A patch clamp study of the nicotinic acetylcholine receptor of bovine adrenomedullary chromaffin cells in culture.

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Abstract

1. Acetylcholine‐induced currents recorded from bovine adrenal medullary chromaffin cells maintained in culture were studied during pressure or ionophoretic applications of ACh, using the ‘whole‐cell’ and ‘outside‐out’ configurations of the patch clamp technique. In standard salines, ACh evoked whole‐cell currents of ‐38 pA to ‐1 nA at ‐60 mV, which had a reversal potential (EACh) of ‐7.1 +/‐ 0.6 mV. The ACh current‐voltage relationship was characteristically linear at negative holding potentials and biphasic at positive holding potentials, displaying a region of almost zero slope conductance between 0 and +40 mV followed by a region of positive slope conductance at more positive potentials. 2. Relative permeation to cations was examined. Substitution of external Na+ by sucrose resulted in a ‐42 mV shift of EACh for a 10‐fold reduction in [Na+]o. Using isotonic substitutions, the permeability ratios (relative to Na+) for monovalent cations were determined to be 1.32 +/‐ 0.02 for Cs+ (n = 11), 1.03 +/‐ 0.02 for Li+ (n = 8) and 0.18 +/‐ 0.02 for Tris+ (n = 7). Elevated external Ca2+ salines were found to shift EACh to more positive potentials, especially in the presence of low external Na+. 3. The nicotinic agonists nicotine, tetramethylammonium and lobeline evoked inward currents in bovine chromaffin cells. In contrast, decamethonium and the muscarinic agonist, methacholine, had no effect. 4. The nicotinic antagonists mecamylamine, trimetaphan, (+)‐tubocurarine and hexamethonium caused dose‐dependent reductions in the amplitude of ACh‐evoked inward currents. The estimated IC50's were 0.25, 0.33, 0.63 and 2.2 microM respectively, for cells voltage clamped at ‐60 mV. High concentrations (> 2 microM) of the muscarinic antagonist, atropine, also produced a dose‐dependent reduction in the amplitude of ACh‐induced currents. 5. Inhibition by trimetaphan was voltage independent. With the other drugs the antagonism was voltage sensitive, increasing with membrane hyperpolarization. The voltage sensitivity was most marked for hexamethonium. Neither hexamethonium nor mecamylamine were found to depress ACh‐evoked outward currents at concentrations which severely depressed inward currents. In addition to its antagonist actions, (+)‐tubocurarine activated unitary currents in these cells and on isolated membrane patches. 6. The results indicate that nicotinic ion channels of bovine chromaffin cells have a similar ionic selectivity to monovalent cations, but that Ca2+ ions permeate the channels to a greater degree than at the motor endplate. The ACh current‐voltage relationship resembles that described for other types of ‘neuronal’ nicotinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Original languageEnglish
Pages (from-to)503-527
Number of pages25
JournalThe Journal of Physiology
Volume455
Issue number1
DOIs
Publication statusPublished - 1 Sep 1992

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Hexamethonium
Chromaffin Cells
Nicotinic Receptors
Trimethaphan
Mecamylamine
Tubocurarine
Monovalent Cations
Cell Culture Techniques
Ion Channels
Drug Antagonism
Lobeline
Nicotinic Agonists
Nicotinic Antagonists
Motor Endplate
Muscarinic Agonists
Muscarinic Antagonists
Membranes
Methacholine Chloride
Patch-Clamp Techniques
Nicotine

