Mathew V. Jones

Desensitized states prolong GABAA channel responses to brief agonist pulses

We studied the role of desensitization at inhibitory synapses by comparing nonequilibrium GABAA channel gating with inhibitory postsynaptic currents (IPSCs). Currents activated by brief pulses of 1-10 mM GABA to outside-out patches from cultured hippocampal neurons mimicked GABA-mediated IPSCs. Although the average open time of single GABAA channels following brief pulses was less than 10 ms, channels entered long (tau = 38-69 ms) closed states and subsequently reopened. Movement through these states resulted in paired-pulse desensitization. The time required for deactivation after removal of agonist also increased in proportion to the extent of desensitization. These results suggest that visits to desensitized states buffer the channel in bound conformations and underlie the expression of long-lasting components of the IPSC. Reopening after GABAA receptor desensitization may thus enhance inhibitory synaptic transmission by prolonging the response to a brief synaptic GABA transient.

The impact of receptor desensitization on fast synaptic transmission.

The role of desensitization of ligand-gated channels at fast chemical synapses has been difficult to establish. Densensitization has been studied traditionally with prolonged agonist exposure, whereas the duration of free neurotransmitter in the synaptic cleft is relatively brief. Studies of acetylcholine-, glutamate- and GABA-gated channels using rapid agonist application now provide a means to assess the effects of densensitization in shaping synaptic responses and in influencing neuronal excitability. These data reveal several strikingly different patterns by which the receptor-specific kinetics of densensitization can determine the size, timecourse and frequency of transmitted signals. Densensitization is thus a surprisingly versatile mechanism for shaping synaptic transmission.

Defining Affinity with the GABAA Receptor

At nicotinic and glutamatergic synapses, the duration of the postsynaptic response depends on the affinity of the receptor for transmitter (Colquhoun et al., 1977; Pan et al., 1993). Affinity is often thought to be determined by the ligand unbinding rate, whereas the binding rate is assumed to be diffusion-limited. In this view, the receptor selects for those ligands that form a stable complex on binding, but binding is uniformly fast and does not itself affect selectivity. We tested these assumptions for the GABAAreceptor by dissecting the contributions of microscopic binding and unbinding kinetics for agonists of equal efficacy but of widely differing affinities. Agonist pulses applied to outside-out patches of cultured rat hippocampal neurons revealed that agonist unbinding rates could not account for affinity if diffusion-limited binding was assumed. However, direct measurement of the instantaneous competition between agonists and a competitive antagonist revealed that binding rates were orders of magnitude slower than expected for free diffusion, being more steeply correlated with affinity than were the unbinding rates. The deviation from diffusion-limited binding indicates that a ligand-specific energy barrier between the unbound and bound states determines GABAA receptor selectivity. This barrier and our kinetic observations can be quantitatively modeled by requiring the participation of movable elements within a flexible GABA binding site.

Reduced cortical inhibition in a mouse model of familial childhood absence epilepsy

Mutations in the GABAA receptor γ2 subunit are associated with childhood absence epilepsy and febrile seizures. To understand better the molecular basis of absence epilepsy in man, we developed a mouse model harboring a γ2 subunit point mutation (R43Q) found in a large Australian family. Mice heterozygous for the mutation demonstrated behavioral arrest associated with 6-to 7-Hz spike-and-wave discharges, which are blocked by ethosuximide, a first-line treatment for absence epilepsy in man. Seizures in the mouse showed an abrupt onset at around age 20 days corresponding to the childhood nature of this disease. Reduced cell surface expression of γ2(R43Q) was seen in heterozygous mice in the absence of any change in α1 subunit surface expression, ruling out a dominant-negative effect. GABAA-mediated synaptic currents recorded from cortical pyramidal neurons revealed a small but significant reduction that was not seen in the reticular or ventrobasal thalamic nuclei. We hypothesize that a subtle reduction in cortical inhibition underlies childhood absence epilepsy seen in humans harboring the R43Q mutation.

Slow Desensitization Regulates the Availability of Synaptic GABA A Receptors

At central synapses, a large and fast spike of neurotransmitter efficiently activates postsynaptic receptors. However, low concentrations of transmitter can escape the cleft and activate presynaptic and postsynaptic receptors. We report here that low concentrations of GABA reduce IPSCs in hippocampal neurons by preferentially desensitizing rather than opening GABAAchannels. GABA transporter blockade also caused desensitization by locally elevating GABA to ∼1 μm. Recovery of the IPSC required several seconds, mimicking recovery of the channel from slow desensitization. These results indicate that low levels of GABA can regulate the amplitude of IPSCs by producing a slow form of receptor desensitization. Accumulation of channels in this absorbing state allows GABAA receptors to detect even a few molecules of GABA in the synaptic cleft.

