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KEY POINTS Physiologically relevant combinations of recombinant GABAA receptor (GABAR) subunits were expressed in HEK293 cells. Using whole-cell voltage clamp and rapid drug application, we measured the GABAR-subtype-specific properties to convey either synaptic or extrasynaptic signalling in a range of physiological contexts. α4βδ GABARs are optimally tuned to submicromolar tonic GABA and transient surges of micromolar GABA concentrations. α5β2γ2l GABARs are better suited to higher tonic GABA levels, but also convey robust responses to brief synaptic and perisynaptic GABA fluctuations. α1β2/3δ GABARs function well at prolonged, micromolar (>2 μm) GABA levels, but not to low tonic (

KEY POINTS: Physiologically relevant combinations of recombinant GABA(A) receptor (GABAR) subunits were expressed in HEK293 cells. Using whole‐cell voltage clamp and rapid drug application, we measured the GABAR‐subtype‐specific properties to convey either synaptic or extrasynaptic signalling in a range of physiological contexts. α4βδ GABARs are optimally tuned to submicromolar tonic GABA and transient surges of micromolar GABA concentrations. α5β2γ2l GABARs are better suited to higher tonic GABA levels, but also convey robust responses to brief synaptic and perisynaptic GABA fluctuations. α1β2/3δ GABARs function well at prolonged, micromolar (>2 μm) GABA levels, but not to low tonic (

A GABA(A) receptor α6 subunit mutation, R46W, was identified as a susceptibility gene that may contribute to the pathogenesis of childhood absence epilepsy (CAE), but the molecular basis for alteration of GABA(A) receptor function is unclear. The R46W mutation is located in a region homologous to a GABA(A) receptor γ2 subunit missense mutation, R82Q, that is associated with CAE and febrile seizures in humans. To determine how this mutation reduces GABAergic inhibition, we expressed wild-type (α6β2γ2L and α6β2δ) and mutant (α6(R46W)β2γ2L and α6(R46W)β2δ) receptors in HEK 293T cells and characterize their whole-cell and single-channel currents, and surface and total levels. We demonstrated that gating and assembly of both α6(R46W)β2γ2L and α6(R46W)β2δ receptors were impaired. Compared to wild-type currents, α6(R46W)β2γ2L and α6(R46W)β2δ receptors had a reduced current density, α6(R46W)β2γ2L currents desensitized to a greater extent and deactivated at a slower rate, α6(R46W)β2δ receptors did not desensitize but deactivated faster and both α6(R46W)β2γ2L and α6(R46W)β2δ single-channel current mean open times and burst durations were reduced. Surface levels of coexpressed α6(R46W), β2 and δ, but not γ2L, subunits were decreased. 'Heterozygous' coexpression of α6(R46W) and α6 subunits with β2 and γ2L subunits produced intermediate macroscopic current amplitudes by increasing incorporation of wild-type and decreasing incorporation of mutant subunits into receptors trafficked to the surface. Finally, these findings suggest that similar to the γ2(R82Q) mutation, the CAE-associated α6(R46W) mutation could cause neuronal disinhibition and thus increase susceptibility to generalized seizures through a reduction of αβγ and αβδ receptor function and expression.

Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking.

The time course of inhibitory postsynaptic currents (IPSCs) reflects GABAA receptor deactivation, the process of current relaxation following transient activation. Fast desensitization has been demonstrated to prolong deactivation, and these processes have been described as being ‘coupled’. However, the relationship between desensitization and deactivation remains poorly understood. We investigated the ‘uncoupling’ of GABAA receptor macroscopic desensitization and deactivation using experimental conditions that affected these two processes differently. Changing agonist affinity preferentially altered deactivation, changing agonist concentration preferentially altered macroscopic desensitization, and a pore domain mutation prolonged deactivation despite blocking fast desensitization. To gain insight into the mechanistic basis for coupling and uncoupling, simulations were used to systematically evaluate the interplay between agonist affinity, gating efficacy, and desensitized state stability in shaping macroscopic desensitization and deactivation. We found that the influence of individual kinetic transitions on macroscopic currents depended not only on model connectivity, but also on the relationship among transitions within a given model. In addition, changing single rate constants differentially affected macroscopic desensitization and deactivation, thus providing parsimonious kinetic explanations for experimentally observed uncoupling. Finally, these findings permitted development of an algorithmic framework for kinetic interpretation of experimental manipulations that alter macroscopic current properties.

