Properties of calcium channels in guinea-pig gastric myocytes

David A Katzka, M. Morad

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The inward membrane current in enzymatically dispersed guinea-pig gastric myocytes was studied using whole-cell voltage clamp technique. Only one inward membrane current was found in gastric myocytes which was identified as the Ca2+ current based on its inhibition by Ni2+, Cd2+ and Co2+, its dependence on [Ca2+](o), and its insensitivity to variations of [Na+](o). Ca2+ current activated at -20 mV, peaked around +10 mV and was markedly enhanced when the holding potential was increased from -40 to -90 mV. The enhancement of I(Ca) at negative holding potentials did not alter the activation threshold of I(Ca). When Ba2+ was substituted for Ca2+, I(Ba) was similarly enhanced at more negative potentials. In cells where internal Ca2+ was bufffered with 10 mM-EGTA, the time course of inactivation was fitted with two exponentials, with time constants: τ(f) = 53.4 ± 18.1 ms and τ(s) = 175.2 ± 46.1 ms. When Ba2+ was the charge carrier through the channel, the time course of inactivation could be fitted often by only one exponential which approximated τ(s) for inactivation of I(Ca). The voltage dependence of steady-state inactivation of Ca2+ channels was not significantly altered when Ba2+ was the charge carrier. Using different buffering systems (EGTA, EDTA and citrate), we found that citrate maintained the I(Ca) and slowed inactivation more effectively than the other buffers tested. Because the calculated change in [Ca2+](i) did not differ significantly between buffer systems, we speculate that suppression of inactivation by citrate is related to increased accessability of the buffer to cytoplasmic Ca2+ near the Ca2+ channel. Changes in [Mg2+](i) affected peak I(Ca) but not the kinetics of inactivation indicating that [Mg2+](i) may regulate the steady-state inactivation or the availability of the Ca2+ channels. The divalent selectivity of the Ca2+ channel had the following sequence: Ba2+ > Ca2+ ≥ Sr2+ >>> Mg2+. In very low extracellular Ca2+ (<10-7 M), the Ca2+ channel conducted Na+. Increasing [H+](o) appeared to differentially affect peak and maintained components of I(Ca). At pH < 6.5, the maintained component of I(Ca) was suppressed more than the peak component indicating possible time-and voltage-dependent inhibition of I(Ca) by protons. Nifedipine, D600 and diltiazem inhibited I(Ca) in a voltage-dependent manner. The order or potency for inhibition of peak I(Ca) was nifedipine ~ D600 >> diltiazem. Diltiazem, unlike the other two drugs, enhanced the inactivation of I(Ca), even when peak I(Ca), was not affected suggesting either a time- and voltage-dependent block of the open channel or possible existence of more than one population of Ca2+ channels with different drug sensitivities. At much lower concentration (100 nM) diltiazem enhanced the peak but not the maintained component of the Ca2+ current. A similar agonistic effect was also recorded for D600. The Ca2+ channel in the guinea-pig gastric myocyte is similar to Ca2+ channels of other tissues in its activation, ionic selectivity and inactivation. The inactivation of the Ca2+ channel is dependent both on voltage and [Ca2+](i). Although some of our data support the possible presence of more than one population of Ca2+ channels, we could provide definitive proof for only one channel type.

Original languageEnglish (US)
Pages (from-to)175-197
Number of pages23
JournalJournal of Physiology
Volume413
StatePublished - 1989
Externally publishedYes

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Calcium Channels
Muscle Cells
Diltiazem
Stomach
Guinea Pigs
Citric Acid
Buffers
Egtazic Acid
Gallopamil
Membranes
Patch-Clamp Techniques
Edetic Acid
Pharmaceutical Preparations
Population

ASJC Scopus subject areas

  • Physiology

Cite this

Properties of calcium channels in guinea-pig gastric myocytes. / Katzka, David A; Morad, M.

In: Journal of Physiology, Vol. 413, 1989, p. 175-197.

