Gustav Akk

Neurosteroid Access to the GABAA Receptor

GABAA receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABAA receptor are important anesthetics, sedatives, anticonvulsants, and anxiolytics. Current views of receptor modulation suggest that many exogenous drugs access and bind to an extracellular receptor domain. Using novel synthetic steroid analogs, we examined the access route for neuroactive steroids, potent GABAA receptor modulators also produced endogenously. Tight-seal recordings, in which direct aqueous drug access to receptor was prevented, demonstrated that steroids can reach the receptor either through plasma membrane lateral diffusion or through intracellular routes. A fluorescent neuroactive steroid accumulated intracellularly, but recordings from excised patches indicated that the intracellular reservoir is not necessary for receptor modulation, although it can apparently equilibrate with the plasma membrane within seconds. A membrane impermeant neuroactive steroid modulated receptor activity only when applied to the inner membrane leaflet, demonstrating that the steroid does not access an extracellular modulatory site. Thus, neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation.

Pregnenolone sulfate block of GABAA receptors: mechanism and involvement of a residue in the M2 region of the α subunit

1 Neurosteroids are produced in the brain, and can have rapid actions on membrane channels of neurons. Pregnenolone sulfate (PS) is a sulfated neurosteroid which reduces the responses of the γ‐aminobutyric acid A (GABAA) receptor. We analysed the actions of PS on single‐channel currents from recombinant GABAA receptors formed from α1, β2 and γ2L subunits. 2 Currents were elicited by a concentration of GABA eliciting a half‐maximal response (50 μm) and a saturating concentration (1 mm). PS reduced the duration of clusters of single‐channel activity at either concentration of GABA. 3 PS had no discernable effect on rapid processes: no effects were apparent on channel opening and closing, nor on GABA affinity, and a rapidly recovering desensitised state was not affected. Instead, PS produced a slowly developing block which occurred at a similar rate for receptors with open or closed channels and with one or two bound GABA molecules. 4 The rate of block was independent of membrane potential, implying that the charged sulfate moiety does not move through the membrane field. 5 Change in a specific residue near the intracellular end of the channel lining portion of the α1 subunit had a major effect on the rate of block. Mutation of the residue α1 V256S reduced the rate of block by 30‐fold. A mutation at the homologous position of the β2 subunit (β2 A252S) had no effect, nor did a complementary mutation in the γ2L subunit (γ2L S266A). It seems likely that this residue is involved in a conformational change underlying block by PS, instead of forming part of the binding site for PS.

Modulation of GABAA receptor channel gating by pentobarbital

1 We have studied the kinetic properties of channel gating of recombinant α1β2γ2L GABAA receptors transiently expressed in human embryonic kidney 293 cells, using the cell‐attached, single‐channel patch‐clamp technique. The receptors were activated by GABA, β‐alanine or piperidine‐4‐sulfonic acid (P4S), and the effects of pentobarbital (PB) on single‐channel activity were examined. 2 At relatively high concentrations of agonist, single‐channel activity occurred in well‐defined clusters. In global terms, PB increased the mean open time for events in clusters, without changing the mean closed time. The addition of PB shifted the curve relating the probability of being open in a cluster (Po) to lower agonist concentrations, and that shift could be accounted for by the changes in mean open time. 3 The intracluster closed‐time histograms contained four components. The durations and relative frequencies of these closed‐dwell components were not affected by the presence of 40 μm PB, at any agonist concentration. The duration of one component was dependent upon the concentration of agonist used to activate the receptor. Accordingly, the inverse of the mean duration of this component will be called the effective opening rate. 4 The channel‐opening rate constant (β) was determined from the value of the effective opening rate at a saturating agonist concentration. β was about 1900 s−1 when the receptors were activated by GABA, 1500 s−1 when activated by β‐alanine, and too low to be determined when P4S was administered. In the presence of 40 μm PB, β was about 1500 s−1 when the receptors were activated by GABA, 1400 s−1 when activated by β‐alanine, and 50 s−1 when activated by P4S. Hence, the potentiating effect of PB is not mediated by a change in β. The concentration of agonist producing a half‐maximal effective opening rate also remained unaffected in the presence of PB, indicating that receptor affinity for agonists is not influenced by PB. 5 The distributions of the intracluster open durations elicited by GABA could be described by the sum of three exponentials, with mean durations of about 0.4, 2.4 and 6.3 ms. The duration and relative frequency of the components did not change with GABA concentration (20 μm to 1 mm). In the presence of 40 μm PB, however, the mean duration of the longest of the open times increased (mean durations of about 0.4, 2.0 and 13 ms). The intracluster open durations elicited by β‐alanine could be described by the sum of two exponential components (1.1 and 3.5 ms). However, in the presence of 40 μm PB the open‐time distribution contained three exponential components (0.2, 2 and 10 ms). Finally, openings elicited by P4S exhibited two components (0.3 and 0.9 ms). In the presence of 40 μm PB, three components could be distinguished (0.5, 2.5 and 13 ms). 6 These observations indicate that the potentiating effect of PB on GABA type A (GABAA) receptors reflects effects on the open state(s) of the receptors. In the case of receptors activated by GABA, the observations are consistent with the idea that the action is the result of PB stabilizing one of the open states. The actions on receptors activated by P4S or β‐alanine are also broadly consistent with this idea. However, the changes in open‐time distributions caused by PB appear to be more complex. Possible explanations of the effects of PB on gating by different agonists are considered.

