The influence of membrane potential on chloride channels activated by GABA in rat cultured hippocampal neurons

Chloride currents were activated by a low concentration of GABA (0.5 m) in neonatal rat hippocampal neurons cultured for up to 14 days. Currents elicited by 0.5 m GABA in neurons, voltage-clamped using the whole-cell technique with pipettes containing 149 mm Cl^–, reversed close to 0 mV whether pipettes contained 144 mm Na⁺ or 140 mm Cs⁺, and were blocked by 100 m bicuculline. Current-voltage curves showed outward rectification. Single channel currents appeared in cell-attached patches when the pipette tip was perfused with pipette solution containing 0.5 m GABA and disappeared when a solution containing 100 m bicuculline plus 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification and, in some patches, had a much lower probability of opening at hyperpolarized potentials. The average chord conductance in 10 patches hyperpolarized by 80 mV was 7.8±1.6 pS (sem) compared with a chord conductance of 34.1±3.5 pS (sem) in the same patches depolarized by 80 mV. Similar single channel currents were also activated in cell-free, inside-out patches in symmetrical chloride solutions when 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification similar to that seen in cell-attached patches, and some channels had a lower probability of opening at hyperpolarized potentials. The average chord conductance in 13 patches hyperpolarized by 80 mV was 11.8±2.3 pS (sem) compared with 42.1±3.1 pS (sem) in the same patches depolarized by 80 mV.We are grateful to B. McLachlan and M. Robertson for their general assistance, to C. McCulloch and M. Smith for writing computer programs and to W. O'Hare for making the pipette injection device.     

Exogenous GABA persistently opens Cl− channels in cultured embryonic rat thalamic neurons

We recorded whole-cell Cl^– currents in cultured embryonic rat thalamic neurons by brief applications of GABA or the structural analogue muscimol. In 17 of 141 neurons (12%) the Cl^– current persisted for a minute or more after the pipette was removed from the bath. Cl^– current never persisted after muscimol exposure even in those cells exhibiting persistent GABA-activated currents (PGC). The half decay times (T₅₀) of PGCs were exponentially and asymptotically related to the duration of GABA exposure and could be interrupted or completely aborted by low-pressure application of saline. PGCs were insensitive to membrane potential, to Tiagabine, a nipecotic acid analogue known to block GABA uptake, and persisted in Ca _o ²⁺ -free medium. Fluctuation analysis revealed that PGCs exhibited inferred Cl^– channel properties whose kinetic components and estimated average elementary conductance showed no significant difference from those estimated during GABA exposure. The relative contribution of low frequency components was consistently reduced and that of high frequency components modestly increased during PGC compared to those recorded during GABA exposure. Taken together, the results suggest the existence of a superficial compartment in these embryonic neurons that can momentarily accumulate and release exogenous GABA.We thank Dr. Wu Ma for advice in isolating thalamic neurons. Tiagabine is a kind gift from Novo Nordisk A/S, Denmark.     

Conductance states activated by glycine and GABA in rat cultured spinal neurones

Summary The conductance properties of single Cl^– channels activated by glycine and gamma-aminobutyric acid (GABA) were examined in rat spinal cord neurones grown in cell culture. The majority (85%) of spinal neurones were sensitive to both glycine and GABA as were most (83%) outside-out patches tested. Glycine and GABA activated multiple conductance state Cl^– channels with linear current-voltage properties when the chloride activities of the solutions bathing both sides of the membrane were similar. Glycine activated six distinct conductance states with conductances of 14, 20, 30, 43, 64 and 93 pS, whereas GABA activated five states with conductances of 13, 20, 29, 39 and 71 pS. The 30 and 43 pS states and the 20 and 29 pS states were observed most frequently with glycine and GABA, respectively. As the values of the glycine- and GABA-activated conductance states form a geometric progression when arranged in ascending order, we concluded that the channels do not consist of a cluster of identical pores. Additional conductance states (50 and 100 pS) were activated by glycine occasionally. The similarity between the conductances of the states activated by the two transmitters is consistent with the proposal that they both activate the same type of Cl^– channel.     

Interaction between pentobarbital and GABA-activated ionic channels in rat cerebellar neurons

Using techniques of voltage clamping at the membrane, intracellular perfusion, and concentration clamping, GABA- and barbiturate-activated currents were investigated in single neurons isolated from the rat cerebellum. The dissociation constant for interaction between GABA and GABA receptors was measured at 3±0.8 × 10^–5 M. The presence of pentobarbital in the bathing solution exerts a potentiating effect on GABA-induced conductance in isolated neurons, shifting the dose-response curve for GABA towards lower concentration values without increasing peak chloride conductance. The concentrations at which GABA effects are potentiated range between 10^–6–10^–4 M. High concentrations of pentobarbital inhibit GABA-activated conductance; at concentrations in excess of 5 × 10^–4 M, it also brings about activation of chloride conductance, depressed by bicuculline and picrotoxin, in the absence of GABA. A short-term increase in membrane conductance is produced by rapid pentobarbital washout.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 1, pp. 93–98, January–February, 1990.     

