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Openings of the rat recombinant alpha 1 homomeric glycine receptor as a function of the number of agonist molecules bound.

Beato M, Groot-Kormelink PJ, Colquhoun D, Sivilotti LG - J. Gen. Physiol. (2002)

Bottom Line: The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations.The time constants for the different components of the burst length distributions were found to have similar values at different concentrations.Durations of adjacent open and shut times were found to be strongly (negatively) correlated.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, The School of Pharmacy, London WC1N 1AX, United Kingdom. m.beato@ucl.ac.uk

ABSTRACT
The functional properties of rat homomeric alpha 1 glycine receptors were investigated using whole-cell and outside-out recording from human embryonic kidney cells transfected with rat alpha1 subunit cDNA. Whole-cell dose-response curves gave EC(50) estimates between 30 and 120 microM and a Hill slope of approximately 3.3. Single channel recordings were obtained by steady-state application of glycine (0.3, 1, or 10 microM) to outside-out patches. Single channel conductances were mostly 60-90 pS, but smaller conductances of approximately 40 pS were also seen (10% of the events) with a relative frequency that did not depend on agonist concentration. The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations. There was also evidence of a previously missed short-lived open state that was more common at lower glycine concentrations. The time constants for the different components of the burst length distributions were found to have similar values at different concentrations. Nevertheless, the mean burst length increased with increasing glycine. This was because the relative area of each burst-length component was concentration dependent and short bursts were favored at lower glycine concentrations. Durations of adjacent open and shut times were found to be strongly (negatively) correlated. Additionally, long bursts were made up of longer than average openings separated by short gaps, whereas short bursts usually consisted of single isolated short openings. The most plausible explanation for these findings is that long bursts are generated when a higher proportion of the five potential agonist binding sites on the receptor is occupied by glycine. On the basis of the concentration dependence and the intraburst structure we provide a preliminary kinetic scheme for the activation of the homomeric glycine receptor, in which any number of glycine molecules from one to five can open the channel, although not with equal efficiency.

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The predictions of the fitted model agree with the observed concentration dependence. A shows the equilibrium occupancy (normalized for visibility) of each state of ligation at the different concentrations tested. For singly liganded molecules, for example, the occupancy of AR + AR* are divided by 1 − pR, where by pR is the occupancy of the resting state, R. B shows the concentration dependence of open times as predicted by the fit (c.f. observed distribution in Fig. 3 D). C shows the predicted burst length distributions at the glycine concentrations tested and should be compared with Fig. 6 C. All calculations are based on the average rate constants as in Fig. 11 F.
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fig12: The predictions of the fitted model agree with the observed concentration dependence. A shows the equilibrium occupancy (normalized for visibility) of each state of ligation at the different concentrations tested. For singly liganded molecules, for example, the occupancy of AR + AR* are divided by 1 − pR, where by pR is the occupancy of the resting state, R. B shows the concentration dependence of open times as predicted by the fit (c.f. observed distribution in Fig. 3 D). C shows the predicted burst length distributions at the glycine concentrations tested and should be compared with Fig. 6 C. All calculations are based on the average rate constants as in Fig. 11 F.

Mentions: Using the average rate constants shown in Fig. 11 F, we calculated the equilibrium occupancy of each of the states in the model. The plot of Fig. 12 A shows the occupancy of each level of ligation (open or closed) for the three concentrations tested. At the lowest concentration most bound receptors are monoliganded and very few were more than diliganded. Even at the highest concentration tested, the fraction of fully-liganded receptors is still quite low.


Openings of the rat recombinant alpha 1 homomeric glycine receptor as a function of the number of agonist molecules bound.

Beato M, Groot-Kormelink PJ, Colquhoun D, Sivilotti LG - J. Gen. Physiol. (2002)

The predictions of the fitted model agree with the observed concentration dependence. A shows the equilibrium occupancy (normalized for visibility) of each state of ligation at the different concentrations tested. For singly liganded molecules, for example, the occupancy of AR + AR* are divided by 1 − pR, where by pR is the occupancy of the resting state, R. B shows the concentration dependence of open times as predicted by the fit (c.f. observed distribution in Fig. 3 D). C shows the predicted burst length distributions at the glycine concentrations tested and should be compared with Fig. 6 C. All calculations are based on the average rate constants as in Fig. 11 F.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233816&req=5

fig12: The predictions of the fitted model agree with the observed concentration dependence. A shows the equilibrium occupancy (normalized for visibility) of each state of ligation at the different concentrations tested. For singly liganded molecules, for example, the occupancy of AR + AR* are divided by 1 − pR, where by pR is the occupancy of the resting state, R. B shows the concentration dependence of open times as predicted by the fit (c.f. observed distribution in Fig. 3 D). C shows the predicted burst length distributions at the glycine concentrations tested and should be compared with Fig. 6 C. All calculations are based on the average rate constants as in Fig. 11 F.
Mentions: Using the average rate constants shown in Fig. 11 F, we calculated the equilibrium occupancy of each of the states in the model. The plot of Fig. 12 A shows the occupancy of each level of ligation (open or closed) for the three concentrations tested. At the lowest concentration most bound receptors are monoliganded and very few were more than diliganded. Even at the highest concentration tested, the fraction of fully-liganded receptors is still quite low.

Bottom Line: The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations.The time constants for the different components of the burst length distributions were found to have similar values at different concentrations.Durations of adjacent open and shut times were found to be strongly (negatively) correlated.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, The School of Pharmacy, London WC1N 1AX, United Kingdom. m.beato@ucl.ac.uk

ABSTRACT
The functional properties of rat homomeric alpha 1 glycine receptors were investigated using whole-cell and outside-out recording from human embryonic kidney cells transfected with rat alpha1 subunit cDNA. Whole-cell dose-response curves gave EC(50) estimates between 30 and 120 microM and a Hill slope of approximately 3.3. Single channel recordings were obtained by steady-state application of glycine (0.3, 1, or 10 microM) to outside-out patches. Single channel conductances were mostly 60-90 pS, but smaller conductances of approximately 40 pS were also seen (10% of the events) with a relative frequency that did not depend on agonist concentration. The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations. There was also evidence of a previously missed short-lived open state that was more common at lower glycine concentrations. The time constants for the different components of the burst length distributions were found to have similar values at different concentrations. Nevertheless, the mean burst length increased with increasing glycine. This was because the relative area of each burst-length component was concentration dependent and short bursts were favored at lower glycine concentrations. Durations of adjacent open and shut times were found to be strongly (negatively) correlated. Additionally, long bursts were made up of longer than average openings separated by short gaps, whereas short bursts usually consisted of single isolated short openings. The most plausible explanation for these findings is that long bursts are generated when a higher proportion of the five potential agonist binding sites on the receptor is occupied by glycine. On the basis of the concentration dependence and the intraburst structure we provide a preliminary kinetic scheme for the activation of the homomeric glycine receptor, in which any number of glycine molecules from one to five can open the channel, although not with equal efficiency.

Show MeSH
Related in: MedlinePlus