Research output: Contribution to journalArticle

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abstract = "The inward membrane current in enzymatically dispersed guinea-pig gastric myocytes was studied using whole-cell voltage clamp technique. Only one inward membrane current was found in gastric myocytes which was identified as the Ca2+ current based on its inhibition by Ni2+, Cd2+ and Co2+, its dependence on [Ca2+](o), and its insensitivity to variations of [Na+](o). Ca2+ current activated at -20 mV, peaked around +10 mV and was markedly enhanced when the holding potential was increased from -40 to -90 mV. The enhancement of I(Ca) at negative holding potentials did not alter the activation threshold of I(Ca). When Ba2+ was substituted for Ca2+, I(Ba) was similarly enhanced at more negative potentials. In cells where internal Ca2+ was bufffered with 10 mM-EGTA, the time course of inactivation was fitted with two exponentials, with time constants: τ(f) = 53.4 ± 18.1 ms and τ(s) = 175.2 ± 46.1 ms. When Ba2+ was the charge carrier through the channel, the time course of inactivation could be fitted often by only one exponential which approximated τ(s) for inactivation of I(Ca). The voltage dependence of steady-state inactivation of Ca2+ channels was not significantly altered when Ba2+ was the charge carrier. Using different buffering systems (EGTA, EDTA and citrate), we found that citrate maintained the I(Ca) and slowed inactivation more effectively than the other buffers tested. Because the calculated change in [Ca2+](i) did not differ significantly between buffer systems, we speculate that suppression of inactivation by citrate is related to increased accessability of the buffer to cytoplasmic Ca2+ near the Ca2+ channel. Changes in [Mg2+](i) affected peak I(Ca) but not the kinetics of inactivation indicating that [Mg2+](i) may regulate the steady-state inactivation or the availability of the Ca2+ channels. The divalent selectivity of the Ca2+ channel had the following sequence: Ba2+ > Ca2+ ≥ Sr2+ >>> Mg2+. In very low extracellular Ca2+ (<10-7 M), the Ca2+ channel conducted Na+. Increasing [H+](o) appeared to differentially affect peak and maintained components of I(Ca). At pH < 6.5, the maintained component of I(Ca) was suppressed more than the peak component indicating possible time-and voltage-dependent inhibition of I(Ca) by protons. Nifedipine, D600 and diltiazem inhibited I(Ca) in a voltage-dependent manner. The order or potency for inhibition of peak I(Ca) was nifedipine ~ D600 >> diltiazem. Diltiazem, unlike the other two drugs, enhanced the inactivation of I(Ca), even when peak I(Ca), was not affected suggesting either a time- and voltage-dependent block of the open channel or possible existence of more than one population of Ca2+ channels with different drug sensitivities. At much lower concentration (100 nM) diltiazem enhanced the peak but not the maintained component of the Ca2+ current. A similar agonistic effect was also recorded for D600. The Ca2+ channel in the guinea-pig gastric myocyte is similar to Ca2+ channels of other tissues in its activation, ionic selectivity and inactivation. The inactivation of the Ca2+ channel is dependent both on voltage and [Ca2+](i). Although some of our data support the possible presence of more than one population of Ca2+ channels, we could provide definitive proof for only one channel type.",
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T1 - Properties of calcium channels in guinea-pig gastric myocytes

AU - Katzka, David A

AU - Morad, M.

PY - 1989

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N2 - The inward membrane current in enzymatically dispersed guinea-pig gastric myocytes was studied using whole-cell voltage clamp technique. Only one inward membrane current was found in gastric myocytes which was identified as the Ca2+ current based on its inhibition by Ni2+, Cd2+ and Co2+, its dependence on [Ca2+](o), and its insensitivity to variations of [Na+](o). Ca2+ current activated at -20 mV, peaked around +10 mV and was markedly enhanced when the holding potential was increased from -40 to -90 mV. The enhancement of I(Ca) at negative holding potentials did not alter the activation threshold of I(Ca). When Ba2+ was substituted for Ca2+, I(Ba) was similarly enhanced at more negative potentials. In cells where internal Ca2+ was bufffered with 10 mM-EGTA, the time course of inactivation was fitted with two exponentials, with time constants: τ(f) = 53.4 ± 18.1 ms and τ(s) = 175.2 ± 46.1 ms. When Ba2+ was the charge carrier through the channel, the time course of inactivation could be fitted often by only one exponential which approximated τ(s) for inactivation of I(Ca). The voltage dependence of steady-state inactivation of Ca2+ channels was not significantly altered when Ba2+ was the charge carrier. Using different buffering systems (EGTA, EDTA and citrate), we found that citrate maintained the I(Ca) and slowed inactivation more effectively than the other buffers tested. Because the calculated change in [Ca2+](i) did not differ significantly between buffer systems, we speculate that suppression of inactivation by citrate is related to increased accessability of the buffer to cytoplasmic Ca2+ near the Ca2+ channel. Changes in [Mg2+](i) affected peak I(Ca) but not the kinetics of inactivation indicating that [Mg2+](i) may regulate the steady-state inactivation or the availability of the Ca2+ channels. The divalent selectivity of the Ca2+ channel had the following sequence: Ba2+ > Ca2+ ≥ Sr2+ >>> Mg2+. In very low extracellular Ca2+ (<10-7 M), the Ca2+ channel conducted Na+. Increasing [H+](o) appeared to differentially affect peak and maintained components of I(Ca). At pH < 6.5, the maintained component of I(Ca) was suppressed more than the peak component indicating possible time-and voltage-dependent inhibition of I(Ca) by protons. Nifedipine, D600 and diltiazem inhibited I(Ca) in a voltage-dependent manner. The order or potency for inhibition of peak I(Ca) was nifedipine ~ D600 >> diltiazem. Diltiazem, unlike the other two drugs, enhanced the inactivation of I(Ca), even when peak I(Ca), was not affected suggesting either a time- and voltage-dependent block of the open channel or possible existence of more than one population of Ca2+ channels with different drug sensitivities. At much lower concentration (100 nM) diltiazem enhanced the peak but not the maintained component of the Ca2+ current. A similar agonistic effect was also recorded for D600. The Ca2+ channel in the guinea-pig gastric myocyte is similar to Ca2+ channels of other tissues in its activation, ionic selectivity and inactivation. The inactivation of the Ca2+ channel is dependent both on voltage and [Ca2+](i). Although some of our data support the possible presence of more than one population of Ca2+ channels, we could provide definitive proof for only one channel type.

