O(2) deprivation inhibits Ca(2+)-activated K(+) channels via cytosolic factors in mice neocortical neurons

Abstract

O(2) 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 O(2). This O(2)-sensitive K(+) channel was identified as a large-conductance Ca(2+)-activated K(+) channel (BK(Ca)), 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 Ca(2+). A low-O(2) medium (PO(2) = 10-20 mmHg) markedly inhibited this BK(Ca) channel open probability in a voltage-dependent manner in cell-attached patches, but not in inside-out patches, indicating that the effect of O(2) deprivation on BK(Ca) channels of mice neocortical neurons was mediated via cytosol-dependent processes. Lowering intracellular pH (pH(i)), or cytosolic addition of the catalytic subunit of a cAMP-dependent protein kinase A in the presence of Mg-ATP, caused a decrease in BK(Ca) channel activity by reducing the sensitivity of this channel to Ca(2+). In contrast, the reducing agents glutathione and DTT increased single BK(Ca) channel open probability without affecting unitary conductance. We suggest that in neocortical neurons, (a) BK(Ca) is modulated by O(2) deprivation via cytosolic factors and cytosol-dependent processes, and (b) the reduction in channel activity during hypoxia is likely due to reduced Ca(2+) sensitivity resulting from cytosolic alternations such as in pH(i) and phosphorylation. Because of their large conductance and prevalence in the neocortex, BK(Ca) channels may be considered as a target for pharmacological intervention in conditions of acute anoxia or ischemia.

Figure 1

Different types of K⁺ channels in isolated neocortical neurons of mice. Currents were recorded from inside-out patches of a neocortical neuron using symmetrical 140 mM KCl on both sides of the membrane at a V_m of –40mV. The channel closed level is indicated by “C”. (a) Example traces of small-conductance K⁺ channel. The amplitude histogram to the left was compiled from 40 seconds of current record digitized at 500 Hz. (b) Example traces and the amplitude histogram of the medium conductance K⁺ channel. (c) Example traces and the amplitude histogram of the large-conductance K⁺ channel. (d) Example traces containing multiple K⁺ channel types.

Figure 2

(a) Single large-conductance K⁺ currents recorded from an inside-out patch, recorded at various holding potentials in the presence of symmetrical 140 mM KCl across the neuronal membrane. (b) The current-voltage relationship of the large-conductance K⁺ channel under conditions already described. The line drawn through these points is a linear regression fit with a conductance of 210 ± 6.9 pS (n = 15). (c) Relationship between open probability and voltage for this large-conductance channel. The line is fitted to a Boltzmann distribution: y = 1/{1 + exp [(V_0.5 – V_m)/k]}, where y is normalized open probability (NP_o/NP_o.max), Vm is membrane potential, V_0.5 is the membrane potential at which NP_o is half of NP_o.max, and k is a constant related to the slope of curve. V_0.5 = –46 mV.

Figure 3

(a) Single-channel recordings of BK_Ca channel activity in the presence of varying concentration of Ca²⁺_i at a V_m of –40 mV. (b) Relationship between Ca²⁺_i and channel open probability at various membrane potentials. Data were fitted to Hill’s equation: y = 1/{1 + ([Ca²⁺]_i/k)^h}, where y is the normalized open probability (NP_o/NP_o.max), [Ca²⁺]_i is intracellular free Ca²⁺ concentration, k is the free Ca²⁺ concentration required for maximum channel activation, and h is the Hill coefficient.

Figure 4

(a) Currents recorded under symmetrical 140 mM KCl condition in an outside-out patch from a neocortical neuron at a V_m of 30 mV. TEA (0–1 mM), from the extracellular side, blocks the BK_Ca channel. (b) Effects of ChTX (100 nM) and IbTX (20 nM), also on the extracellular side of the BK_Ca channels. Currents were also recorded in an outside-out patch at a V_m of 30 mV. (c) Effect of DTX-I on single BK_Ca channels. Currents were recorded in an inside-out patch under symmetrical 140 mM KCl condition at a V_m of –40 mV. DTX-I (200–400 nM) induced a subconductance state with 72% of normal open-state current. Amplitude histogram to the upper trace was compiled from 60 seconds (DTX-I 200 nM) of current record digitized at 500 Hz. Amplitude peaks are identified as closed state (c), substate (s), or open state (o).