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title = "A patch clamp study of the nicotinic acetylcholine receptor of bovine adrenomedullary chromaffin cells in culture.",
abstract = "1. Acetylcholine‐induced currents recorded from bovine adrenal medullary chromaffin cells maintained in culture were studied during pressure or ionophoretic applications of ACh, using the ‘whole‐cell’ and ‘outside‐out’ configurations of the patch clamp technique. In standard salines, ACh evoked whole‐cell currents of ‐38 pA to ‐1 nA at ‐60 mV, which had a reversal potential (EACh) of ‐7.1 +/‐ 0.6 mV. The ACh current‐voltage relationship was characteristically linear at negative holding potentials and biphasic at positive holding potentials, displaying a region of almost zero slope conductance between 0 and +40 mV followed by a region of positive slope conductance at more positive potentials. 2. Relative permeation to cations was examined. Substitution of external Na+ by sucrose resulted in a ‐42 mV shift of EACh for a 10‐fold reduction in [Na+]o. Using isotonic substitutions, the permeability ratios (relative to Na+) for monovalent cations were determined to be 1.32 +/‐ 0.02 for Cs+ (n = 11), 1.03 +/‐ 0.02 for Li+ (n = 8) and 0.18 +/‐ 0.02 for Tris+ (n = 7). Elevated external Ca2+ salines were found to shift EACh to more positive potentials, especially in the presence of low external Na+. 3. The nicotinic agonists nicotine, tetramethylammonium and lobeline evoked inward currents in bovine chromaffin cells. In contrast, decamethonium and the muscarinic agonist, methacholine, had no effect. 4. The nicotinic antagonists mecamylamine, trimetaphan, (+)‐tubocurarine and hexamethonium caused dose‐dependent reductions in the amplitude of ACh‐evoked inward currents. The estimated IC50's were 0.25, 0.33, 0.63 and 2.2 microM respectively, for cells voltage clamped at ‐60 mV. High concentrations (> 2 microM) of the muscarinic antagonist, atropine, also produced a dose‐dependent reduction in the amplitude of ACh‐induced currents. 5. Inhibition by trimetaphan was voltage independent. With the other drugs the antagonism was voltage sensitive, increasing with membrane hyperpolarization. The voltage sensitivity was most marked for hexamethonium. Neither hexamethonium nor mecamylamine were found to depress ACh‐evoked outward currents at concentrations which severely depressed inward currents. In addition to its antagonist actions, (+)‐tubocurarine activated unitary currents in these cells and on isolated membrane patches. 6. The results indicate that nicotinic ion channels of bovine chromaffin cells have a similar ionic selectivity to monovalent cations, but that Ca2+ ions permeate the channels to a greater degree than at the motor endplate. The ACh current‐voltage relationship resembles that described for other types of ‘neuronal’ nicotinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)",
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A patch clamp study of the nicotinic acetylcholine receptor of bovine adrenomedullary chromaffin cells in culture. / Nooney, J. M.; Peters, J. A.; Lambert, J. J.

In: The Journal of Physiology, Vol. 455, No. 1, 01.09.1992, p. 503-527.

Research output: Contribution to journalArticle

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T1 - A patch clamp study of the nicotinic acetylcholine receptor of bovine adrenomedullary chromaffin cells in culture.

AU - Nooney, J. M.

AU - Peters, J. A.

AU - Lambert, J. J.