Up-regulation of the GABAA receptor alpha4 subunit subtype has been consistently shown in multiple animal models of chronic epilepsy. This isoform is expressed in both thalamus and hippocampus and is likely to play a significant role in regulating corticothalamic and hippocampal rhythms. However, little is known about its physiological properties, thus limiting understanding of the role of alpha4 subtype-containing GABAA receptors in normal and abnormal physiology. We used rapid GABA application to recombinant GABAA receptors expressed in HEK293T cells to compare the macroscopic kinetic properties of alpha4beta3gamma2L receptors to those of the more widely distributed alpha1beta3gamma2L receptors. These receptor currents had similar peak current amplitudes and GABA EC50 values. However, alpha4beta3gamma2L currents activated more slowly when exposed to submaximal GABA concentrations, had more fast desensitization (tau = 15-100 ms), and had less residual current during long GABA applications. In addition, alpha4beta3gamma2L currents deactivated more slowly than alpha1beta3gamma2L currents. Peak currents evoked by repetitive, brief GABA applications were more strongly attenuated for alpha4beta3gamma2L currents than alpha1beta3gamma2L currents. Moreover, the time required to recover from desensitization was prolonged in alpha4beta3gamma2L currents compared to alpha1beta3gamma2L currents. We also found that exposure to prolonged low levels of GABA, similar to those that might be present in the extrasynaptic space, greatly suppressed the response of alpha4beta3gamma2L currents to higher concentrations of GABA, while alpha1beta3gamma2L currents were less affected by exposure to low levels of GABA. Taken together, these data suggest that alpha4beta3gamma2L receptors have unique kinetic properties that limit the range of GABA applications to which they can respond maximally. While similar to alpha1beta3gamma2L receptors in their ability to respond to brief and low frequency synaptic inputs, alpha4beta3gamma2L receptors are less efficacious when exposed to prolonged tonic GABA or during repetitive stimulation, as may occur during learning and seizures.

1. To determine their contributions to GABAA receptor (GABAR) channel properties, rat gamma 2L and delta subunits were acutely co-expressed with alpha 1 and beta 3 subtypes in mouse L929 fibroblasts to produce alpha 1 beta 3, alpha 1 beta 3 delta or alpha 1 beta 3 gamma 2L GABAR isoforms. 2. With whole-cell recording, the alpha 1 beta 3 isoform had relatively high sensitivity to GABA (EC50 2.1 microM) and low maximum current amplitude. The alpha 1 beta 1 delta isoform had similar sensitivity to GABA (EC50 2.8 microM) and low current amplitude. The alpha 1 beta 3 gamma 2L isoform had lower sensitivity to GABA (EC50 11.6 microM) and higher maximum current amplitude. 3. The single channel conductance of alpha 1 beta 3 channels was low (13 pS) compared with that of alpha 1 beta 3 delta and alpha 1 beta 3 gamma 2L channels (27 pS). 4. The single channel kinetic properties of the channels also differed. The alpha 1 beta 3 gamma 2L channel exhibited three open states, while the alpha 1 beta 3 and alpha 1 beta 3 delta channels exhibited only two open states with mean dwell times similar to those of the two shorter open states of the alpha 1 beta 3 gamma 2L channel. All three channels exhibited at least five closed states. Bursts of alpha 1 beta 3 delta channels consisted primarily of only one or two openings, while those of alpha 1 beta 3 channels contained multiple openings. alpha 1 beta 3 gamma 2L channels exhibited burst kinetics typical for native GABARs with several long openings per burst. 5. These results show that the alpha 1 beta 3 heterodimer formed a GABA-sensitive channel with complex gating kinetics. Addition of a delta subunit to form the alpha 1 beta 3 delta heterotrimer altered the single channel conductance and the kinetic properties of the closed components, but did not affect the GABA sensitivity of the receptor nor the open state kinetics. In contrast, addition of a gamma subunit (gamma 2L subtype) to produce the alpha 1 beta 3 gamma 2L heterotrimer affected the GABA sensitivity, channel conductance and kinetic properties of both open and closed states.

1. The effects of the benzodiazepine receptor agonist, diazepam (DZ), and the inverse agonist, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), on gamma-aminobutyric acid (GABAA) receptor single channel currents were characterized. Outside-out patches were obtained from somata of cultured mouse spinal cord neurones and voltage clamped at -75 mV (ECl = 0 mV). 2. GABA (2 microM) alone or with DZ (20-1000 nM) or DMCM (20-100 nM) was applied to patches by pressure ejection from blunt micropipettes. DZ enhanced GABAA receptor currents with an inverted U-shaped concentration-response curve. Mean steady-state currents were increased by low concentrations of DZ (20-50 nM). At higher concentrations of DZ, the enhancement was diminished. Mean steady-state currents were decreased by DMCM at all concentrations. 3. GABAA receptor channels opened most frequently to a 27 pS main conductance level and less frequently to a 19 pS subconductance level. Neither DZ nor DMCM altered the proportion of time spent at either of the conductance levels. The kinetic properties of the main conductance level were studied. 4. Neither DZ nor DMCM altered the mean GABAA receptor channel open or burst durations. Sums of three exponential functions were required to fit best open and burst duration-frequency histograms for GABA alone or with DZ or DMCM. No significant changes in the three time constants or areas of the three exponential functions for open or burst duration histograms were produced by DZ or DMCM. 5. With increasing concentrations of DZ up to 50 nM, GABA evoked an increased frequency of channel openings and bursts. With higher DZ concentrations, the magnitudes of the increase in channel opening and burst frequencies were reduced. At all concentrations of DMCM, GABA evoked a decreased frequency of channel openings and bursts. 6. Closed duration-frequency histograms for GABA alone or with DZ or DMCM were best fitted by sums of at least six exponential functions. The three shortest closed duration time constants were unchanged by DZ or DMCM. The three longest closed duration time constants were altered by DZ and DMCM, consistent with alterations in opening frequency. 7. DZ increased and DMCM decreased steady-state GABAA receptor current by increasing or decreasing channel opening frequency without altering mean channel open duration. We propose that DZ and DMCM alter GABAA receptor current by acting reciprocally to increase or decrease only, respectively, the apparent agonist association rate at the first of two proposed GABA binding steps without altering channel gating.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking.

