Rundown of N-methyl-D-aspartate channels during whole-cell recording in rat hippocampal neurons: Role of Ca2+ and ATP

C. Rosenmund, Gary Westbrook

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114 Citations (Scopus)

Abstract

1. N-methyl-D-aspartate (NMDA) channel activity was studied on cultured rat hippocampal neurons in whole-cell voltage-clamp mode. NMDA responses were evoked by rapid application of NMDA and the cytosol was modified using pipette dialysis and intracellular perfusion. 2. In the presence of 2 mM [Ca2+](o) with 2.4 mM BAPTA (1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) and 0.4 mM Ca2+ in the whole-cell pipette, the response evoked by regular applications of 10 μM NMDB gradually decreased during prolonged whole-cell recording. After 25 min the peak current was reduced to 56 ± 1.6% of control. Channel 'rundown' could be prevented by inclusion of an ATP regenerating solution in the pipette. 3. Rundown did not occur in Ca2+-free medium even in the absence of added ATP regenerating solution. Rundown was also prevented by increasing [BBPTA](i) to 10 mM whereas raising [Ca2+](i) by inhibiting the Na+-Ca2+ exchanger or by perfusing the patch pipette with high [Ca2+](i) (15-1000 μM) reversibly inhibited the NMDA current. By contrast, the rundown of kainate responses was Ca2+-independent. 4. The rate and reversibility of rundown was use-dependent. Rundown did not occur with infrequent NMDA applications (0.2/min). Following channel rundown in Ca2+-containing medium, a 5 min pause in agonist applications or adding ATP regenerating solution by intracellular perfusion resulted in complete recovery. However, rundown did not recover following large currents evoked by 300 μM NMDA or when 10 μM EGTA was used as the intracellular buffer. Protease inhibitors did not prevent irreversible rundown. 5. ATP-γ-S (4 mM) was less effective than the ATP regenerating solution in preventing rundown. Likewise, intracellular dialysis with alkaline phosphatase, phosphatase 1 or calcineurin did not induce rundown and addition of phosphatase inhibitors also did not block rundown. Thus receptor dephosphorylation did not appear to be primarily responsible for channel rundown. B. The mean open time and unitary conductance of the NMDA channel were unaffected by rundown as estimated by fluctuation analysis. The conductance was 42.8 ± 2.9 nS before and 43.7 ± 2.8 nS after rundown. The mean open times were 17.3 and 4.0 ms before and 15.9 and 4.0 ms after rundown. However the open probability was reduced following rundown as determined by the onset of MK-801 block of steady-state NMDA currents. 7. Our results suggest that an increase in intracellular calcium leads to channel rundown during whole-cell recording by reducing the open probability of the NMDA channel. Although high concentrations of ATP prevented rundown, we suggest that this action is not a direct result of receptor or phsyphorylation.

Original languageEnglish (US)
Pages (from-to)705-729
Number of pages25
JournalJournal of Physiology
Volume470
StatePublished - 1993

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Patch-Clamp Techniques
N-Methylaspartate
Adenosine Triphosphate
Neurons
Phosphoric Monoester Hydrolases
Dialysis
Perfusion
Ethane
Dizocilpine Maleate
Calcineurin
Kainic Acid
Egtazic Acid
Protease Inhibitors
Cytosol
Alkaline Phosphatase
Buffers
Calcium
Acids

