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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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Effect of holding potential on EPSC properties.(A). Representative tracings showing the effect of voltage on the EPSC I–V relationship in a neuron maintained at −40 (a) or −90 mV (b) holding potential. EPSCs were evoked in either case at test command potentials over the same −30/−100 mV membrane potential range in 10 mV steps. Peak EPSC amplitude of the tracings was fitted by the Goldman current equation over the whole voltage range tested, providing an estimate of EACh as derived from the PK/PNa ratio (−14.5 mV for VH-40 and +3.1 mV for VH-90). (B). Mean I–V relationship for peak EPSC amplitude. Data from 10 neurons held at −40 (filled circles) and subsequently at −90 mV (open circles) holding potential. Analysis indicates a shift of the mean PK/PNa ratio from 1.40±0.13 (VH-40) to 0.92±0.10 (VH-90).
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pone-0017318-g001: Effect of holding potential on EPSC properties.(A). Representative tracings showing the effect of voltage on the EPSC I–V relationship in a neuron maintained at −40 (a) or −90 mV (b) holding potential. EPSCs were evoked in either case at test command potentials over the same −30/−100 mV membrane potential range in 10 mV steps. Peak EPSC amplitude of the tracings was fitted by the Goldman current equation over the whole voltage range tested, providing an estimate of EACh as derived from the PK/PNa ratio (−14.5 mV for VH-40 and +3.1 mV for VH-90). (B). Mean I–V relationship for peak EPSC amplitude. Data from 10 neurons held at −40 (filled circles) and subsequently at −90 mV (open circles) holding potential. Analysis indicates a shift of the mean PK/PNa ratio from 1.40±0.13 (VH-40) to 0.92±0.10 (VH-90).

Mentions: The I–V curve of EPSCs elicited over the −30/−100 mV voltage range has been measured in neurons held at −40 mV (VH-40); each neuron was thereafter brought to and held at −90 mV for at least 3 min (VH-90), and the pulse sequence was repeated from this new holding level; a third trial was performed after returning to −40 mV (VH-40R). Typical EPSC families recorded in the same neuron, starting from the two holding potentials, are illustrated in Figure 1A; the mean peak EPSC amplitude measured in a 10-neuron sample is plotted in Figure 1B against the membrane level at which synaptic currents were evoked. Both EPSC amplitude and relative slope of the I–V curve were affected by increased membrane negativity: the PK/PNa ratio decreased from 1.40±0.13 at VH-40 to 0.92±0.10 at VH-90, the mean virtual EACh shifted from −15.7±1.0 mV to −5.9±2.0 mV, while the mean synaptic conductance, gsyn (evaluated from the slope of the I–V relationship), decreased from 0.48±0.4 µS to 0.38±0.5 µS. The entire −40/−90/−40 mV cycle was tested in 5 neurons to demonstrate that the voltage effects were reversible after the steady-state was regained at the starting membrane potential (−14.3±1.7, −4.2±3.8, −16.2±1.6 mV for the EACh at VH-40, VH-90 and VH-40R, respectively; 0.53±0.06, 0.44±0.01, 0.53±0.08 µS for gsyn).


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 holding potential on EPSC properties.(A). Representative tracings showing the effect of voltage on the EPSC I–V relationship in a neuron maintained at −40 (a) or −90 mV (b) holding potential. EPSCs were evoked in either case at test command potentials over the same −30/−100 mV membrane potential range in 10 mV steps. Peak EPSC amplitude of the tracings was fitted by the Goldman current equation over the whole voltage range tested, providing an estimate of EACh as derived from the PK/PNa ratio (−14.5 mV for VH-40 and +3.1 mV for VH-90). (B). Mean I–V relationship for peak EPSC amplitude. Data from 10 neurons held at −40 (filled circles) and subsequently at −90 mV (open circles) holding potential. Analysis indicates a shift of the mean PK/PNa ratio from 1.40±0.13 (VH-40) to 0.92±0.10 (VH-90).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045433&req=5

pone-0017318-g001: Effect of holding potential on EPSC properties.(A). Representative tracings showing the effect of voltage on the EPSC I–V relationship in a neuron maintained at −40 (a) or −90 mV (b) holding potential. EPSCs were evoked in either case at test command potentials over the same −30/−100 mV membrane potential range in 10 mV steps. Peak EPSC amplitude of the tracings was fitted by the Goldman current equation over the whole voltage range tested, providing an estimate of EACh as derived from the PK/PNa ratio (−14.5 mV for VH-40 and +3.1 mV for VH-90). (B). Mean I–V relationship for peak EPSC amplitude. Data from 10 neurons held at −40 (filled circles) and subsequently at −90 mV (open circles) holding potential. Analysis indicates a shift of the mean PK/PNa ratio from 1.40±0.13 (VH-40) to 0.92±0.10 (VH-90).
Mentions: The I–V curve of EPSCs elicited over the −30/−100 mV voltage range has been measured in neurons held at −40 mV (VH-40); each neuron was thereafter brought to and held at −90 mV for at least 3 min (VH-90), and the pulse sequence was repeated from this new holding level; a third trial was performed after returning to −40 mV (VH-40R). Typical EPSC families recorded in the same neuron, starting from the two holding potentials, are illustrated in Figure 1A; the mean peak EPSC amplitude measured in a 10-neuron sample is plotted in Figure 1B against the membrane level at which synaptic currents were evoked. Both EPSC amplitude and relative slope of the I–V curve were affected by increased membrane negativity: the PK/PNa ratio decreased from 1.40±0.13 at VH-40 to 0.92±0.10 at VH-90, the mean virtual EACh shifted from −15.7±1.0 mV to −5.9±2.0 mV, while the mean synaptic conductance, gsyn (evaluated from the slope of the I–V relationship), decreased from 0.48±0.4 µS to 0.38±0.5 µS. The entire −40/−90/−40 mV cycle was tested in 5 neurons to demonstrate that the voltage effects were reversible after the steady-state was regained at the starting membrane potential (−14.3±1.7, −4.2±3.8, −16.2±1.6 mV for the EACh at VH-40, VH-90 and VH-40R, respectively; 0.53±0.06, 0.44±0.01, 0.53±0.08 µS for gsyn).

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