Physiological role of dendrotoxin-sensitive K+ channels in the rat cerebellar Purkinje neurons

To understand the contribution of potassium (K+) channels, particularly alpha-dendrotoxin (D-type)-sensitive K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits), to the generation of neuronal spike output we must have detailed information of the functional role of these channels in the neuronal membrane. Conventional intracellular recording methods in current clamp mode were used to identify the role of alpha-dendrotoxin (alpha-DTX)-sensitive K+ channel currents in shaping the spike output and modulation of neuronal properties of cerebellar Purkinje neurons (PCs) in slices. Addition of alpha-DTX revealed that D-type K+ channels play an important role in the shaping of Purkinje neuronal firing behavior. Repetitive firing capability of PCs was increased following exposure to artificial cerebrospinal fluid (aCSF) containing alpha-DTX, so that in response to the injection of 0.6 nA depolarizing current pulse of 600 ms, the number of action potentials insignificantly increased from 15 in the presence of 4-AP to 29 action potentials per second after application of DTX following pretreatment with 4-AP. These results indicate that D-type K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits) may contribute to the spike frequency adaptation in PCs. Our findings suggest that the activation of voltage-dependent K+ channels (D and A types) markedly affect the firing pattern of PCs.     

On the kinetics of large-conductance glutamate-receptor ion channels in rat cerebellar granule neurons

Ion channels activated by glutamate, aspartate, and N-methyl-D-aspartate (NMDA) have been investigated in outside-out patches from cultured cerebellar granule neurons of the rat. Openings of these channels occur in bursts, within which the individual openings are separated by brief shuttings or gaps. The shut-time distributions obtained with each agonist were fitted with four exponential components. The briefest two components were considered as 'gaps within bursts'. Their mean time-constants were: glutamate, 58.0 microseconds and 592 microseconds; aspartate, 31.3 microseconds and 644 microseconds; NMDA, 40.5 microseconds and 903 microseconds. Distributions of burst durations were fitted with three exponential components. The mean time-constants obtained for the longest two components were: glutamate, 1.33 ms and 10.5 ms; aspartate, 2.15 ms and 10.3 ms; NMDA, 2.42 ms and 10.5 ms. Evidence is given that these two components of burst duration reflect the gating kinetics of 50 pS openings and not the fact that each agonist produces openings to more than one conductance level. Not only do openings occur in bursts, but these bursts were observed to occur in clusters, which can be hundreds of milliseconds long. We discuss the relation between the kinetics of single-channel openings observed in patches and the spectral components detected in whole-cell current noise.     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

Ion channels activated by light in Limulus ventral photoreceptors

The light-activated conductance of Limulus ventral photoreceptors was studied using the patch-clamp technique. Channels (40 pS) were observed whose probability of opening was greatly increased by light. In some cells the latency of channel activation was nearly the same as that of the macroscopic response, while in other cells the channel latency was much greater. Like the macroscopic conductance, channel activity was reduced by light adaptation but enhanced by the intracellular injection of the calcium chelator EGTA. The latter observation indicates that channel activation was not a secondary result of the light-induced rise in intracellular calcium. A two-microelectrode voltage-clamp method was used to measure the voltage dependence of the light-activated macroscopic conductance. It was found that this conductance is constant over a wide voltage range more negative than zero, but it increases markedly at positive voltages. The single channel currents measured over this same voltage range show that the single channel conductance is independent of voltage, but that channel gating properties are dependent on voltage. Both the mean channel open time and the opening rate increase at positive voltages. These properties change in a manner consistent with the voltage dependence of the macroscopic conductance. The broad range of similarities between the macroscopic and single channel currents supports the conclusion that the 40-pS channel that we have observed is the principal channel underlying the response to light in these photoreceptors.     

Ion channels in rabbit cultured fibroblasts

Large outward currents are recorded with the whole-cell patch-clamp technique on depolarization of rabbit cultured fibroblasts. Our findings suggest that these outward currents consist of two voltage-dependent components, one of which also depends on cytoplasmic calcium concentration. Total replacement of external Cl- by the large anion ascorbate does not affect the amplitude of the currents, indicating that both components must be carried by K+. Consistent with these findings with whole-cell currents, in single channel recordings from fibroblasts we found that most patches contain high-conductance potassium-selective channels whose activation depends on both membrane potential and the calcium concentration at the cytoplasmic surface of the membrane. In a smaller number of patches, a second population of high-conductance calcium-independent potassium channels is observed having different voltage-dependence. The calcium- and voltage-dependence suggest that these two channels correspond with the two components of outward current seen in the whole-cell recordings. The single channel conductance of both channels in symmetrical KCl (150 mM) is 260-270 pS. Both channels are highly selective for K+ over both Na+ and Cl-. The conductance of the channels when outward current is carried by Rb+ is considerably smaller than when it is carried by K+. Some evidence is adduced to support the hypothesis that these potassium channel populations may be involved in the control of cell proliferation.     

