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Changes in cationic selectivity of the nicotinic channel at the rat ganglionic synapse: a role for chloride ions?

Sacchi O, Rossi ML, Canella R, Fesce R - PLoS ONE (2011)

Bottom Line: Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV.Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o).A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Evolution, Section of Physiology and Biophysics, Ferrara University, Ferrara, Italy. sho@unife.it

ABSTRACT
The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.

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Cumulative effects of αBgTx application and [Cl−]o reduction.(A). I–V relationship of EPSC peak amplitude in control (filled circles; PK/PNa ratio  = 1.52±0.11), after 200 nM αBgTx application (squares; PK/PNa ratio  = 2.22±0.13) and after cumulative isethionate replacement (open circles; PK/PNa ratio  = 2.38±0.07), substituting for 136 mM NaCl. Goldman equation curves are fitted to the mean values from the same 6-neuron pool. (B). I–V curves of the difference currents showing the effect of αBgTx (filled triangles; note the largely positive equilibrium potential) and of the subsequent application of the low-chloride solution in the presence of the toxin (open triangles). Control values (circles) and the EPSC peak amplitudes generating the difference currents are the same as in (A). Zero crossing points of the fits indicate the corresponding EACh estimates.
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pone-0017318-g003: Cumulative effects of αBgTx application and [Cl−]o reduction.(A). I–V relationship of EPSC peak amplitude in control (filled circles; PK/PNa ratio  = 1.52±0.11), after 200 nM αBgTx application (squares; PK/PNa ratio  = 2.22±0.13) and after cumulative isethionate replacement (open circles; PK/PNa ratio  = 2.38±0.07), substituting for 136 mM NaCl. Goldman equation curves are fitted to the mean values from the same 6-neuron pool. (B). I–V curves of the difference currents showing the effect of αBgTx (filled triangles; note the largely positive equilibrium potential) and of the subsequent application of the low-chloride solution in the presence of the toxin (open triangles). Control values (circles) and the EPSC peak amplitudes generating the difference currents are the same as in (A). Zero crossing points of the fits indicate the corresponding EACh estimates.

Mentions: Manipulation of [Cl−]o or of the nicotinic receptor by αBgTx resulted in phenomenologically similar modifications of the synaptic current, since both treatments similarly affected both channel permeation and kinetics. We verified the possible interactions between the two treatments by applying sequentially and cumulatively, in either order, the chloride reduction (136 mM Na-isethionate substitution for NaCl) and 200 nM αBgTx. Data from 6 neurons, in which the isethionate-solution was followed by the αBgTx application, are presented in Figure 3 and Table 1. The same table also reports the results of the mirror experiment, in which the αBgTx treatment was followed by the [Cl−]o reduction (n = 7). The effects appear to be partly overlapping and complementary. By comparing the data from the two experiments, the following conclusions can be drawn: 1) αBgTx or external chloride reduction modify the nicotinic cationic selectivity to approximately the same extent (PK/PNa ratios move from 1.5 to about 2.2); 2) once the permeability properties of the channel are modified by either αBgTx or isethionate, the subsequent application of the second treatment (isethionate or αBgTx) does not further modify the channel properties significantly, indicating mutual occlusion of the effects; 3) the effects on gsyn are instead additive: external chloride reduction decreases the momentary available gsyn by the same amount (about −20%), independently of the application order; the same holds true for αBgTx (about −10%); 4) the ultimate cumulative effect (both in terms of cation selectivity and total conductance) is quantitatively the same, independent of the order in which the two treatments are applied.


Changes in cationic selectivity of the nicotinic channel at the rat ganglionic synapse: a role for chloride ions?

Sacchi O, Rossi ML, Canella R, Fesce R - PLoS ONE (2011)

Cumulative effects of αBgTx application and [Cl−]o reduction.(A). I–V relationship of EPSC peak amplitude in control (filled circles; PK/PNa ratio  = 1.52±0.11), after 200 nM αBgTx application (squares; PK/PNa ratio  = 2.22±0.13) and after cumulative isethionate replacement (open circles; PK/PNa ratio  = 2.38±0.07), substituting for 136 mM NaCl. Goldman equation curves are fitted to the mean values from the same 6-neuron pool. (B). I–V curves of the difference currents showing the effect of αBgTx (filled triangles; note the largely positive equilibrium potential) and of the subsequent application of the low-chloride solution in the presence of the toxin (open triangles). Control values (circles) and the EPSC peak amplitudes generating the difference currents are the same as in (A). Zero crossing points of the fits indicate the corresponding EACh estimates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045433&req=5

pone-0017318-g003: Cumulative effects of αBgTx application and [Cl−]o reduction.(A). I–V relationship of EPSC peak amplitude in control (filled circles; PK/PNa ratio  = 1.52±0.11), after 200 nM αBgTx application (squares; PK/PNa ratio  = 2.22±0.13) and after cumulative isethionate replacement (open circles; PK/PNa ratio  = 2.38±0.07), substituting for 136 mM NaCl. Goldman equation curves are fitted to the mean values from the same 6-neuron pool. (B). I–V curves of the difference currents showing the effect of αBgTx (filled triangles; note the largely positive equilibrium potential) and of the subsequent application of the low-chloride solution in the presence of the toxin (open triangles). Control values (circles) and the EPSC peak amplitudes generating the difference currents are the same as in (A). Zero crossing points of the fits indicate the corresponding EACh estimates.
Mentions: Manipulation of [Cl−]o or of the nicotinic receptor by αBgTx resulted in phenomenologically similar modifications of the synaptic current, since both treatments similarly affected both channel permeation and kinetics. We verified the possible interactions between the two treatments by applying sequentially and cumulatively, in either order, the chloride reduction (136 mM Na-isethionate substitution for NaCl) and 200 nM αBgTx. Data from 6 neurons, in which the isethionate-solution was followed by the αBgTx application, are presented in Figure 3 and Table 1. The same table also reports the results of the mirror experiment, in which the αBgTx treatment was followed by the [Cl−]o reduction (n = 7). The effects appear to be partly overlapping and complementary. By comparing the data from the two experiments, the following conclusions can be drawn: 1) αBgTx or external chloride reduction modify the nicotinic cationic selectivity to approximately the same extent (PK/PNa ratios move from 1.5 to about 2.2); 2) once the permeability properties of the channel are modified by either αBgTx or isethionate, the subsequent application of the second treatment (isethionate or αBgTx) does not further modify the channel properties significantly, indicating mutual occlusion of the effects; 3) the effects on gsyn are instead additive: external chloride reduction decreases the momentary available gsyn by the same amount (about −20%), independently of the application order; the same holds true for αBgTx (about −10%); 4) the ultimate cumulative effect (both in terms of cation selectivity and total conductance) is quantitatively the same, independent of the order in which the two treatments are applied.

Bottom Line: Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV.Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o).A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Evolution, Section of Physiology and Biophysics, Ferrara University, Ferrara, Italy. sho@unife.it

ABSTRACT
The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.

Show MeSH
Related in: MedlinePlus