-currents include two components. One is extremely fast, approximately 0.2 ms, at all voltages. The other is slow, 2-3 ms at all voltages. Surprisingly, the slow component includes a process which strongly depends on voltage and can be as fast as 0.3 ms at + 40 mV. The reason that it does not affect the overall time constant of the slow component is that it carries very little charge. The two fast processes are suitable candidates to link between charge movement and agonist binding affinity under physiological conditions.
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20107506
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Figure 1. Characteristic features of AA-currents and GCs.(A) AA-currents recorded from m2R expressing oocytes following depolarizing pulses to the indicated potentials from a holding potential of , notice the different scales. The arrows in (A) indicate the bumps observed in the AA-currents. Upper panel - the experimental protocol. Symmetric capacitive currents were subtracted by using pulses of from a holding potential of . (B) Superposition of the results in (A) where each graph is normalized to the peak amplitude of its fast component. (C) recordings of GCs from oocytes expressing the Shaker channel, taken with permission from Bezanilla et al. [7], notice the different scales.
Figure 2. The effect of the subtraction holding potential on the AA-currents kinetics.The AA-currents were subtracted by using from a holding potential of (), () and (). (A) Left panel, On currents elicited following depolarizing pulse from to . Right panel, Off currents elicited in the return to the holding potential (). The graphs are normalized, each to the peak amplitude of its fast component. (B) Time constants of the fast component of the AA-currents. (C) Time constants of the slow component of the AA-currents. (D) Time constants of the bump rising phase. The results in B, C and D are presented as mean SD (nâ=â5â60).
Figure 3. Effect of temperature on AA-currents kinetics.(A) Currents recorded at two temperatures, , left panel and , right panel following depolarizing pulses to the indicated potentials from . Arrows indicate the bump in the Off responses. BâD, time-constants of various features of the Off responses. (B) Time constants of the fast component. (C) Time constants of the slow component. (D) Time constants of the bump rising phase. The results in B, C and D are presented as mean SD (nâ=â36).
Figure 4. Comparing simulation and experimental results employing the standard protocol ( pulse duration, holding potential).Average SD (nâ=â5) currents at indicated depolarizing pulses, model, gray lines, experiments, black lines. The experimental currents are normalized, each to the peak amplitude of its fast component and the simulations are normalized, each to the peak amplitude of the corresponding experimental fast component (see text for details).
Figure 5. Comparing average (nâ=â5) simulation and experimental results employing the standard protocol.(A) Time constants of the various transitions, (circles), (triangles) and (diamonds). Here and below, open symbols correspond to the model and filled symbols to the experiments. (B) curves of the total charge (diamonds), the fast component (squares) and the slow component (triangles). (C) and (D) Average occupancies of the fast component states and (gray lines), the values of and (black lines) and the simulated normalized charge carried by the fast component (squares). In (C) the rate constants were estimated under the constraint of exponential dependency on voltage [7], while in (D) the rate constants were estimated after relaxing this constraint (Table S1 and Table S2 respectively).
Figure 6. How the model generates the AA-currents features.Occupancies of relevant states of the model during the On (left column) and the Off (right column) responses employing the standard protocol. The simulation results were obtained using the unconstraint parameters (Table S2).
Figure 7. Kinetics of the AA-currents and curves employing the reverse protocol.(A) Kinetics of the experimental and simulation results. The currents are normalized as describe in Figure 4. Here and in B, experiments, black lines and simulations gray lines. (B) curves of the total charge. Experimental and simulation results are presented as mean SD (nâ=â5). The simulation results were obtained using the unconstraint parameters (Table S2).
Figure 8. The effect of pulse duration on the characteristics of the AA-currents.(A) Experimental (right panel) and simulation (left panel) results employing the standard protocol with pulse duration of (black lines), (dark gray lines) and (light gray lines). The currents are normalized as describe in Figure 4. (B) Corresponding curves of the total charge, pulse durations of (diamonds), (squares) and (triangles), experimental (black lines) and model (gray lines). Experimental and average simulation AA-currents are presented as mean (A) or mean SD (B) (nâ=â5). The simulation results were obtained using the unconstraint parameters (Table S2).
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