Single-channel currents activated by low intracellular pH in cultured hippocampal neurons of rat.

Patch clamp technique was applied to the plasma membrane of cultured hippocampal neurons of rat. Elementary currents of a cation-selective channel were elicited by low intracellular pH (pHi 3.5-4.5). Channel activity starts with 1-2 min delay from the application of low pHi, and persists upon restoration of physiological pH conditions. The channel has a conductance of approx. 110 pS in symmetrical 300 mM NaCl, and is strongly selective for cations over anions. The channel is active over the whole voltage range tested (from +75 mV to -75 mV). Mean open time is function of voltage, increasing with depolarization. Low pH applied extracellularly did not activate the channel.     

Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle

The conductance and selectivity of the Ca-activated K channel in cultured rat muscle was studied. Shifts in the reversal potential of single channel currents when various cations were substituted for Ki+ were used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The selectivity was Tl+ greater than K+ greater than Rb+ greater than NH4+, with permeability ratios of 1.2, 1.0, 0.67, and 0.11. Na+, Li+, and Cs+ were not measurably permeant, with permeabilities less than 0.05 that of K+. Currents with the various ions were typically less than expected on the basis of the permeability ratios, which suggests that the movement of an ion through the channel was not independent of the other ions present. For a fixed activity of Ko+ (77 mM), plots of single channel conductance vs. activity of Ki+ were described by a two-barrier model with a single saturable site. This observation, plus the finding that the permeability ratios of Rb+ and NH+4 to K+ did not change with ion concentration, is consistent with a channel that can contain a maximum of one ion at any time. The empirically determined dissociation constant for the single saturable site was 100 mM, and the maximum calculated conductance for symmetrical solutions of K+ was 640 pS. TEAi+ (tetraethylammonium ion) reduced single channel current amplitude in a voltage-dependent manner. This effect was accounted for by assuming voltage-dependent block by TEA+ (apparent dissociation constant of 60 mM at 0 mV) at a site located 26% of the distance across the membrane potential, starting at the inner side. TEAo+ was much more effective in reducing single channel currents, with an apparent dissociation constant of approximately 0.3 mM.     

Differentiated expression inXenopus oocytes of calcium channels from rat cerebellar and forebrain neurons

Calcium channels were expressed inXenopus laevis oocytes by means of matrix RNA (mRNA) extracted from the cerebellum (RNAc) and forebrain (RNAfb). In these oocytes, inward barium currents,I _Ba, evoked by 40 mM Ba²⁺ were investigated using a double microelectrode technique. Currents expressed after injection of both RNAc and RNAfb (further referred to as RNAc- and RNAfb-expressed currents) showed a voltage-dependent characteristic typical of high-threshold calcium channels of mammalian neurons. The threshold of activation was about –40 mV, the maximum amplitude was observed at +20 mV and reversal potential at +60 mV. In both groups of oocytes, no expression of low- or high-threshold calcium channels of other types was observed. Although in both cases the expression ofI _Ba had similar macrokinetics, characteristics of their stationary inactivation differed. The half-inactivation potential ranged between –32 and –16 mV, and the slope factor was 28 and 16.6 mV in RNAfb- and RNAc-injected oocytes, respectively. In both cases,I _Ba were insensitive to dihydropyridines; however their relation to other pharmacological agents was different. RNAfb-expressedI _Ba was completely blocked by Cd²⁺ (K _d=10 µM) and depressed up to 70% by -conotoxin (1 µM), being insensitive to either whole spider toxin fromAgelenopsis aperta venom or to its FTX fraction. On the contrary, RNAc-expressedI _Ba was more sensitive to Cd²⁺ (K _d=0.1 µM), stable to -conotoxin, and suppressed up to 75–90% by wholeA. aperta toxin in a dilution of 1:10000, and by FTX at a concentration of 0.5 µM. The findings allow us to suggest that the forebrain and cerebellum of mammals are the structures, whose mRNA differ and provide predominant expression of voltage-dependent calcium channels of N- and P-types, respectively.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 427–436, November–December, 1994.     

Junctional and extrajunctional membrane channels activated by GABA in locust muscle fibres

Iontophoretic application of GABA to voltage-clamped locust muscle fibres has demonstrated the presence of both extrajunctional and junctional GABA receptors. Extrajunctional GABA receptors are distinct from extrajunctional glutamate receptors which also occur in these muscle fibres. Inward GABA currents are nonlinearly dependent on membrane potential. Analysis of membrane current noise produced by iontophoretic GABA application shows that for junctional and extrajunctional GABA receptors the mean channel lifetime is 3-4 ms and the single-channel conductance is approximately 22 pS at - 80 mV (T = 21 degrees C). The mean lifetime as previously demonstrated for glutamate-sensitive excitatory channels in locust muscle fibres.     

