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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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Related in: MedlinePlus

mEPSP I–V curves are influenced by the impermeant chloride ion.(A). I–V relationship of mEPSPs recorded, under two-electrode current clamp conditions at different holding potentials in the −40/−90 mV range, in 4 neurons exposed to an external solution made hyperosmotic (718 mosmol/kg) with 0.37 M sucrose. Quantum size was calculated in the different tracings as described in Figure 4B and C, and normalized to the value measured at −90 mV (−90 mV mean value  = 1.6±0.2 mV). The I–V curve of EPSPs (continuous line) was virtually superimposable to the normalized I–V curve of control EPSCs (dotted line; same data, normalized, as in Figure 1B). Hypertonicity does not seem to affect the I–V relation. (B). mEPSP I–V relationship (continuous line) evaluated as in (A) in a 6-neuron sample exposed to enriched K+ solution (−90 mV quantum size  = 1.3±0.3 mV). Control EPSC I–V relationship is shown as in (A) (dotted line), for comparison. The different slope of the curves points to a rightward shift of the  potential in high [K+]e. (C). I–V relationship of quantal events recorded in a single neuron at different holding levels while exposed to a K+-enriched solution in which 136 mM isethionate had been substituted for an isoosmolar amount of NaCl (data normalized to the −100 mV value  = 3.2 mV). The dotted line shows the control EPSC I–V curve (normalized), for comparison. The general behavior appears to have reverted back to control: note that the cation composition of the bathing solution in (B) and (C) was the same, despite the large differences in the presumable  potential of the mEPSP. Point numbers indicate the order in which the different test levels were successively imposed.
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pone-0017318-g005: mEPSP I–V curves are influenced by the impermeant chloride ion.(A). I–V relationship of mEPSPs recorded, under two-electrode current clamp conditions at different holding potentials in the −40/−90 mV range, in 4 neurons exposed to an external solution made hyperosmotic (718 mosmol/kg) with 0.37 M sucrose. Quantum size was calculated in the different tracings as described in Figure 4B and C, and normalized to the value measured at −90 mV (−90 mV mean value  = 1.6±0.2 mV). The I–V curve of EPSPs (continuous line) was virtually superimposable to the normalized I–V curve of control EPSCs (dotted line; same data, normalized, as in Figure 1B). Hypertonicity does not seem to affect the I–V relation. (B). mEPSP I–V relationship (continuous line) evaluated as in (A) in a 6-neuron sample exposed to enriched K+ solution (−90 mV quantum size  = 1.3±0.3 mV). Control EPSC I–V relationship is shown as in (A) (dotted line), for comparison. The different slope of the curves points to a rightward shift of the potential in high [K+]e. (C). I–V relationship of quantal events recorded in a single neuron at different holding levels while exposed to a K+-enriched solution in which 136 mM isethionate had been substituted for an isoosmolar amount of NaCl (data normalized to the −100 mV value  = 3.2 mV). The dotted line shows the control EPSC I–V curve (normalized), for comparison. The general behavior appears to have reverted back to control: note that the cation composition of the bathing solution in (B) and (C) was the same, despite the large differences in the presumable potential of the mEPSP. Point numbers indicate the order in which the different test levels were successively imposed.

Mentions: The accuracy of the present EPSC I–V curves is based on the assumption that a constant amount of ACh is released by each supramaximal preganglionic stimulus. In principle, the assumption is tenable, based on the observation that the ACh volley output and the overall EPSC amplitude and properties remain reasonably constant over time. Nonetheless, the EPSC is intrinsically a compound phenomenon, strictly related to the summed effect of quantal transmitter packets, whose single size is not readily measurable. We tested whether some of the results described under voltage-clamp conditions would be confirmed under current-clamp, considering the single quantal units, the mEPSPs. Current clamp was preferred because of the large intrinsic noise of the two-electrode voltage-clamp technique, which makes the analysis of the small unitary currents inaccurate. Application of solutions made hyperosmotic (with sucrose; final value: 718 mosmol/kg; n = 4) or with increased [K+]o (35 mM; n = 6) raised the otherwise negligible mEPSP emission rate. A sequence of mEPSPs was recorded while injecting current so to drive the membrane potential for short periods (20–60 s) at various levels in the −30/−100 mV voltage range, in random sequence and more than once, when possible, in the same experiment, from a holding potential of −50 mV. After each episode, the neuron was returned and maintained at −50 mV for 2 min before applying the subsequent voltage step; the voltage-dependent [Cl−]i shifts, which obligatorily accompany membrane potential migrations [16], were thus minimized. Quantal emission rate was constant, independent of the type of stimulation and of the postsynaptic membrane potential level; it was higher in the K+-enriched solution (30–65 mEPSP/s) than in hyperosmotic conditions (27–32 mEPSP/s). Typical recordings at two different test potentials are shown in Figure 4A, together with the mEPSP amplitude and interval distributions (Fig. 4B and C). The mean amplitude varied almost linearly with voltage (Fig. 4Ba,Ca and Fig. 5), as expected (the passive neuron properties are reasonably constant over this voltage range, especially during short time periods), and the random nature of the presynaptic release mechanism was not affected by the postsynaptic membrane potential (Fig. 4Bb and Cb). The current-voltage relations were not fit with Goldman current equations under these conditions, because the size of the mEPSP is not expected to be linear with the conductance, especially as membrane potential approaches the potential. The comparisons were performed semi-quantitatively by normalizing the values measured in each experiment to the mean mEPSP amplitude at −90 mV and fitting a linear regression curve. The dotted line in each panel of Figure 5 displays the control EPSC I–V curve, similarly normalized, as a visual control.