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N2 - 1. Acetylcholine‐induced currents recorded from bovine adrenal medullary chromaffin cells maintained in culture were studied during pressure or ionophoretic applications of ACh, using the ‘whole‐cell’ and ‘outside‐out’ configurations of the patch clamp technique. In standard salines, ACh evoked whole‐cell currents of ‐38 pA to ‐1 nA at ‐60 mV, which had a reversal potential (EACh) of ‐7.1 +/‐ 0.6 mV. The ACh current‐voltage relationship was characteristically linear at negative holding potentials and biphasic at positive holding potentials, displaying a region of almost zero slope conductance between 0 and +40 mV followed by a region of positive slope conductance at more positive potentials. 2. Relative permeation to cations was examined. Substitution of external Na+ by sucrose resulted in a ‐42 mV shift of EACh for a 10‐fold reduction in [Na+]o. Using isotonic substitutions, the permeability ratios (relative to Na+) for monovalent cations were determined to be 1.32 +/‐ 0.02 for Cs+ (n = 11), 1.03 +/‐ 0.02 for Li+ (n = 8) and 0.18 +/‐ 0.02 for Tris+ (n = 7). Elevated external Ca2+ salines were found to shift EACh to more positive potentials, especially in the presence of low external Na+. 3. The nicotinic agonists nicotine, tetramethylammonium and lobeline evoked inward currents in bovine chromaffin cells. In contrast, decamethonium and the muscarinic agonist, methacholine, had no effect. 4. The nicotinic antagonists mecamylamine, trimetaphan, (+)‐tubocurarine and hexamethonium caused dose‐dependent reductions in the amplitude of ACh‐evoked inward currents. The estimated IC50's were 0.25, 0.33, 0.63 and 2.2 microM respectively, for cells voltage clamped at ‐60 mV. High concentrations (> 2 microM) of the muscarinic antagonist, atropine, also produced a dose‐dependent reduction in the amplitude of ACh‐induced currents. 5. Inhibition by trimetaphan was voltage independent. With the other drugs the antagonism was voltage sensitive, increasing with membrane hyperpolarization. The voltage sensitivity was most marked for hexamethonium. Neither hexamethonium nor mecamylamine were found to depress ACh‐evoked outward currents at concentrations which severely depressed inward currents. In addition to its antagonist actions, (+)‐tubocurarine activated unitary currents in these cells and on isolated membrane patches. 6. The results indicate that nicotinic ion channels of bovine chromaffin cells have a similar ionic selectivity to monovalent cations, but that Ca2+ ions permeate the channels to a greater degree than at the motor endplate. The ACh current‐voltage relationship resembles that described for other types of ‘neuronal’ nicotinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

AB - 1. Acetylcholine‐induced currents recorded from bovine adrenal medullary chromaffin cells maintained in culture were studied during pressure or ionophoretic applications of ACh, using the ‘whole‐cell’ and ‘outside‐out’ configurations of the patch clamp technique. In standard salines, ACh evoked whole‐cell currents of ‐38 pA to ‐1 nA at ‐60 mV, which had a reversal potential (EACh) of ‐7.1 +/‐ 0.6 mV. The ACh current‐voltage relationship was characteristically linear at negative holding potentials and biphasic at positive holding potentials, displaying a region of almost zero slope conductance between 0 and +40 mV followed by a region of positive slope conductance at more positive potentials. 2. Relative permeation to cations was examined. Substitution of external Na+ by sucrose resulted in a ‐42 mV shift of EACh for a 10‐fold reduction in [Na+]o. Using isotonic substitutions, the permeability ratios (relative to Na+) for monovalent cations were determined to be 1.32 +/‐ 0.02 for Cs+ (n = 11), 1.03 +/‐ 0.02 for Li+ (n = 8) and 0.18 +/‐ 0.02 for Tris+ (n = 7). Elevated external Ca2+ salines were found to shift EACh to more positive potentials, especially in the presence of low external Na+. 3. The nicotinic agonists nicotine, tetramethylammonium and lobeline evoked inward currents in bovine chromaffin cells. In contrast, decamethonium and the muscarinic agonist, methacholine, had no effect. 4. The nicotinic antagonists mecamylamine, trimetaphan, (+)‐tubocurarine and hexamethonium caused dose‐dependent reductions in the amplitude of ACh‐evoked inward currents. The estimated IC50's were 0.25, 0.33, 0.63 and 2.2 microM respectively, for cells voltage clamped at ‐60 mV. High concentrations (> 2 microM) of the muscarinic antagonist, atropine, also produced a dose‐dependent reduction in the amplitude of ACh‐induced currents. 5. Inhibition by trimetaphan was voltage independent. With the other drugs the antagonism was voltage sensitive, increasing with membrane hyperpolarization. The voltage sensitivity was most marked for hexamethonium. Neither hexamethonium nor mecamylamine were found to depress ACh‐evoked outward currents at concentrations which severely depressed inward currents. In addition to its antagonist actions, (+)‐tubocurarine activated unitary currents in these cells and on isolated membrane patches. 6. The results indicate that nicotinic ion channels of bovine chromaffin cells have a similar ionic selectivity to monovalent cations, but that Ca2+ ions permeate the channels to a greater degree than at the motor endplate. The ACh current‐voltage relationship resembles that described for other types of ‘neuronal’ nicotinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

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DO - 10.1113/jphysiol.1992.sp019314

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