1. Single-channel kinetics of steroid enhancement of single gamma-aminobutyric acidA (GABA) receptor currents obtained from somata of mouse spinal cord neurones in culture were investigated using the excised outside-out patch-clamp recording technique. GABA (2 microM) and GABA (2 microM) plus androsterone (5 alpha-androstan-3 alpha-ol-17-one, AND, 10 nM-10 microM) or pregnanolone (5 beta-pregnan-3 alpha-ol-20-one, PRE, 100 nM-10 microM) applied by pressure ejection from micropipettes evoked inward currents when patches were voltage clamped at -75 mV in symmetrical chloride solutions. Averaged GABA receptor currents were increased in the presence of the steroids. 2. GABA receptor currents were recorded with at least two conductance levels, a predominant or main-conductance level of about 28 pS (which contributed 96% of the current evoked) and a minor or sub-conductance level of about 20 pS. The current amplitudes of the two conductance levels were unchanged by the steroids. The gating (opening and closing) kinetics of both of the conductance levels were analysed. Findings for the main-conductance level are summarized below. 3. Both steroids increased the average GABA receptor channel open duration. Consistent with the increased GABA receptor channel average open duration, the steroids shifted frequency histograms of GABA receptor channel open durations to longer durations. Three exponential functions were required to fit best the frequency histograms of GABA open durations, consistent with at least three kinetic open states of the main-conductance level. Time constants obtained from the GABA receptor channel open-duration frequency histograms were unchanged in the presence of the steroids. The basis for the increased average GABA receptor channel open durations by the steroids was due to an increased relative proportion of the two longer open-duration time constants. The GABA receptor channel average open durations were increased by AND and PRE in a concentration-dependent manner by shifting the proportion of openings to the longer open time constants. At a concentration of 10 microM, the prolongation of the average open duration was decreased, suggesting that the GABA receptor channel was blocked by these steroids. 4. GABA receptor channel opening frequency was increased and average channel-closed duration was decreased by AND or PRE. Consistent with this, areas of the frequency histograms of channel closed durations were shifted to shorter durations. Closed frequency distributions were fitted best with five to six exponential functions, suggesting that the channel had multiple kinetic closed states. The three briefest time constants were not greatly altered by the steroids.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The kinetic properties of the two most frequent conductance states of glycine receptor channels from somata of mouse spinal cord neurones in cell culture were investigated using the outside-out patch clamp technique. At low concentrations of glycine (0.5, 1 and 2 microM), single-channel currents were recorded with two predominant amplitudes corresponding to a dominant or main-conductance state of about 42 pS and a sub-conductance state of about 27 pS. Both conductance states opened singly and in bursts of several openings. Total current evoked and single-channel opening frequency increased as glycine concentration was increased from 0.5 to 2 microM. 2. For both conductance states mean open times were increased and open time frequency histograms were shifted to longer times as glycine concentration was increased from 0.5 to 2 microM. For both conductance states, three exponential components were required to fit best open time frequency distribution histograms at all glycine concentrations (0.5, 1 and 2 microM). The time constants of the exponential components for each conductance state were not significantly different across concentration, suggesting that the main- and sub-conductance states of the channel each opened into at least three open states. For the main-conductance state, the time constants were 1.09 +/- 0.09, 4.06 +/- 0.26 and 9.79 +/- 0.30 ms. For the sub-conductance state, the time constants were 0.55 +/- 0.04, 2.64 +/- 0.35 and 8.57 +/- 1.08 ms. The increase in long open times with concentration was due primarily to a shift in relative frequency of occurrence of openings from the shortest to the two longest open states. 3. Closed time frequency distributions of closures between main-conductance state openings, closures between sub-conductance state openings and closures between both main- and sub-conductance state openings were fitted with multiple exponential components, suggesting that the channel had several closed states. The two shortest time constants (0.16 +/- 0.01 and 1.26 +/- 0.13 ms) did not vary significantly with concentration (0.5-2 microM) or method of analysis. The longer time constant varied with concentration. 4. Bursts were defined as groups of openings surrounded by closures greater than a critical closed time. For both conductances states, mean burst durations were increased and burst duration frequency histograms were shifted to longer times as glycine concentration was increased from 0.5 to 2 miroM. Burst duration frequency histograms contained four exponential components for the main-conductance state and three exponential components for the sub-conductance state.(ABSTRACT TRUNCATED AT 400 WORDS)

