Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of [Cl−]o modifications and of pharmacological treatments on EPSC I–V curves.(A). Mean I–V relationship of EPSCs evoked before (circles) and after substitution of 136 mM isethionate for an isoosmotic amount of NaCl (squares). Analysis indicates a shift of the PK/PNa ratio from 1.57±0.09 to 2.26±0.08 in low chloride solution. Peak amplitude difference values (triangles) are used to build the I–V curve of the current cancelled by the treatment: note its largely positive equilibrium potential. (B). Effect of 200 nM αBgTx application. The I–V curves of the EPSC peak amplitude before (circles) and after toxin treatment (squares) are shown. The difference current relationship (triangles) was drawn as in (A). (C). The ratio EPSC-charge/EPSC-decay time constant is fitted against test potential (same data as in B). (D). Effect of the selective nicotinic antagonist F3 (10 µM; squares) vs. controls (circles); triangles show the corresponding difference plot of the EPSC amplitudes.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3045433&req=5

pone-0017318-g002: Effect of [Cl−]o modifications and of pharmacological treatments on EPSC I–V curves.(A). Mean I–V relationship of EPSCs evoked before (circles) and after substitution of 136 mM isethionate for an isoosmotic amount of NaCl (squares). Analysis indicates a shift of the PK/PNa ratio from 1.57±0.09 to 2.26±0.08 in low chloride solution. Peak amplitude difference values (triangles) are used to build the I–V curve of the current cancelled by the treatment: note its largely positive equilibrium potential. (B). Effect of 200 nM αBgTx application. The I–V curves of the EPSC peak amplitude before (circles) and after toxin treatment (squares) are shown. The difference current relationship (triangles) was drawn as in (A). (C). The ratio EPSC-charge/EPSC-decay time constant is fitted against test potential (same data as in B). (D). Effect of the selective nicotinic antagonist F3 (10 µM; squares) vs. controls (circles); triangles show the corresponding difference plot of the EPSC amplitudes.

Mentions: The brief conduction time in preganglionic fibers results in a quasi synchronous ACh release at the presynaptic terminals following stimulation, so that the EPSC time course is strictly related to the interaction of ACh with the nicotinic receptors. The synaptic current onset was not sensitive to the postganglionic membrane potential (either holding or test potentials) and the EPSC time-to-peak remained remarkably constant at 2.1±0.1 ms (n = 11). The synaptic current decay was described by a single exponential function of time with a mild voltage dependence on command voltage. The EPSC decay time constant, but not its voltage dependence on the command potential, was sensitive to the holding potential. The average decay constant significantly decreased when currents were elicited from hyperpolarized neurons: the mean time constants in neurons held at −40 mV were 6.3±0.5 and 8.2±0.6 ms for −40 mV and −90 mV command potentials, respectively, whereas in neurons held at −90 mV the corresponding values were 4.8±0.4 and 6.4±0.5 ms (n = 10). Since the decay time constant reflects the mean open time of the nicotinic channel, its modifications affect the overall synaptic charge. In fact, while the peak synaptic current, given the existing density of receptors, mostly depends on the magnitude of synchronous quantal emission and the driving force for the permeable ion species, the total synaptic charge is also affected by the time the nicotinic channel remains open. This offers an alternative approach to estimate the driving force and EACh. The current through the receptor was thus evaluated from the same current tracings as in Figure 1B by computing the ratio synaptic-charge/τEPSC and plotted versus the test potential. These new point estimates (np-40mV = −17.6 mV; np-90mV = +0.4 mV) were very similar to those extracted from the EPSC peak amplitudes in Figure 1B. The voltage effect on the synaptic-charge/voltage curve proved to be fully reversible (n = 4). A similar analysis is applied in Figure 2C to a different neuron sample.


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)

Effect of [Cl−]o modifications and of pharmacological treatments on EPSC I–V curves.(A). Mean I–V relationship of EPSCs evoked before (circles) and after substitution of 136 mM isethionate for an isoosmotic amount of NaCl (squares). Analysis indicates a shift of the PK/PNa ratio from 1.57±0.09 to 2.26±0.08 in low chloride solution. Peak amplitude difference values (triangles) are used to build the I–V curve of the current cancelled by the treatment: note its largely positive equilibrium potential. (B). Effect of 200 nM αBgTx application. The I–V curves of the EPSC peak amplitude before (circles) and after toxin treatment (squares) are shown. The difference current relationship (triangles) was drawn as in (A). (C). The ratio EPSC-charge/EPSC-decay time constant is fitted against test potential (same data as in B). (D). Effect of the selective nicotinic antagonist F3 (10 µM; squares) vs. controls (circles); triangles show the corresponding difference plot of the EPSC amplitudes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045433&req=5

pone-0017318-g002: Effect of [Cl−]o modifications and of pharmacological treatments on EPSC I–V curves.(A). Mean I–V relationship of EPSCs evoked before (circles) and after substitution of 136 mM isethionate for an isoosmotic amount of NaCl (squares). Analysis indicates a shift of the PK/PNa ratio from 1.57±0.09 to 2.26±0.08 in low chloride solution. Peak amplitude difference values (triangles) are used to build the I–V curve of the current cancelled by the treatment: note its largely positive equilibrium potential. (B). Effect of 200 nM αBgTx application. The I–V curves of the EPSC peak amplitude before (circles) and after toxin treatment (squares) are shown. The difference current relationship (triangles) was drawn as in (A). (C). The ratio EPSC-charge/EPSC-decay time constant is fitted against test potential (same data as in B). (D). Effect of the selective nicotinic antagonist F3 (10 µM; squares) vs. controls (circles); triangles show the corresponding difference plot of the EPSC amplitudes.
Mentions: The brief conduction time in preganglionic fibers results in a quasi synchronous ACh release at the presynaptic terminals following stimulation, so that the EPSC time course is strictly related to the interaction of ACh with the nicotinic receptors. The synaptic current onset was not sensitive to the postganglionic membrane potential (either holding or test potentials) and the EPSC time-to-peak remained remarkably constant at 2.1±0.1 ms (n = 11). The synaptic current decay was described by a single exponential function of time with a mild voltage dependence on command voltage. The EPSC decay time constant, but not its voltage dependence on the command potential, was sensitive to the holding potential. The average decay constant significantly decreased when currents were elicited from hyperpolarized neurons: the mean time constants in neurons held at −40 mV were 6.3±0.5 and 8.2±0.6 ms for −40 mV and −90 mV command potentials, respectively, whereas in neurons held at −90 mV the corresponding values were 4.8±0.4 and 6.4±0.5 ms (n = 10). Since the decay time constant reflects the mean open time of the nicotinic channel, its modifications affect the overall synaptic charge. In fact, while the peak synaptic current, given the existing density of receptors, mostly depends on the magnitude of synchronous quantal emission and the driving force for the permeable ion species, the total synaptic charge is also affected by the time the nicotinic channel remains open. This offers an alternative approach to estimate the driving force and EACh. The current through the receptor was thus evaluated from the same current tracings as in Figure 1B by computing the ratio synaptic-charge/τEPSC and plotted versus the test potential. These new point estimates (np-40mV = −17.6 mV; np-90mV = +0.4 mV) were very similar to those extracted from the EPSC peak amplitudes in Figure 1B. The voltage effect on the synaptic-charge/voltage curve proved to be fully reversible (n = 4). A similar analysis is applied in Figure 2C to a different neuron sample.

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