Polyamine uptake in cultured cerebellar granule neurons

In the present study, we have examined the transport of polyamines in cultured cerebellar granule cells. Our results suggest the existence of two different transporters for polyamines in these neurons. Putrescine and spermidine uptake (K ap m = 2.17 and 1.39 microM, respectively), were affected when extracellular sodium was replaced with choline (about 30% inhibition over controls) or sucrose (about 2.5-fold potentiation over controls). By contrast, the substitution of sodium by choline or sucrose did not modify spermine uptake (K ap m = 13.53 microM) in cerebellar granule cells. Accordingly, alteration of membrane potential with ouabain was able to block putrescine (50% inhibition) and spermidine (60% inhibition) uptake but not spermine uptake. These results indicate that putrescine and spermidine transport in cerebellar granule cells is membrane potential dependent, whereas spermine uptake is not modulated by membrane potential.     

Characterization of glutamate toxicity in cultured rat cerebellar granule neurons at reduced temperature

We have defined conditions whereby glutamate becomes toxic to isolated cerebellar granule neurons in a physiologic salt solution (pH 7.4). In the presence of a physiologic Mg⁺⁺ concentration, acute glutamate excitotoxicity manifests only when the temperature was reduced from 37°C to 22°C. In contrast to glutamate, N-methyl-D-aspartate (NMDA) was non-toxic at either temperature at concentrations as high as 1 mM. Glycine strongly potentiated both the potency and efficacy of glutamate but revealed only a modest NMDA response. The non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxalinedione (CNQX), potently protected against glutamate challenge, although the contribution of antagonism at strychnine-insensitive glycine sites could not be excluded. To further characterize the non-NMDA receptor contribution to the excitotoxic response, the promiscuity of glutamate interaction with ionotropic receptors was simulated by exposing neurons to NMDA in the presence of non-NMDA receptor agonists. NMDA toxicity was potentiated four- to sevenfold when non-NMDA receptors were coactivated by a subtoxic concentration of AMPA, kainate, or domoate. These results suggest that non-NMDA receptor activation participates in the mechanism of acute glutamate toxicity by producing neuronal depolarization (via sodium influx), which in turn promotes the release of the voltage-dependent magnesium blockade of NMDA receptor ion channels. © 1997 John Wiley & Sons, Inc.     

GABA-induced potassium channels in cultured neurons

When gamma-aminobutyric acid (GABA) or baclofen were applied to cultured rat hippocampal neurons, single-channel potassium currents appeared after a delay of 30 s or more in patches of membrane on the cell surface isolated from the agonists by the recording pipette. The appearance of currents in patches not exposed to agonist, the delay in their appearance and the suppression of currents in cells pre-incubated with pertussis toxin indicate the involvement of an intracellular second messenger system. The channels were associated with a GABAB receptor rather than a GABAA receptor as they were blocked by baclofen, a GABAB antagonist, but were not affected by bicuculline, a GABAA antagonist. A feature of the single channel currents was their variable amplitude: they had a maximum conductance of ca. 70 pS and displayed many lower conductance states that were integral multiples of 5-6 pS. In several cells exposed to GABA or baclofen, first small currents and then progressively larger currents appeared: current amplitude was a multiple of an elementary current. It is suggested that binding of GABA to GABAB receptors activates a second messenger system causing opening of oligomeric potassium channels.     

GAD and GABA in an enriched population of cultured GABAergic neurons from rat cerebral cortex

To study various aspects of GABAergic metabolism in an easily accessible system, dissociated cells from postnatal rat cerebral cortex were cultured in a serum-based medium and characterized morphologically and biochemically. The majority (70–90%) of the neurons were GABAergic as determined by three double-labeling procedures. The specific activity of glutamine synthetase in the cultures was 4–5% of the levels in rat astrocyte cultures and intact rat brain, indicating that glia were a minor component. The developmental increase of GABA levels preceded the increase of GAD activity in both immunocytochemical and biochemical experiments. GABA turnover rates also increased with culture age and were 20–30% of GAD activity. Four anti-GAD antibodies, which recognize GAD subunits with differing molecular masses to varying degrees, were used to stain cultured neurons and make immunoblots. Immunoblots showed that the neurons contained two major subunits of GAD which differed in mass by 2 kDa. All four antibodies immunostained both neuronal perikarya and neurites but one antibody, which on the immunoblots predominantly labeled the GAD protein with the lower molecular weight, showed a somewhat more pronounced punctate staining, possibly indicating a principal localization to neurites.