ASJC Scopus subject areas

  • Physiology

Cite this

@article{f0f64652135d493cbf031ce7d4a4c160,
title = "Rundown of N-methyl-D-aspartate channels during whole-cell recording in rat hippocampal neurons: Role of Ca2+ and ATP",
abstract = "1. N-methyl-D-aspartate (NMDA) channel activity was studied on cultured rat hippocampal neurons in whole-cell voltage-clamp mode. NMDA responses were evoked by rapid application of NMDA and the cytosol was modified using pipette dialysis and intracellular perfusion. 2. In the presence of 2 mM [Ca2+](o) with 2.4 mM BAPTA (1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) and 0.4 mM Ca2+ in the whole-cell pipette, the response evoked by regular applications of 10 μM NMDB gradually decreased during prolonged whole-cell recording. After 25 min the peak current was reduced to 56 ± 1.6{\%} of control. Channel 'rundown' could be prevented by inclusion of an ATP regenerating solution in the pipette. 3. Rundown did not occur in Ca2+-free medium even in the absence of added ATP regenerating solution. Rundown was also prevented by increasing [BBPTA](i) to 10 mM whereas raising [Ca2+](i) by inhibiting the Na+-Ca2+ exchanger or by perfusing the patch pipette with high [Ca2+](i) (15-1000 μM) reversibly inhibited the NMDA current. By contrast, the rundown of kainate responses was Ca2+-independent. 4. The rate and reversibility of rundown was use-dependent. Rundown did not occur with infrequent NMDA applications (0.2/min). Following channel rundown in Ca2+-containing medium, a 5 min pause in agonist applications or adding ATP regenerating solution by intracellular perfusion resulted in complete recovery. However, rundown did not recover following large currents evoked by 300 μM NMDA or when 10 μM EGTA was used as the intracellular buffer. Protease inhibitors did not prevent irreversible rundown. 5. ATP-γ-S (4 mM) was less effective than the ATP regenerating solution in preventing rundown. Likewise, intracellular dialysis with alkaline phosphatase, phosphatase 1 or calcineurin did not induce rundown and addition of phosphatase inhibitors also did not block rundown. Thus receptor dephosphorylation did not appear to be primarily responsible for channel rundown. B. The mean open time and unitary conductance of the NMDA channel were unaffected by rundown as estimated by fluctuation analysis. The conductance was 42.8 ± 2.9 nS before and 43.7 ± 2.8 nS after rundown. The mean open times were 17.3 and 4.0 ms before and 15.9 and 4.0 ms after rundown. However the open probability was reduced following rundown as determined by the onset of MK-801 block of steady-state NMDA currents. 7. Our results suggest that an increase in intracellular calcium leads to channel rundown during whole-cell recording by reducing the open probability of the NMDA channel. Although high concentrations of ATP prevented rundown, we suggest that this action is not a direct result of receptor or phsyphorylation.",
author = "C. Rosenmund and Gary Westbrook",
year = "1993",
language = "English (US)",
volume = "470",
pages = "705--729",
journal = "Journal of Physiology",
issn = "0022-3751",
publisher = "Wiley-Blackwell",

}

TY - JOUR

T1 - Rundown of N-methyl-D-aspartate channels during whole-cell recording in rat hippocampal neurons

T2 - Role of Ca2+ and ATP

AU - Rosenmund, C.

