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Single channel analysis of conductance and rectification in cation-selective, mutant glycine receptor channels.

Moorhouse AJ, Keramidas A, Zaykin A, Schofield PR, Barry PH - J. Gen. Physiol. (2002)

Bottom Line: This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one.The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed.These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of New South Wales, Sydney 2052, Australia.

ABSTRACT
Members of the ligand-gated ion channel superfamily mediate fast synaptic transmission in the nervous system. In this study, we investigate the molecular determinants and mechanisms of ion permeation and ion charge selectivity in this family of channels by characterizing the single channel conductance and rectification of alpha1 homomeric human glycine receptor channels (GlyRs) containing pore mutations that impart cation selectivity. The A-1'E mutant GlyR and the selectivity double mutant ([SDM], A-1'E, P-2' Delta) GlyR, had mean inward chord conductances (at -60 mV) of 7 pS and mean outward conductances of 11 and 12 pS (60 mV), respectively. This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one. An additional mutation to neutralize the ring of positive charge at the extracellular mouth of the channel (SDM+R19'A GlyR) made the conductance-voltage relationship linear (14 pS at both 60 and -60 mV). When this external charged ring was made negative (SDM+R19'E GlyR), the inward conductance was further increased (to 22 pS) and now became sensitive to external divalent cations (being 32 pS in their absence). The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed. Mean outward conductances in the SDM+R19'A and SDM+R19'E GlyRs were increased when internal divalent cations were absent, consistent with the intracellular end of the pore being flanked by fixed negative charges. This supports our hypothesis that the ion charge selectivity mutations have inverted the electrostatic profile of the pore by introducing a negatively charged ring at the putative selectivity filter. These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels.

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Representative single channel currents, and corresponding all-point amplitude histograms, from excised outside-out patches containing WT GlyRs. All currents were recorded in control 145 mM NaCl solutions with divalent cations present (see materials and methods). (A) Outward currents recorded at a membrane potential of 60 mV. (B) Inward currents recorded at a membrane potential of −60 mV. In both A and B, the top panel shows ∼8 s of continuous current recordings, whereas the lower panels show an ∼0.8 s subset of this recording (the borders of which are indicated by the broken lines) expanded at higher resolution. Note the different scale bars for the top and bottom panels. C and D show the all-point amplitude histograms (bin widths 0.07–0.10 pA) compiled from longer segments (∼2 min) from the same patches, at −60 mV (C) and 60 mV (D). The overlying curves are the sum of two Gaussian distributions fitted to the histograms using a least squares method, the means of which were used to calculate the conductance values for this patch, which are displayed above the histograms.
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fig3: Representative single channel currents, and corresponding all-point amplitude histograms, from excised outside-out patches containing WT GlyRs. All currents were recorded in control 145 mM NaCl solutions with divalent cations present (see materials and methods). (A) Outward currents recorded at a membrane potential of 60 mV. (B) Inward currents recorded at a membrane potential of −60 mV. In both A and B, the top panel shows ∼8 s of continuous current recordings, whereas the lower panels show an ∼0.8 s subset of this recording (the borders of which are indicated by the broken lines) expanded at higher resolution. Note the different scale bars for the top and bottom panels. C and D show the all-point amplitude histograms (bin widths 0.07–0.10 pA) compiled from longer segments (∼2 min) from the same patches, at −60 mV (C) and 60 mV (D). The overlying curves are the sum of two Gaussian distributions fitted to the histograms using a least squares method, the means of which were used to calculate the conductance values for this patch, which are displayed above the histograms.

Mentions: Outside-out membrane patches excised from 293 cells expressing WT GlyRs displayed clear single channel openings, at both −60 and 60 mV, in response to a low (∼1 μM) concentration of glycine (Fig. 3) . At both potentials, the GlyRs open in clear bursts, with the channel passing considerable current at each opening. Note the higher open probability at the positive potentials. The open channel current at each potential was derived from the mean of Gaussian fits to amplitude histograms, as illustrated in Figs. 3, C and D. At membrane potentials between −50 and −60 mV the dominant conductance level in six patches ranged from 74 to 100 pS (mean of 86.5 ± 4.1 pS; Table II), whereas for the same six patches at membrane potentials between 50 and 60 mV, the main conductance level ranged from 54 to 68 pS (62.2 ± 2.3 pS; Table II). The extent of rectification in a single patch was quantified by dividing the conductance obtained at −50 or −60 mV by that obtained at the corresponding positive membrane potential. This rectification index (R.I.) ranged from 1.10 to 1.64 (1.40 ± 0.08; Table II), indicating that the WT GlyR shows inward rectification of the open channel current.