GABA(A) receptor fast desensitization is thought to shape the time course of individual IPSCs. Although GABA(A) receptors also exhibit slower phases of desensitization, the possible role of slow desensitization in modifying synaptic function is poorly understood. In transiently transfected human embryonic kidney (HEK293T) cells, rat alpha1beta3delta and alpha1beta3gamma2L GABA(A) receptors showed distinct desensitization patterns during long (28 s) concentration jumps using a saturating (1 mM) GABA concentration. alpha1beta3gamma2L receptors desensitized extensively (approximately 90%), with four phases (tau(1) approximately 20 ms, tau(2) approximately 400 ms, tau(3) approximately 2 s, tau(4) approximately 10 s), while alpha1beta3delta receptors desensitized slowly and less extensively (approximately 35 %), with one or two slow phases with time constants similar to tau(3) and tau(4) of alpha1beta3gamma2L receptors. To determine the structural basis of subunit-specific desensitization, delta-gamma2L chimera subunits were expressed with alpha1 and beta3 subunits. Replacing the entire N-terminus of the gamma2L subunit with delta subunit sequence did not alter the number of phases or the extent of desensitization. Although extension of delta subunit sequence into transmembrane domain 1 (TM1) abolished the fast and intermediate components of desensitization, the two slow phases still accounted for substantial current loss (approximately 65 %). However, when delta subunit sequence was extended through TM2, the extent of desensitization was significantly decreased and indistinguishable from that of alpha1beta3delta receptors. The importance of TM2 sequence was confirmed by introducing gamma2 subunit TM2 residues into the delta subunit, which significantly increased the extent of desensitization, without introducing either the fast or intermediate desensitization phases. However, introducing delta subunit TM2 sequence into the gamma2L subunit had minimal effect on the rates or extent of desensitization. The results suggest that distinct delta subunit structures are responsible for its unique desensitization properties: lack of fast and intermediate desensitization and small contribution of the slow phases of desensitization. Finally, to investigate the possible role of slow desensitization in synaptic function, we used a pulse train protocol. We observed inhibition of peak current amplitude that depended on the frequency and duration of GABA pulses for receptors exhibiting extensive desensitization, whether fast phases were present or not. The minimally desensitizing alpha1beta3delta receptor exhibited negligible inhibition during pulse trains. Because receptors that desensitized without the fast and intermediate phases showed pulse train inhibition, we concluded that receptors can accumulate in slowly equilibrating desensitized states during repetitive receptor activation. These results may indicate a previously unrecognized role for the slow phases of desensitization for synaptic function under conditions of repeated GABA(A) receptor activation.

The sensitivity of GABAA receptors (GABARs) to Zn2+ inhibition depends on subunit composition. The predominant neuronal forms of mammalian GABARs, αβγ and αβδ, are differentially sensitive to Zn2+ inhibition; αβγ receptors are substantially less sensitive than αβδ receptors. Recently, functional domains involved in Zn2+ sensitivity have been identified in α and β subunits. Our aim in the present study was to localize functional domains of low Zn2+ sensitivity within γ2L subunits. Chimeric subunits were constructed by progressively replacing the rat γ2L subunit sequence with that of the rat δ subunit sequence. Whole-cell currents were recorded from mouse L929 fibroblasts coexpressing wild-type rat α1 and β3 subunits with a chimeric δ-γ2L subunit. Unlike α and β subunits, the γ2L subunit was found to contain a determinant of low Zn2+ sensitivity in the N-terminal extracellular region. In addition, we identified determinants in the M2 segment and the M2-M3 loop of the γ2L subunit that are homologous to those found in β and α subunits. We postulate that the interface between the latter two domains, which may form the outer vestibule of the channel, represents a single functional domain modulating Zn2+ sensitivity. Thus, the Zn2+ sensitivity of ternary GABARs appears to be determined by two functional domains, one in the N-terminal extracellular region and one near the outer mouth of the channel. The γ-aminobutyric acid type A receptor (GABAR) is the major inhibitory neurotransmitter receptor in the mammalian central nervous system. Functional mammalian GABARs are thought to be pentameric combinations of homologous subunits drawn from seven different families with multiple subtypes: α(1-6), β(1-3), γ(1-3), δ, e, π and θ (Macdonald & Olsen, 1994; Davies et al. 1997; Hedblom & Kirkness, 1997; Bonnert et al. 1999). Like other members of the ligand-gated ion channel gene superfamily, GABAR subunits have a putative membrane topology consisting of a large N-terminus, four membrane-spanning segments (M1-M4), one extracellular and two intracellular loops connecting the membrane-spanning segments (M1-M2, M2-M3, M3-M4), and an extracellular C-terminus. It has been suggested that neuronal GABARs are predominantly ternary αβγ and αβδ combinations (Angelotti et al. 1993; McKernan & Whiting, 1996). The divalent cation Zn2+ has been proposed to be an endogenous modulator of synaptic transmission (Smart et al. 1994; Harrison & Gibbons, 1994). Its ability to inhibit native GABARs, however, varies with neuronal type, age and activity (Smart et