Ca2+- and voltage-dependent gating of Ca2+- and ATP-sensitive cationic channels in brain capillary endothelium

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Abstract

Biophysical properties of the Ca2+-activated nonselective cation channel expressed in brain capillaries were studied in inside-out patches from primary cultures of rat brain microvascular endothelial cells. At -40 mV membrane potential, open probability (Po) was activated by cytosolic [Ca2+] <1 μM and was half-maximal at ∼20 μM. Increasing [Ca2+] stimulated opening rate with little effect on closing rate. At constant [Ca2+], Po was voltage-dependent, and effective gating charge corresponded to 0.6 ∼ 0.1 unitary charges. Depolarization accelerated opening and slowed closing, thereby increasing apparent affinity for Ca2+. Within ∼1 min of excision, Po declined to a lower steady state with decreased sensitivity toward activating Ca2+ when studied at a fixed voltage, and toward activating voltage when studied at a fixed [Ca2+]. Deactivated channels opened ∼5-fold slower and closed ∼10-fold faster. The sulfhydryl-reducing agent dithiotreitol (1 mM) completely reversed acceleration of closing rate but failed to recover opening rate. Single-channel gating was complex; distributions of open and closed dwell times contained at least four and five exponential components, respectively. The longest component of the closed-time distribution was markedly sensitive to both [Ca2+] and voltage. We conclude that the biophysical properties of gating of this channel are remarkably similar to those of large-conductance Ca2+-activated K+ channels.

Original languageEnglish
Pages (from-to)313-327
Number of pages15
JournalBiophysical Journal
Volume85
Issue number1
Publication statusPublished - Jul 1 2003

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Vascular Endothelium
Adenosine Triphosphate
Brain
Calcium-Activated Potassium Channels
Reducing Agents
Membrane Potentials
Cations
Endothelial Cells

ASJC Scopus subject areas

  • Biophysics

Cite this

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abstract = "Biophysical properties of the Ca2+-activated nonselective cation channel expressed in brain capillaries were studied in inside-out patches from primary cultures of rat brain microvascular endothelial cells. At -40 mV membrane potential, open probability (Po) was activated by cytosolic [Ca2+] <1 μM and was half-maximal at ∼20 μM. Increasing [Ca2+] stimulated opening rate with little effect on closing rate. At constant [Ca2+], Po was voltage-dependent, and effective gating charge corresponded to 0.6 ∼ 0.1 unitary charges. Depolarization accelerated opening and slowed closing, thereby increasing apparent affinity for Ca2+. Within ∼1 min of excision, Po declined to a lower steady state with decreased sensitivity toward activating Ca2+ when studied at a fixed voltage, and toward activating voltage when studied at a fixed [Ca2+]. Deactivated channels opened ∼5-fold slower and closed ∼10-fold faster. The sulfhydryl-reducing agent dithiotreitol (1 mM) completely reversed acceleration of closing rate but failed to recover opening rate. Single-channel gating was complex; distributions of open and closed dwell times contained at least four and five exponential components, respectively. The longest component of the closed-time distribution was markedly sensitive to both [Ca2+] and voltage. We conclude that the biophysical properties of gating of this channel are remarkably similar to those of large-conductance Ca2+-activated K+ channels.",
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N2 - Biophysical properties of the Ca2+-activated nonselective cation channel expressed in brain capillaries were studied in inside-out patches from primary cultures of rat brain microvascular endothelial cells. At -40 mV membrane potential, open probability (Po) was activated by cytosolic [Ca2+] <1 μM and was half-maximal at ∼20 μM. Increasing [Ca2+] stimulated opening rate with little effect on closing rate. At constant [Ca2+], Po was voltage-dependent, and effective gating charge corresponded to 0.6 ∼ 0.1 unitary charges. Depolarization accelerated opening and slowed closing, thereby increasing apparent affinity for Ca2+. Within ∼1 min of excision, Po declined to a lower steady state with decreased sensitivity toward activating Ca2+ when studied at a fixed voltage, and toward activating voltage when studied at a fixed [Ca2+]. Deactivated channels opened ∼5-fold slower and closed ∼10-fold faster. The sulfhydryl-reducing agent dithiotreitol (1 mM) completely reversed acceleration of closing rate but failed to recover opening rate. Single-channel gating was complex; distributions of open and closed dwell times contained at least four and five exponential components, respectively. The longest component of the closed-time distribution was markedly sensitive to both [Ca2+] and voltage. We conclude that the biophysical properties of gating of this channel are remarkably similar to those of large-conductance Ca2+-activated K+ channels.

AB - Biophysical properties of the Ca2+-activated nonselective cation channel expressed in brain capillaries were studied in inside-out patches from primary cultures of rat brain microvascular endothelial cells. At -40 mV membrane potential, open probability (Po) was activated by cytosolic [Ca2+] <1 μM and was half-maximal at ∼20 μM. Increasing [Ca2+] stimulated opening rate with little effect on closing rate. At constant [Ca2+], Po was voltage-dependent, and effective gating charge corresponded to 0.6 ∼ 0.1 unitary charges. Depolarization accelerated opening and slowed closing, thereby increasing apparent affinity for Ca2+. Within ∼1 min of excision, Po declined to a lower steady state with decreased sensitivity toward activating Ca2+ when studied at a fixed voltage, and toward activating voltage when studied at a fixed [Ca2+]. Deactivated channels opened ∼5-fold slower and closed ∼10-fold faster. The sulfhydryl-reducing agent dithiotreitol (1 mM) completely reversed acceleration of closing rate but failed to recover opening rate. Single-channel gating was complex; distributions of open and closed dwell times contained at least four and five exponential components, respectively. The longest component of the closed-time distribution was markedly sensitive to both [Ca2+] and voltage. We conclude that the biophysical properties of gating of this channel are remarkably similar to those of large-conductance Ca2+-activated K+ channels.

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