Figure 5

(a) Effect of hypoxia on single BK_Ca 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 V_m 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 BK_Ca channel under ionic condition just described. The line is fitted to a Boltzmann distribution. V_0.5 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 NP_o. 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, NP_o was markedly reduced to about 43% of control level. Reoxygenation led to partial recovery. In inside-out recordings (filled circles), NP_o was not significantly affected during hypoxia. (d) Continuous recording of a single BK_Ca 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 V_m 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.

Figure 6

Cytosolic pH modulates NP_o of BK_Ca channels in inside-out membrane patches from neocortical neurons. (a) Example from a patch. Currents were recorded at a V_m of –40mV. Changing the pH of the bath solution (intracellular side of the membrane) leads to marked changes in NP_o. (b) Relationship between pH_i and channel NP_o at a V_m of –40 mV. Data were fitted to Hill’s equation. The half-inhibition pH value was 6.42.

Figure 7

Reversibility of the pH effect on the BK_Ca channels. Currents were recorded from an inside-out patch of a neocortical neuron, using symmetrical 140 mM KCl on both sides of the membrane, V_m of –30 mV. (a) Single-channel trace showing inhibition by pH 5.6 and complete reactivation at pH 7.2. (b) Similar experiment, but where the reactivation was done by the addition of 5 mM Ca²⁺. (c) Mean change of NP_o, measured at different conditions. (d) Effect of pH (6.0 and 7.2) on the relation of Ca²⁺ and NP_o. Data were acquired from inside-out patches of neocortical neurons using symmetrical 140 mM KCl on both sides of the membrane, V_m of –40 mV, and fitted to Hill’s equation.

Figure 8

Effect of the catalytic subunit of PKA on the BK_Ca channel. Currents were recorded from inside-out patches of neocortical neurons, using symmetrical 140 mM KCl on both sides of the membrane, V_m of –30 mV. (a) Single-channel traces showing the inhibition by PKA 20 U/mL and reactivation upon washing out. (b) Similar experiment, where the reactivation was done by the addition of 5 mM Ca²⁺. (c) Mean change in NP_o, measured at different conditions. (d) Effect of 20 U/mL PKA on the relation between Ca²⁺ and NP_o. Data were acquired from inside-out patches of neocortical neurons using symmetrical 140 mM KCl on both sides of the membrane, V_m of –40 mV and fitted to Hill’s equation.

Figure 9

Effects of PKA and PKC activators on BK_Ca channel currents. Currents were recorded from a cell-attached patch of a neocortical neuron with high-KCl (140 mM) solution in the pipette and bathing outside solution, at a V_m of –20 mV. (a) Application of 0.1 mM db cAMP markedly decreased the single BK_Ca channel activity and recovered after washout. (b) Application of 100 nM PMA had no apparent effect on BK_Ca channel activity. (c) Mean change in NP_o, measured in different conditions.

Figure 10

Effect of the redox agents on the BK_Ca channel. Currents were recorded from inside-out patches of neocortical neurons, using symmetrical 140 mM KCl on both sides of the membrane, V_m of –30 mV. (a) Application of 1 mM GSH markedly increased the single BK_Ca channel activity, and this activity persisted after washout. (b) Application of 1 mM GSSG had no apparent effect on BK_Ca channel activity. (c) The increase in channel activity caused by GSH (1 mM) recovered to control level when GSSG was applied. (d) Reducing agent DTT (1 mM) markedly augmented BK_Ca channel activity, which was reversed by the oxidizing agent DTNB (1 mM). (e) DTNB (1 mM) had no significant effect on the BK_Ca channel. (f) Mean change in NP_o, measured at different conditions.