al. 1994; Kapur & Macdonald, 1997). Studies of recombinant receptors indicate that the inhibitory activity of Zn2+ on GABAR currents depends on subunit composition (Draguhn et al. 1990; Smart et al. 1991). Binary αβ GABARs have a high sensitivity to Zn2+ with IC50 values ranging from 0.1 to 1 μm (Draguhn et al. 1990; Smart et al. 1991; Wooltorton et al. 1997; Horenstein & Akabas, 1998). In ternary αβX GABARs (where X is δ, γ, e or π), Zn2+ sensitivity is decreased. Receptors containing π subunits have a Zn2+ IC50 of ≈2 μm (Neelands & Macdonald, 1999), receptors containing δ subunits have Zn2+ IC50 values ranging from 5 to 16 μm (Saxena & Macdonald, 1996; Krishek et al. 1998), and receptors containing e subunits have a Zn2+ IC50 of ≈40 μm (Whiting et al. 1997; Neelands et al. 1999). Incorporation of a γ subunit, however, has an even greater effect on GABAR Zn2+ sensitivity, resulting in IC50 values that range from 20 to 600 μm (Saxena & Macdonald, 1996; Burgard et al. 1996; Krishek et al. 1998). Functional domains involved in Zn2+ sensitivity have been identified in two GABAR subunit families, α and β. The extracellular end of the M2 segment in the β subunit has been shown to be a major determinant of Zn2+ sensitivity in β homomers and αβ receptors (Wooltorton et al. 1997; Horenstein & Akabas, 1998). The M2-M3 extracellular loop of the α6 subtype has been shown to confer higher Zn2+ sensitivity to α6β3γ2L GABAR isoforms as compared to α1β3γ2L receptors (Fisher & Macdonald, 1998). In addition, in GABAC receptors, which are structurally homologous to GABARs, the N-terminus has an essential role in conferring Zn2+ sensitivity to ρ1 homomers (Wang et al. 1995). Such functional domains, however, have yet to be identified for other GABAR subunit families including δ and γ. To determine which domains of the γ2L subunit subtype are involved in conferring low Zn2+ sensitivity to GABARs, we constructed GABAR chimeras by replacing varying lengths of the rat γ2L subunit with the homologous sequence of the rat δ subunit. These chimeric subunits were coexpressed in mouse L929 cells with wild-type α1 and β3 subunits, and whole-cell currents were elicited by application of GABA. The relative Zn2+ sensitivities of GABARs containing the chimeric subunits revealed that two different and non-contiguous γ2L subunit domains, one in the N-terminal extracellular region and one near the outer mouth of the channel, contributed to the regulation of Zn2+ sensitivity.

GABAA receptors (GABARs) mediate the majority of fast inhibitory neurotransmission in the mammalian brain. Functional GABARs are ligand-gated chloride ion channels composed of five individual subunits. These subunits derive from six identified families, many with multiple subtypes (α1-6, β1-3, γ1-3, δ, e and π). When expressed in Xenopus oocytes or mammalian cells, different GABAR subunit combinations form receptors with unique pharmacological and biophysical properties (Macdonald & Olsen, 1994). These subunits do not assemble randomly, however, for while αβ subunit combinations readily express in mammalian cells, addition of a γ subunit drives expression of αβγ isoforms (Angelotti & Macdonald, 1993). The majority of native receptors are believed to be formed by combinations of αβγ and αβδ subunits (McKernan & Whiting, 1996), although the recently characterized e and π subunits may substitute for γ or δ subunits in some instances (Hedblom & Kirkness, 1997; Davies et al. 1997). In the rat, the γ2 subtype becomes the dominant γ subunit expressed at later developmental stages, and mRNA and membrane protein for this subtype are expressed in most brain regions. In contrast, the δ subunit is restricted only to a few cell populations in the postnatal rat that include thalamic relay neurons, cerebellar granule neurons and dentate granule neurons of the hippocampus (Laurie et al. 1992a,b; Wisden et al. 1992; Sperk et al. 1997). While the δ subunit has been shown to combine preferentially with the α6 subtype in cerebellar granule neurons (Jones et al. 1997), the GABAR subtypes that it combines with in dentate granule neurons remain unknown. The potential importance of hippocampal δ subunit-containing GABARs is underscored, however, by δ subunit knockout mice that exhibit spontaneous seizures (Olsen et al. 1997). For this investigation, we chose the α1β3γ2L and α1β3δ GABAR isoforms to determine the roles of γ and δ subunits in shaping GABAR currents. α1β3γ2L and α1β3δ GABAR whole-cell currents have been characterized previously in L929 fibroblasts, where incorporation of the δ subunit resulted in higher apparent GABA affinity, slower and less complete whole-cell current desensitization, and smaller whole-cell currents compared with receptors containing the γ2 subtype (Saxena & Macdonald, 1994). α1β3 currents also desensitized more rapidly than α1β3δ currents, although both faster (Fisher & Macdonald, 1997) and slower desensitization (Dominguez-Perrot et al. 1996) relative to α1β3γ2L currents have been reported. In addition, α1β3 single channels had a smaller main channel conductance level (13 pS), while α1β3γ2L and α1β3δ channels had a similar larger main conductance level (27 pS). The γ2L subtype, however, conferred a change on the open and closed properties of the receptor, leading to a tendency for longer duration openings and longer bursts of openings (Fisher & Macdonald, 1997). While suggesting major differences in channel gating and desensitization, these analyses did not resolve the rapid phases of activation, desensitization