O2 deprivation inhibits Ca2+-activated K+ channels via cytosolic factors in mice neocortical neurons
Huajun Liu, Edward Moczydlowski, Gabriel G. Haddad
Huajun Liu, Edward Moczydlowski, Gabriel G. Haddad
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O2 deprivation inhibits Ca2+-activated K+ channels via cytosolic factors in mice neocortical neurons

Abstract

O2 deprivation induces membrane depolarization in mammalian central neurons. It is possible that this anoxia-induced depolarization is partly mediated by an inhibition of K+ channels. We therefore performed experiments using patch-clamp techniques and dissociated neurons from mice neocortex. Three types of K+ channels were observed in both cell-attached and inside-out configurations, but only one of them was sensitive to lack of O2. This O2-sensitive K+ channel was identified as a large-conductance Ca2+-activated K+ channel (BKCa), as it exhibited a large conductance of 210 pS under symmetrical K+ (140 mM) conditions, a strong voltage-dependence of activation, and a marked sensitivity to Ca2+. A low-O2 medium (PO2 = 10–20 mmHg) markedly inhibited this BKCa channel open probability in a voltage-dependent manner in cell-attached patches, but not in inside-out patches, indicating that the effect of O2 deprivation on BKCa channels of mice neocortical neurons was mediated via cytosol-dependent processes. Lowering intracellular pH (pHi), or cytosolic addition of the catalytic subunit of a cAMP-dependent protein kinase A in the presence of Mg-ATP, caused a decrease in BKCa channel activity by reducing the sensitivity of this channel to Ca2+. In contrast, the reducing agents glutathione and DTT increased single BKCa channel open probability without affecting unitary conductance. We suggest that in neocortical neurons, (a) BKCa is modulated by O2 deprivation via cytosolic factors and cytosol-dependent processes, and (b) the reduction in channel activity during hypoxia is likely due to reduced Ca2+ sensitivity resulting from cytosolic alternations such as in pHi and phosphorylation. Because of their large conductance and prevalence in the neocortex, BKCa channels may be considered as a target for pharmacological intervention in conditions of acute anoxia or ischemia.

Authors

Huajun Liu, Edward Moczydlowski, Gabriel G. Haddad

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Figure 5

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(a) Effect of hypoxia on single BKCa channel in a cell-attached patch fr...
(a) Effect of hypoxia on single BKCa channel in a cell-attached patch from a neocortical neuron. Current was recorded with high-KCl (140 mM) solution in the pipette and physiological solution in the bath at a Vm of –30 mV. The channel closed level is indicated by “C”. Three parts of the compressed trace are shown as indicated by numbers 1–3 (fast time resolution). (b) Effect of hypoxia on the voltage activation of BKCa channel under ionic condition just described. The line is fitted to a Boltzmann distribution. V0.5 shifted from –42.4 ± 4.8 mV to –18.6 ± 3.5 mV after exposure of hypoxia for 10 minutes. (c) Time course of the hypoxia-induced effect on NPo. In cell-attached recordings (filled squares), channel inhibition started about 5 minutes after the onset of hypoxia, and a maximum inhibition was reached in about 10 minutes. After that time, NPo was markedly reduced to about 43% of control level. Reoxygenation led to partial recovery. In inside-out recordings (filled circles), NPo was not significantly affected during hypoxia. (d) Continuous recording of a single BKCa channel current from an inside-out patch of a neocortical neuron during hypoxia, using a symmetrical 140 mM KCl on both sides of the membrane, with a Vm of –30 mV. The channel closed level is indicated by “C”. Two parts of the compressed trace (indicated by the numbers 1 and 2) are shown below at fast time resolution.