Presynaptic, Activity-Dependent Modulation of Cannabinoid Type 1 Receptor-Mediated Inhibition of GABA Release

Endocannabinoid signaling couples activity-dependent rises in postsynaptic Ca2+ levels to decreased presynaptic GABA release. Here, we present evidence from paired recording experiments that cannabinoid-mediated inhibition of GABA release depends on the firing rates of the presynaptic interneurons. Low-frequency action potentials in post hoc identified cholecystokinin-positive CA1 basket cells elicited IPSCs in the postsynaptic pyramidal cells that, as expected, were fully abolished by the exogenous application of the cannabinoid receptor agonist WIN55,212-2 [R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone monomethanesulfonate] at 5 μm. However, the presynaptic basket cells recovered from the cannabinoid agonist-induced inhibition of GABA release when the presynaptic firing rate was increased to ≥20 Hz. Pharmacological experiments showed that the recovered transmission was exclusively dependent on presynaptic N-type Ca2+ channels. Furthermore, the increased presynaptic firing could also overcome even complete depolarization-induced suppression of inhibition, indicating that the magnitude of DSI markedly depends on the activity levels of basket cells. These results reveal a new locus of activity-dependent modulation for endocannabinoid signaling and suggest that endocannabinoid-mediated inhibition of GABA release may differ in distinct behavioral states.

Altered kinetics and benzodiazepine sensitivity of a GABAA receptor subunit mutation [γ2(R43Q)] found in human epilepsy

The γ-aminobutyric acid type A (GABAA) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABAA receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABAA γ2-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABAA receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from α1β2γ2 or α1β2γ2(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imidazopyridine zolpidem. These results provide evidence of impaired GABAA receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

Microscopic Kinetics and Energetics Distinguish GABAA Receptor Agonists from Antagonists

Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABA(A) and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABA(A) receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.

An Arginine Involved in GABA Binding and Unbinding But Not Gating of the GABAA Receptor

GABAA receptor function can be conceptually divided into interactions between ligand and receptor (binding) and the opening and closing of the ligand-bound channel (gating). The relationship between binding, gating, and receptor structure remains unclear. Studies of mutations have identified many amino acid residues that contribute to the GABAbinding site. Most of these studies assayed changes in GABA dose–response curves, which are macroscopic measures that depend on the interplay of many processes and cannot resolve individual microscopic transitions. Understanding the microscopic basis of binding and gating is critical, because kinetic transition rates predict how receptors will behave at synapses. Furthermore, microscopic rates are directly related to the molecular interactions underlying receptor function. Here, we focused on a residue (β2-R207) previously identified as lining the GABA-binding site that, when mutated to cysteine, greatly reduces apparent GABA affinity and was predicted to affect both binding and gating. To better understand the role of β2-R207, we expressed α1β2 and α1β2-R207C receptors in human embryonic kidney 293 cells and studied receptor kinetics using fast solution applications. The mutation accelerated deactivation by 10-fold, without altering desensitization in the presence of saturating GABA. Maximum open probability and single-channel open times were also unaltered by the mutation, but the GABA-binding rate was reduced eightfold. Therefore, the effects of this mutation in a predicted binding site residue are solely attributable to changes in GABA-binding and unbinding kinetics, with no changes in channel gating. Because β2-R207 stabilizes GABA in the binding pocket, it may directly contact the GABA molecule.

Slow GABA Transient and Receptor Desensitization Shape Synaptic Responses Evoked by Hippocampal Neurogliaform Cells

The kinetics of GABAergic synaptic currents can vary by an order of magnitude depending on the cell type. The neurogliaform cell (NGFC) has recently been identified as a key generator of slow GABAA receptor-mediated volume transmission in the isocortex. However, the mechanisms underlying slow GABAA receptor-mediated IPSCs and their use-dependent plasticity remain unknown. Here, we provide experimental and modeling data showing that hippocampal NGFCs generate an unusually prolonged (tens of milliseconds) but low-concentration (micromolar range) GABA transient, which is responsible for the slow response kinetics and which leads to a robust desensitization of postsynaptic GABAA receptors. This strongly contributes to the use-dependent synaptic depression elicited by various patterns of NGFC activity including the one detected during theta network oscillations in vivo. Synaptic depression mediated by NGFCs is likely to play an important modulatory role in the feedforward inhibition of CA1 pyramidal cells provided by the entorhinal cortex.