and deactivation of GABAR currents. These rapid kinetic properties are critical to understanding the potential synaptic roles of the α1β3γ2L and α1β3δ GABAR isoforms. In previous studies of native receptors, rapid application of GABA to outside-out membrane patches containing many GABARs reproduced the rapid activation and deactivation of IPSCs (Maconochie et al. 1994; Jones & Westbrook, 1995; Tia et al. 1996; Galaretta & Hestrin, 1997; Mellor & Randall, 1997, 1998). Also, with this rapid application protocol, it was demonstrated that GABAR desensitization was an important factor in shaping the deactivation time course of macropatch responses (Jones & Westbrook, 1995, 1996). In addition, macropatch deactivation kinetics were altered by allosteric modulators of GABARs such as benzodiazepines (Lavoie & Twyman, 1996; Mellor & Randall, 1997) and the anaesthetic propofol (Zhu & Vicini, 1997), as well as by intracellular phosphatase activity (Jones & Westbrook, 1997). Moreover, different recombinant GABAR isoforms displayed unique rapid kinetic properties (Verdoorn, 1994; Tia et al. 1996; Lavoie et al. 1997) that probably contribute to the diversity in GABAergic synaptic responses. In addition to predicting the synaptic behaviour of recombinant GABAR isoforms, rapid kinetic analysis of macroscopic currents may serve as a bridge between single-channel and whole-cell analysis, allowing for the development of more comprehensive kinetic models of GABAR behaviour that incorporate desensitization (Macdonald & Twyman, 1992). For this study, we implemented a GABA application system that allowed very rapid solution exchange (10–90 % rise time < 400 μs) during electrophysiological recordings from outside-out membrane patches containing multiple receptor channels. Using this technique, we determined the rapid activation, desensitization and deactivation kinetics of α1β3γ2L and α1β3δ GABAR currents and used these kinetic data in combination with steady-state single-channel analysis to develop comprehensive models of GABAR kinetic behaviour for these isoforms. In some instances, the α1β3 isoform was examined so that contributions of the γ and δ subunits could be more thoroughly assessed.

1. Reduction by penicillin of single gamma-aminobutyric acidA (GABAA) receptor currents from somata of mouse spinal cord neurones in culture was investigated using the excised outside-out patch-clamp recording technique. 2. GABA (2 microM) alone or with penicillin (100-5000 microM) applied by pressure ejection from micropipettes evoked inward currents when patches were voltage-clamped at -75 mV in symmetrical chloride solutions. Averaged GABA receptor currents were decreased in the presence of penicillin. 3. GABA receptor currents were recorded with at least two conductance states, a more frequent or main-conductance state of about 27 pS and a less frequent sub-conductance state of about 19-20 pS. The conductances of the two states were unchanged in the presence of penicillin. The kinetic properties of the main-conductance state were analysed and are summarized below. 4. Penicillin produced a concentration-dependent reduction of GABA receptor open properties by reduction of average GABA receptor channel open duration and an increase in channel opening frequency. 5. Penicillin shifted frequency histograms of GABA receptor channel open durations to shorter durations in a concentration-dependent manner. Three exponential functions were required to fit best the frequency histograms of open durations, suggesting that the channel had at least three open states. Penicillin produced a concentration-dependent reduction in the time constants obtained from the open duration frequency histograms. 6. Frequency histograms of GABA receptor channel closed durations could be fitted with five to seven exponential functions, suggesting that the channel had multiple closed states. In the presence of increased concentration of penicillin, there was a reduction in the relative frequency of brief gaps and the appearance of new closed time constants. 7. With increased penicillin concentration, GABA receptor channel burst frequency was unchanged, burst durations were increased, the number of openings per burst was increased and the per cent time open within a burst was decreased. 8. The results suggested that penicillin produced simple open channel blockade of the GABA receptor channel. However, the experimental results also suggested that the association with and, perhaps, the dissociation of the blocker from its binding site were dependent upon the kinetic state of the open channel. Penicillin had faster association and slower dissociation rates when the channel was in unstable, brief open kinetic state than when the channel was in a more stable, longer open kinetic state. Possible models for penicillin reduction of single GABA receptor currents were simulated by computer and analysed.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The whole-cell variation of the patch clamp technique was used to study the effect of the purified catalytic subunit of the cyclic AMP-dependent protein kinase (A kinase catalytic subunit: AK-C) on the calcium current components of acutely dissociated rat nodose ganglion neurones. 