Desensitization of ligand-gated ion channels plays a critical role for the information transfer between neurons. The current view on γ-aminobutyric acid (GABA)A and glycine receptors includes significant rapid components of desensitization as well as cross-desensitization between the two receptor types. Here, we analyze the mechanism of apparent cross-desensitization between native GABAA and glycine receptors in rat central neurons and quantify to what extent the current decay in the presence of ligand is a result of desensitization versus changes in intracellular Cl− concentration ([Cl−]i). We show that apparent cross-desensitization of currents evoked by GABA and by glycine is caused by changes in [Cl−]i. We also show that changes in [Cl−]i are critical for the decay of current in the presence of either GABA or glycine, whereas changes in conductance often play a minor role only. Thus, the currents decayed significantly quicker than the conductances, which decayed with time constants of several seconds and in some cells did not decay below the value at peak current during 20-s agonist application. By taking the cytosolic volume into account and numerically computing the membrane currents and expected changes in [Cl−]i, we provide a theoretical framework for the observed effects. Modeling diffusional exchange of Cl− between cytosol and patch pipettes, we also show that considerable changes in [Cl−]i may be expected and cause rapidly decaying current components in conventional whole cell or outside-out patch recordings. The findings imply that a reevaluation of the desensitization properties of GABAA and glycine receptors is needed.

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A Ca2+‐activated K+ current was identified in neurons from the rat medial preoptic nucleus. Its functional role for the resting potential and for impulse generation was characterised by using the reversible blocking agent bicuculline methiodide. Acutely dissociated neurons were studied by perforated‐patch recordings. The effect of bicuculline methiodide was investigated under voltage‐clamp conditions to clearly identify the current affected. At membrane potentials > ‐50 mV, bicuculline methiodide rapidly (< 1 s) and reversibly blocked a steady outward current. Half‐saturating concentration was 12 μm. The current amplitude increased with potential in the range ‐50 to 0 mV. The bicuculline‐sensitive current was identified as an apamin‐sensitive, Ca2+‐dependent K+ current. It was neither affected by the GABAA receptor blocker picrotoxin (100 μm) nor by a changed pipette Cl− concentration, but was affected by substitution of extracellular K+ for Na+. The current was dependent on extracellular Ca2+ and was sensitive to 1 μm apamin but not to 200 nm charybdotoxin. A role for the Ca2+‐dependent K+ current in setting the resting potential and controlling spontaneous firing frequency was observed under current‐clamp conditions. Bicuculline methiodide (100 μm) induced a positive shift (5 ± 1 mV; n= 18 of resting potential in all neurons tested. In the majority of spontaneously firing neurons, the firing frequency was reversibly affected, either increased or decreased depending on the cell, by bicuculline methiodide.

The aim of the present study was to clarify the identity of slow spontaneous currents, the underlying mechanism and possible role for impulse generation in neurons of the rat medial preoptic nucleus (MPN). Acutely dissociated neurons were studied with the perforated patch-clamp technique. Spontaneous outward currents, at a frequency of approximately 0.5 Hz and with a decay time constant of approximately 200 ms, were frequently detected in neurons when voltage-clamped between approximately -70 and -30 mV. The dependence on extracellular K(+) concentration was consistent with K(+) as the main charge carrier. We concluded that the main characteristics were similar to those of spontaneous miniature outward currents (SMOCs), previously reported mainly for muscle fibers and peripheral nerve. From the dependence on voltage and from a pharmacological analysis, we concluded that the currents were carried through small-conductance Ca(2+)-activated (SK) channels, of the SK3 subtype. From experiments with ryanodine, xestospongin C, and caffeine, we concluded that the spontaneous currents were triggered by Ca(2+) release from intracellular stores via ryanodine receptor channels. An apparent voltage dependence was explained by masking of the spontaneous currents as a consequence of steady SK-channel activation at membrane potentials > -30 mV. Under current-clamp conditions, corresponding transient hyperpolarizations occasionally exceeded 10 mV in amplitude and reduced the frequency of spontaneous impulses. In conclusion, MPN neurons display spontaneous hyperpolarizations triggered by Ca(2+) release via ryanodine receptors and SK3-channel activation. Thus such events may affect impulse firing of MPN neurons.

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The present study was conducted to characterize possible rapid effects of 17-β-estradiol on voltage-gated K+ channels in preoptic neurons and, in particular, to identify the mechanisms by which 17-β-estradiol affects the K+ channels. Whole-cell currents from dissociated rat preoptic neurons were studied by perforated-patch recording. 17-β-estradiol rapidly (within seconds) and reversibly reduced the K+ currents, showing an EC50 value of 9.7 µM. The effect was slightly voltage dependent, but independent of external Ca2+, and not sensitive to an estrogen-receptor blocker. Although 17-α-estradiol also significantly reduced the K+ currents, membrane-impermeant forms of estradiol did not reduce the K+ currents and other estrogens, testosterone and cholesterol were considerably less effective. The reduction induced by estradiol was overlapping with that of the KV-2-channel blocker r-stromatoxin-1. The time course of K+ current in 17-β-estradiol, with a time-dependent inhibition and a slight dependence on external K+, suggested an open-channel block mechanism. The properties of block were predicted from a computational model where 17-β-estradiol binds to open K+ channels. It was concluded that 17-β-estradiol rapidly reduces voltage-gated K+ currents in a way consistent with an open-channel block mechanism. This suggests a new mechanism for steroid action on ion channels.

The relation between current and voltage, I-V relation, is central to functional analysis of membrane ion channels. A commonly used method, since the introduction of the voltage-clamp technique, to establish the I-V relation depends on the interpolation of current amplitudes recorded at different steady voltages. By a theoretical computational approach as well as by experimental recordings from GABAA-receptor mediated currents in mammalian central neurons, we here show that this interpolation method may give reversal potentials and conductances that do not reflect the properties of the channels studied under conditions when ion flux may give rise to concentration changes. Therefore, changes in ion concentrations may remain undetected and conclusions on changes in conductance, such as during desensitization, may be mistaken. In contrast, an alternative experimental approach, using rapid voltage ramps, enable I-V relations that much better reflect the properties of the studied ion channels.

Maintenance of a low intraneuronal Cl– concentration, [Cl–]i, is critical for inhibition in the CNS. Here, the contribution of passive, conductive Cl– flux to recovery of [Cl–]i after a high load was analyzed in mature central neurons from rat. A novel method for quantifying the resting Cl– conductance, important for [Cl–]i recovery, was developed and the possible contribution of GABAA and glycine receptors and of ClC-2 channels to this conductance was analyzed. The hypothesis that spontaneous, action potential-independent release of GABA is important for [Cl–]i recovery was tested. [Cl–]i was examined by gramicidin-perforated patch recordings in medial preoptic neurons. Cells were loaded with Cl– by combining GABA or glycine application with a depolarized voltage, and the time course of [Cl–]i was followed by measurements of the Cl– equilibrium potential, as obtained from the current recorded during voltage ramps combined with GABA or glycine application. The results show that passive Cl– flux contributes significantly, in the same order of magnitude as does K+-Cl– cotransporter 2 (KCC2), to [Cl–]i recovery and that Cl– conductance accounts for ∼ 6% of the total resting conductance. A major fraction of this resting Cl– conductance is picrotoxin (PTX)-sensitive and likely due to open GABAA receptors, but ClC-2 channels do not contribute. The results also show that when the decay of GABAA receptor-mediated miniature postsynaptic currents (minis) is slowed by the neurosteroid allopregnanolone, such minis may significantly quicken [Cl–]i recovery, suggesting a possible steroid-regulated role for minis in the control of Cl– homeostasis.