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)

mEPSP I–V curves are influenced by the impermeant chloride ion.(A). I–V relationship of mEPSPs recorded, under two-electrode current clamp conditions at different holding potentials in the −40/−90 mV range, in 4 neurons exposed to an external solution made hyperosmotic (718 mosmol/kg) with 0.37 M sucrose. Quantum size was calculated in the different tracings as described in Figure 4B and C, and normalized to the value measured at −90 mV (−90 mV mean value  = 1.6±0.2 mV). The I–V curve of EPSPs (continuous line) was virtually superimposable to the normalized I–V curve of control EPSCs (dotted line; same data, normalized, as in Figure 1B). Hypertonicity does not seem to affect the I–V relation. (B). mEPSP I–V relationship (continuous line) evaluated as in (A) in a 6-neuron sample exposed to enriched K+ solution (−90 mV quantum size  = 1.3±0.3 mV). Control EPSC I–V relationship is shown as in (A) (dotted line), for comparison. The different slope of the curves points to a rightward shift of the  potential in high [K+]e. (C). I–V relationship of quantal events recorded in a single neuron at different holding levels while exposed to a K+-enriched solution in which 136 mM isethionate had been substituted for an isoosmolar amount of NaCl (data normalized to the −100 mV value  = 3.2 mV). The dotted line shows the control EPSC I–V curve (normalized), for comparison. The general behavior appears to have reverted back to control: note that the cation composition of the bathing solution in (B) and (C) was the same, despite the large differences in the presumable  potential of the mEPSP. Point numbers indicate the order in which the different test levels were successively imposed.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017318-g005: mEPSP I–V curves are influenced by the impermeant chloride ion.(A). I–V relationship of mEPSPs recorded, under two-electrode current clamp conditions at different holding potentials in the −40/−90 mV range, in 4 neurons exposed to an external solution made hyperosmotic (718 mosmol/kg) with 0.37 M sucrose. Quantum size was calculated in the different tracings as described in Figure 4B and C, and normalized to the value measured at −90 mV (−90 mV mean value  = 1.6±0.2 mV). The I–V curve of EPSPs (continuous line) was virtually superimposable to the normalized I–V curve of control EPSCs (dotted line; same data, normalized, as in Figure 1B). Hypertonicity does not seem to affect the I–V relation. (B). mEPSP I–V relationship (continuous line) evaluated as in (A) in a 6-neuron sample exposed to enriched K+ solution (−90 mV quantum size  = 1.3±0.3 mV). Control EPSC I–V relationship is shown as in (A) (dotted line), for comparison. The different slope of the curves points to a rightward shift of the potential in high [K+]e. (C). I–V relationship of quantal events recorded in a single neuron at different holding levels while exposed to a K+-enriched solution in which 136 mM isethionate had been substituted for an isoosmolar amount of NaCl (data normalized to the −100 mV value  = 3.2 mV). The dotted line shows the control EPSC I–V curve (normalized), for comparison. The general behavior appears to have reverted back to control: note that the cation composition of the bathing solution in (B) and (C) was the same, despite the large differences in the presumable potential of the mEPSP. Point numbers indicate the order in which the different test levels were successively imposed.
Mentions: The accuracy of the present EPSC I–V curves is based on the assumption that a constant amount of ACh is released by each supramaximal preganglionic stimulus. In principle, the assumption is tenable, based on the observation that the ACh volley output and the overall EPSC amplitude and properties remain reasonably constant over time. Nonetheless, the EPSC is intrinsically a compound phenomenon, strictly related to the summed effect of quantal transmitter packets, whose single size is not readily measurable. We tested whether some of the results described under voltage-clamp conditions would be confirmed under current-clamp, considering the single quantal units, the mEPSPs. Current clamp was preferred because of the large intrinsic noise of the two-electrode voltage-clamp technique, which makes the analysis of the small unitary currents inaccurate. Application of solutions made hyperosmotic (with sucrose; final value: 718 mosmol/kg; n = 4) or with increased [K+]o (35 mM; n = 6) raised the otherwise negligible mEPSP emission rate. A sequence of mEPSPs was recorded while injecting current so to drive the membrane potential for short periods (20–60 s) at various levels in the −30/−100 mV voltage range, in random sequence and more than once, when possible, in the same experiment, from a holding potential of −50 mV. After each episode, the neuron was returned and maintained at −50 mV for 2 min before applying the subsequent voltage step; the voltage-dependent [Cl−]i shifts, which obligatorily accompany membrane potential migrations [16], were thus minimized. Quantal emission rate was constant, independent of the type of stimulation and of the postsynaptic membrane potential level; it was higher in the K+-enriched solution (30–65 mEPSP/s) than in hyperosmotic conditions (27–32 mEPSP/s). Typical recordings at two different test potentials are shown in Figure 4A, together with the mEPSP amplitude and interval distributions (Fig. 4B and C). The mean amplitude varied almost linearly with voltage (Fig. 4Ba,Ca and Fig. 5), as expected (the passive neuron properties are reasonably constant over this voltage range, especially during short time periods), and the random nature of the presynaptic release mechanism was not affected by the postsynaptic membrane potential (Fig. 4Bb and Cb). The current-voltage relations were not fit with Goldman current equations under these conditions, because the size of the mEPSP is not expected to be linear with the conductance, especially as membrane potential approaches the potential. The comparisons were performed semi-quantitatively by normalizing the values measured in each experiment to the mean mEPSP amplitude at −90 mV and fitting a linear regression curve. The dotted line in each panel of Figure 5 displays the control EPSC I–V curve, similarly normalized, as a visual control.

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