2. The transient low-threshold calcium current component (T) was stable during whole-cell recording. In contrast, currents containing the transient high-threshold (N) and slowly inactivating high-threshold (L) current components declined steadily after stabilization of the currents during the first 5-7 min of recording. When AK-C was included in the recording pipette at physiological concentrations (50 micrograms/ml, approximately 1 microM), currents containing the N- and L-components increased in magnitude beginning 7-9 min after patch rupture, but there was no effect on the isolated T-current. The current-voltage relation of the T-current component was similar to controls, but the current-voltage relation for the N- and L-current components was shifted slightly to more depolarized clamp potentials (Vc), approximately 10 mV. 3. The effect of AK-C on currents containing the N- and L-currents was concentration dependent. There was no effect of 0.1 microgram/ml AK-C, the lowest concentration tested. Currents evoked from holding potentials (Vh) = -80 mV increased 5-10% during a 20 min recording in the presence of 1 microgram/ml AK-C and 30-35% in the presence of 50 micrograms/ml AK-C. In contrast, currents evoked from Vh = -40 mV increased 5-10% in the presence of either 1 or 50 micrograms/ml AK-C. The increase in current magnitude was associated with an increased rate of current inactivation and was evident particularly in currents evoked from Vh = -80 mV. 4. These effects were blocked by prior incubation of AK-C (1 microgram/ml) with a specific peptide inhibitor (protein kinase inhibitor peptide, PKIP; 0.2 mg/ml). 5. We evoked calcium currents using very long (1 s) voltage commands and modelled the traces using a multiexponential function in order to determine the effects of AK-C on the N- and L-current components. The (curve-fitted) N- and L-current components each declined approximately 50% during a 20 min recording in control neurones.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Using the single-electrode voltage clamp technique, three calcium current components were recorded at 35 degrees C from mouse dorsal root ganglion (DRG) neurones in culture. A transient low-threshold calcium current (T current) was recorded at clamp potentials (Vc) positive to -60 mV. Holding potentials (Vh) at or negative to -90 mV were required to fully remove inactivation. A large transient high-threshold calcium current component (N current) was recorded at Vc positive to -40 mV. Vh at or negative to -80 mV removed all steady-state inactivation. A slowly inactivating high-threshold calcium current component (L current) was recorded at Vc positive to -30 mV. Inactivation was removed by Vh at or negative to -60 mV. When currents were evoked at Vc positive to -20 mV from Vh negative to -60 mV, all three calcium current components were present. 2. Pentobarbitone (500 microM) had no effect on the isolated T current, but reduced the isolated L current 50-100% when evoked at Vc of -20 to 0 mV from Vh of -50 mV. Pentobarbitone had voltage-dependent effects on calcium currents containing all three calcium current components. Pentobarbitone produced small and equal reductions of the peak and late (greater than or equal to 300 ms) calcium currents evoked at -20 to 0 mV from Vh at or negative to -80 mV, but at more positive Vh there was a greater reduction in the peak current. The rate of current inactivation was increased in the presence of pentobarbitone. 3. Current-voltage plots were constructed from currents recorded in the absence and presence of 500 microM-pentobarbitone. Pentobarbitone reduced the magnitude of the calcium current without affecting the voltage dependence of the current-voltage relation. 4. Calcium current traces were fitted with a multiexponential function to determine the amplitudes and inactivation time constants (tau i) of the three calcium current components. Inactivation time constants decreased with more positive Vc for all three calcium current components. Pentobarbitone reduced only those tau i corresponding to the N current. 5. Recovery from inactivation of the N current was determined using a two-pulse protocol. In control neurones, recovery from inactivation occurring at 0 mV was slower at Vh = -65 mV than at Vh = -80 mV. In the presence of pentobarbitone, recovery from inactivation was faster, and occurred at a similar rate at both potentials. 6. Steady-state inactivation curves for the N current were derived from neurones in the absence and presence of pentobarbitone.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The kinetic properties of the main conductance state of gamma-aminobutyric acidA (GABA) receptor channels from somata of mouse spinal cord neurones in cell culture were investigated using patch clamp techniques. 2. Whole-cell GABA receptor currents increased in a concentration-dependent manner from 0.5 to 5 microM. 3. Single-channel currents were recorded with a main conductance state of 27.2 pS and a less frequent conductance state of 15.9 pS. The main conductance state opened singly and in bursts of several openings. 4. Mean open times of GABA receptor main conductance currents were increased and open-time frequency histograms were shifted to longer times as GABA concentration was increased from 0.5 to 5 microM. Three exponential functions were required to fit the histograms at all GABA concentrations, suggesting that the channel opened into at least three open states (O1, O2 and O3). The three functions had the same time constants (1.0 +/- 0.2, 3.7 +/- 0.4 and 11.3 +/- 0.5 ms; mean +/- S.D.) at each concentration. The increase in long open times with concentration was due to a shift in relative frequency of occurrence of openings from the shortest (O1) to the two longest (O2 and O3) open states. 