AB - The inward membrane current in enzymatically dispersed guinea-pig gastric myocytes was studied using whole-cell voltage clamp technique. Only one inward membrane current was found in gastric myocytes which was identified as the Ca2+ current based on its inhibition by Ni2+, Cd2+ and Co2+, its dependence on [Ca2+](o), and its insensitivity to variations of [Na+](o). Ca2+ current activated at -20 mV, peaked around +10 mV and was markedly enhanced when the holding potential was increased from -40 to -90 mV. The enhancement of I(Ca) at negative holding potentials did not alter the activation threshold of I(Ca). When Ba2+ was substituted for Ca2+, I(Ba) was similarly enhanced at more negative potentials. In cells where internal Ca2+ was bufffered with 10 mM-EGTA, the time course of inactivation was fitted with two exponentials, with time constants: τ(f) = 53.4 ± 18.1 ms and τ(s) = 175.2 ± 46.1 ms. When Ba2+ was the charge carrier through the channel, the time course of inactivation could be fitted often by only one exponential which approximated τ(s) for inactivation of I(Ca). The voltage dependence of steady-state inactivation of Ca2+ channels was not significantly altered when Ba2+ was the charge carrier. Using different buffering systems (EGTA, EDTA and citrate), we found that citrate maintained the I(Ca) and slowed inactivation more effectively than the other buffers tested. Because the calculated change in [Ca2+](i) did not differ significantly between buffer systems, we speculate that suppression of inactivation by citrate is related to increased accessability of the buffer to cytoplasmic Ca2+ near the Ca2+ channel. Changes in [Mg2+](i) affected peak I(Ca) but not the kinetics of inactivation indicating that [Mg2+](i) may regulate the steady-state inactivation or the availability of the Ca2+ channels. The divalent selectivity of the Ca2+ channel had the following sequence: Ba2+ > Ca2+ ≥ Sr2+ >>> Mg2+. In very low extracellular Ca2+ (<10-7 M), the Ca2+ channel conducted Na+. Increasing [H+](o) appeared to differentially affect peak and maintained components of I(Ca). At pH < 6.5, the maintained component of I(Ca) was suppressed more than the peak component indicating possible time-and voltage-dependent inhibition of I(Ca) by protons. Nifedipine, D600 and diltiazem inhibited I(Ca) in a voltage-dependent manner. The order or potency for inhibition of peak I(Ca) was nifedipine ~ D600 >> diltiazem. Diltiazem, unlike the other two drugs, enhanced the inactivation of I(Ca), even when peak I(Ca), was not affected suggesting either a time- and voltage-dependent block of the open channel or possible existence of more than one population of Ca2+ channels with different drug sensitivities. At much lower concentration (100 nM) diltiazem enhanced the peak but not the maintained component of the Ca2+ current. A similar agonistic effect was also recorded for D600. The Ca2+ channel in the guinea-pig gastric myocyte is similar to Ca2+ channels of other tissues in its activation, ionic selectivity and inactivation. The inactivation of the Ca2+ channel is dependent both on voltage and [Ca2+](i). Although some of our data support the possible presence of more than one population of Ca2+ channels, we could provide definitive proof for only one channel type.

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