Single channel analysis of conductance and rectification in cation-selective, mutant glycine receptor channels.

Moorhouse AJ, Keramidas A, Zaykin A, Schofield PR, Barry PH - J. Gen. Physiol. (2002)

Representative single channel currents, and corresponding all-point amplitude histograms, from excised outside-out patches containing WT GlyRs. All currents were recorded in control 145 mM NaCl solutions with divalent cations present (see materials and methods). (A) Outward currents recorded at a membrane potential of 60 mV. (B) Inward currents recorded at a membrane potential of −60 mV. In both A and B, the top panel shows ∼8 s of continuous current recordings, whereas the lower panels show an ∼0.8 s subset of this recording (the borders of which are indicated by the broken lines) expanded at higher resolution. Note the different scale bars for the top and bottom panels. C and D show the all-point amplitude histograms (bin widths 0.07–0.10 pA) compiled from longer segments (∼2 min) from the same patches, at −60 mV (C) and 60 mV (D). The overlying curves are the sum of two Gaussian distributions fitted to the histograms using a least squares method, the means of which were used to calculate the conductance values for this patch, which are displayed above the histograms.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233819&req=5

fig3: Representative single channel currents, and corresponding all-point amplitude histograms, from excised outside-out patches containing WT GlyRs. All currents were recorded in control 145 mM NaCl solutions with divalent cations present (see materials and methods). (A) Outward currents recorded at a membrane potential of 60 mV. (B) Inward currents recorded at a membrane potential of −60 mV. In both A and B, the top panel shows ∼8 s of continuous current recordings, whereas the lower panels show an ∼0.8 s subset of this recording (the borders of which are indicated by the broken lines) expanded at higher resolution. Note the different scale bars for the top and bottom panels. C and D show the all-point amplitude histograms (bin widths 0.07–0.10 pA) compiled from longer segments (∼2 min) from the same patches, at −60 mV (C) and 60 mV (D). The overlying curves are the sum of two Gaussian distributions fitted to the histograms using a least squares method, the means of which were used to calculate the conductance values for this patch, which are displayed above the histograms.
Mentions: Outside-out membrane patches excised from 293 cells expressing WT GlyRs displayed clear single channel openings, at both −60 and 60 mV, in response to a low (∼1 μM) concentration of glycine (Fig. 3) . At both potentials, the GlyRs open in clear bursts, with the channel passing considerable current at each opening. Note the higher open probability at the positive potentials. The open channel current at each potential was derived from the mean of Gaussian fits to amplitude histograms, as illustrated in Figs. 3, C and D. At membrane potentials between −50 and −60 mV the dominant conductance level in six patches ranged from 74 to 100 pS (mean of 86.5 ± 4.1 pS; Table II), whereas for the same six patches at membrane potentials between 50 and 60 mV, the main conductance level ranged from 54 to 68 pS (62.2 ± 2.3 pS; Table II). The extent of rectification in a single patch was quantified by dividing the conductance obtained at −50 or −60 mV by that obtained at the corresponding positive membrane potential. This rectification index (R.I.) ranged from 1.10 to 1.64 (1.40 ± 0.08; Table II), indicating that the WT GlyR shows inward rectification of the open channel current.

Bottom Line: This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one.The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed.These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of New South Wales, Sydney 2052, Australia.

ABSTRACT
Members of the ligand-gated ion channel superfamily mediate fast synaptic transmission in the nervous system. In this study, we investigate the molecular determinants and mechanisms of ion permeation and ion charge selectivity in this family of channels by characterizing the single channel conductance and rectification of alpha1 homomeric human glycine receptor channels (GlyRs) containing pore mutations that impart cation selectivity. The A-1'E mutant GlyR and the selectivity double mutant ([SDM], A-1'E, P-2' Delta) GlyR, had mean inward chord conductances (at -60 mV) of 7 pS and mean outward conductances of 11 and 12 pS (60 mV), respectively. This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one. An additional mutation to neutralize the ring of positive charge at the extracellular mouth of the channel (SDM+R19'A GlyR) made the conductance-voltage relationship linear (14 pS at both 60 and -60 mV). When this external charged ring was made negative (SDM+R19'E GlyR), the inward conductance was further increased (to 22 pS) and now became sensitive to external divalent cations (being 32 pS in their absence). The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed. Mean outward conductances in the SDM+R19'A and SDM+R19'E GlyRs were increased when internal divalent cations were absent, consistent with the intracellular end of the pore being flanked by fixed negative charges. This supports our hypothesis that the ion charge selectivity mutations have inverted the electrostatic profile of the pore by introducing a negatively charged ring at the putative selectivity filter. These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels.

Show MeSH
Related in: MedlinePlus