5. Closed-time distributions of closures between main conductance state openings were fitted with multiple exponential functions, suggesting that the channel had several closed states. The two shortest time constants (0.24 +/- 0.03 and 2.0 +/- 0.3 ms) were concentration independent (0.5 to 5 microM). Three longer time constants decreased as concentration increased. 6. Bursts were defined as groups of openings surrounded by closures greater than a critical closed time (tc = 5 ms). Mean burst durations were increased and burst duration frequency histograms were shifted to longer times as GABA concentration was increased from 0.5 to 5 microM. Burst-duration frequency histograms were best fitted with three exponential functions. The time constants were concentration independent and were 1.0 +/- 0.2, 5.5 +/- 0.2 and 29.8 +/- 1.6 ms. The increase in burst duration with concentration was due to a relative shift from short duration bursts to longer duration bursts. 7. The shortest burst time constant was similar to the shortest open time constant suggesting that there was a population of single openings of short duration. The two longest burst time constants were longer than the two longest open time constants, suggesting that the bursts from the two longest burst components were composed of two or more openings.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Barbiturate regulation of the kinetic properties of gamma-aminobutyric acidA (GABA) receptor channel chloride currents from somata of mouse spinal cord neurones were investigated using whole-cell and excised outside-out patch-clamp recording techniques. 2. GABA (2 microM), GABA (2 microM) plus phenobarbitone (PhB) (500 microM) and GABA (2 microM) plus pentobarbitone (PB) (50 microM), applied by pressure ejection from blunt perfusion micropipettes, evoked inward chloride currents when neurones or patches were voltage clamped at -75 mV and the chloride equilibrium potential was 0 mV. GABA receptor channel currents were increased by PhB and PB. 3. Single GABA receptor channel currents were recorded with a main conductance state of 27 pS and a less frequent subconductance state of 16.5 pS. The conductances of the two states were unchanged by the barbiturates. 4. The main conductance state kinetics were analysed. GABA alone or with the barbiturates gated the channel open singly and in groups of openings. 5. The barbiturates increased GABA receptor channel mean open time and shifted frequency histograms of channel open times to longer times. 6. Three exponential functions were required to fit the frequency histograms of GABA receptor channel open times, suggesting that the channel has at least three open states (O1, O2, O3). The time constants for the exponential functions (0.9, 2.7 and 7.8 ms, respectively) were unchanged by the barbiturates. The increases in mean open times and the shifts of the open-time frequency histograms by the barbiturates were due to a reduction in relative frequency of occurrence of the two short open states (O1 and O2) and to an increase in the relative frequency of occurrence of the longest open state (O3). 7. Frequency histograms of GABA receptor channel closed times were fitted with five exponential functions, suggesting that the channel has multiple closed states. None of the time constants nor areas of the exponential functions were significantly changed by the barbiturates. 8. For analysis, a burst was defined as openings surrounded by closures greater than a critical closed time, tc, of 5 ms. For GABA (2 microM), frequency histograms of GABA receptor channel bursts were fitted with three exponential functions, suggesting that the channel has three burst states (B1, B2, B3). The B1 burst state was probably a single opening to the O1 open state while the B2 and B3 burst states were probably composed of multiple openings to the O2 and O3 open states.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine and several of its analogues produced a concentration-dependent shortening of calcium-dependent action potential (c.a.p.) duration of mouse dorsal root ganglion (d.r.g.) neurones in dissociated cell culture. The following rank order of potency was obtained: N6-(L-phenylisopropyl)adenosine greater than N6-(D-phenylisopropyl)adenosine greater than N6-cyclohexyladenosine greater than 2-chloroadenosine much greater than 1-methylisoguanosine greater than adenosine. Effects of adenosine agonists on c.a.p. duration were blocked by methylxanthine adenosine antagonists. Adenosine monophosphate (AMP) and cyclic AMP shortened c.a.p.s in d.r.g. neurones, while ATP also depolarized cells. Voltage-clamp analysis revealed that the effect arose from reduction of a voltage-dependent calcium conductance. Adenosine agonists reduced depolarization-evoked inward currents but did not alter membrane conductance following blockade of calcium channels by cadmium. Additionally, adenosine reduced the instantaneous current-voltage slope (chord conductance) during step commands that produced maximal activation of voltage-dependent calcium conductance. If effects of adenosine on neuronal somata and synaptic terminals are similar, adenosine agonists may inhibit neurotransmitter release in the central nervous system by inhibiting a voltage-dependent calcium conductance. Since effects of adenosine agonists did not correspond with their relative potencies as modulators of adenylate cyclase activity or inhibitors of neurotransmitter release in peripheral tissues, a novel adenosine receptor may be involved in regulation of this conductance.