AU - Westbrook, Gary

PY - 1993

Y1 - 1993

N2 - 1. N-methyl-D-aspartate (NMDA) channel activity was studied on cultured rat hippocampal neurons in whole-cell voltage-clamp mode. NMDA responses were evoked by rapid application of NMDA and the cytosol was modified using pipette dialysis and intracellular perfusion. 2. In the presence of 2 mM [Ca2+](o) with 2.4 mM BAPTA (1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) and 0.4 mM Ca2+ in the whole-cell pipette, the response evoked by regular applications of 10 μM NMDB gradually decreased during prolonged whole-cell recording. After 25 min the peak current was reduced to 56 ± 1.6% of control. Channel 'rundown' could be prevented by inclusion of an ATP regenerating solution in the pipette. 3. Rundown did not occur in Ca2+-free medium even in the absence of added ATP regenerating solution. Rundown was also prevented by increasing [BBPTA](i) to 10 mM whereas raising [Ca2+](i) by inhibiting the Na+-Ca2+ exchanger or by perfusing the patch pipette with high [Ca2+](i) (15-1000 μM) reversibly inhibited the NMDA current. By contrast, the rundown of kainate responses was Ca2+-independent. 4. The rate and reversibility of rundown was use-dependent. Rundown did not occur with infrequent NMDA applications (0.2/min). Following channel rundown in Ca2+-containing medium, a 5 min pause in agonist applications or adding ATP regenerating solution by intracellular perfusion resulted in complete recovery. However, rundown did not recover following large currents evoked by 300 μM NMDA or when 10 μM EGTA was used as the intracellular buffer. Protease inhibitors did not prevent irreversible rundown. 5. ATP-γ-S (4 mM) was less effective than the ATP regenerating solution in preventing rundown. Likewise, intracellular dialysis with alkaline phosphatase, phosphatase 1 or calcineurin did not induce rundown and addition of phosphatase inhibitors also did not block rundown. Thus receptor dephosphorylation did not appear to be primarily responsible for channel rundown. B. The mean open time and unitary conductance of the NMDA channel were unaffected by rundown as estimated by fluctuation analysis. The conductance was 42.8 ± 2.9 nS before and 43.7 ± 2.8 nS after rundown. The mean open times were 17.3 and 4.0 ms before and 15.9 and 4.0 ms after rundown. However the open probability was reduced following rundown as determined by the onset of MK-801 block of steady-state NMDA currents. 7. Our results suggest that an increase in intracellular calcium leads to channel rundown during whole-cell recording by reducing the open probability of the NMDA channel. Although high concentrations of ATP prevented rundown, we suggest that this action is not a direct result of receptor or phsyphorylation.

AB - 1. N-methyl-D-aspartate (NMDA) channel activity was studied on cultured rat hippocampal neurons in whole-cell voltage-clamp mode. NMDA responses were evoked by rapid application of NMDA and the cytosol was modified using pipette dialysis and intracellular perfusion. 2. In the presence of 2 mM [Ca2+](o) with 2.4 mM BAPTA (1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) and 0.4 mM Ca2+ in the whole-cell pipette, the response evoked by regular applications of 10 μM NMDB gradually decreased during prolonged whole-cell recording. After 25 min the peak current was reduced to 56 ± 1.6% of control. Channel 'rundown' could be prevented by inclusion of an ATP regenerating solution in the pipette. 3. Rundown did not occur in Ca2+-free medium even in the absence of added ATP regenerating solution. Rundown was also prevented by increasing [BBPTA](i) to 10 mM whereas raising [Ca2+](i) by inhibiting the Na+-Ca2+ exchanger or by perfusing the patch pipette with high [Ca2+](i) (15-1000 μM) reversibly inhibited the NMDA current. By contrast, the rundown of kainate responses was Ca2+-independent. 4. The rate and reversibility of rundown was use-dependent. Rundown did not occur with infrequent NMDA applications (0.2/min). Following channel rundown in Ca2+-containing medium, a 5 min pause in agonist applications or adding ATP regenerating solution by intracellular perfusion resulted in complete recovery. However, rundown did not recover following large currents evoked by 300 μM NMDA or when 10 μM EGTA was used as the intracellular buffer. Protease inhibitors did not prevent irreversible rundown. 5. ATP-γ-S (4 mM) was less effective than the ATP regenerating solution in preventing rundown. Likewise, intracellular dialysis with alkaline phosphatase, phosphatase 1 or calcineurin did not induce rundown and addition of phosphatase inhibitors also did not block rundown. Thus receptor dephosphorylation did not appear to be primarily responsible for channel rundown. B. The mean open time and unitary conductance of the NMDA channel were unaffected by rundown as estimated by fluctuation analysis. The conductance was 42.8 ± 2.9 nS before and 43.7 ± 2.8 nS after rundown. The mean open times were 17.3 and 4.0 ms before and 15.9 and 4.0 ms after rundown. However the open probability was reduced following rundown as determined by the onset of MK-801 block of steady-state NMDA currents. 7. Our results suggest that an increase in intracellular calcium leads to channel rundown during whole-cell recording by reducing the open probability of the NMDA channel. Although high concentrations of ATP prevented rundown, we suggest that this action is not a direct result of receptor or phsyphorylation.

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