Voltage dependence of mouse acetylcholine receptor gating: different charge movements in di‐, mono‐ and unliganded receptors.

1. The voltage dependence of binding and gating in wild‐type and mutant recombinant mouse nicotinic acetylcholine receptors (AChRs) was examined at the single‐channel level. 2. The closing rate constant of diliganded receptors decreased e‐fold with approximately 66 mV hyperpolarization in both wild‐type (adult and embryonic) and mutant receptors. The opening rate constant of a mutant receptor (alpha Y93F) was not voltage dependent. 3. The voltage dependence of closing in monoliganded receptors was examined in several receptors having a mutation in the binding site (alpha G153S) or pore region (alpha L251C and epsilon T264P). The closing rate constant of these monoliganded receptors decreased e‐fold with approximately 124 mV hyperpolarization. 4. The voltage dependence of closing and opening in unliganded receptors was examined in two receptors having a mutation in the pore region (alpha L251C and epsilon T264P). Neither the closing nor the opening rate constants of unliganded receptors were voltage dependent. 5. If z if the amount of charge that moves during channel closure and delta is the distance (as a fraction of the electric field) that the charge moves, we conclude that z delta = 0.4 in diliganded receptors, 0.2 in monoliganded receptors, and 0.0 in unliganded receptors. It is likely that charges on the protein, rather than the agonist molecule, move z delta = 0.2 after each ACh molecule has bound. 6. The results suggest that unliganded openings arise from a local, concerted change in the structure of the pore (channel opening) that does not involve the net movement of charged residues. We speculate that as a consequence of agonist binding, charged moieties in the protein change their disposition so that they move with respect to the electric field when the channel gates. The results are consistent with the idea that there is semi‐independent movement of distinct domains during AChR gating.

Neuroactive steroids have multiple actions to potentiate GABAA receptors

The effects of neuroactive steroids on the function of GABAA receptors were studied using cell‐attached records of single channel activity recorded from HEK293 cells transfected with α1 β2 γ2L subunits. Activity was elicited with a half‐maximal (50 μm) concentration of GABA. Two steroids were studied in detail: ACN ((3α,5α,17β)‐3‐hydroxyandrostane‐17‐carbonitrile) and B285 ((3α,5β,17β)‐3‐hydroxy‐18‐norandrostane‐17‐carbonitrile). Four effects on channel activity were seen, two on open time distributions and two on closed times. When clusters of openings were elicited in the absence of steroid, the open time distribution contained three components. ACN produced concentration‐dependent alterations in the open time distribution. The prevalence of the longest duration class of open times was increased from about 15% to about 40% (EC50 about 180 nm ACN), while the duration of the longest class increased from 7.4 ms to 27 ms (EC50 about 35 nm ACN). B285 also increased the prevalence of the longest duration open times (EC50 about 18 nm B285) but increased the duration only at concentrations close to 10 μm. The differences in the actions of these two steroids suggest that the effects on proportion and duration of the long duration open time component are produced by independent mechanisms and that there are separate recognition sites for the steroids which are associated with the two functional actions. The closed time distributions also showed three components in the absence of steroid. The rate of occurrence of the two brief duration closed time components decreased with increasing ACN, with an EC50 of about 50 nm ACN. In contrast, B285 did not reduce the rate of occurrence of the brief closings until high concentrations were applied. However, both B285 and ACN reduced the rate of occurrence of the activation‐related closed state selectively, with comparable IC50 concentrations (about 40 nm ACN, 20 nm B285). As in the case for action on open times these data suggest that there are two recognition sites and two independent mechanisms, perhaps the sites and mechanisms associated with actions on open times. The presence of  1 μm ACN had no effect on the estimated channel opening rate or on the apparent affinity of the receptor for GABA. Mutation of the carboxy terminus of the γ2 subunit, but not the α1 or β2 subunits, abolished the ability of ACN to increase the duration of OT3 but had no effect on the reduction of the rate of occurrence of the activation‐related closed state. These observations are also consistent with the idea that there is more than one distinguishable steroid recognition site on the GABAA receptor.

Activation of GABAA receptors containing the α4 subunit by GABA and pentobarbital

The activation properties of GABAA receptors containing α4β2γ2 and α4β2δ subunits were examined in the presence of GABA or pentobarbital. The receptors were expressed transiently in HEK 293 cells, and the electrophysiological experiments were carried out using cell‐attached single‐channel patch clamp or whole‐cell macroscopic recordings. The data show that GABA is a stronger activator of α4β2γ2 receptors than α4β2δ receptors. Single‐channel clusters were recorded from α4β2γ2 receptors in the presence of 10–5000 μm GABA. The maximal intracluster open probability was 0.35, with a half‐maximal response elicited by 32 μm GABA. Simultaneous kinetic analysis of single‐channel currents obtained at various GABA concentrations yields a channel opening rate constant of 250 s−1, and a KD of 20 μm. In contrast, only isolated openings were observed in the presence of GABA for the α4β2δ receptor. Pentobarbital was a strong activator of both α4β2γ2 and α4β2δ receptors. The maximal cluster open probability, recorded in the presence of 100 μm pentobarbital, was 0.7. At higher pentobarbital concentrations, the cluster open probability was reduced, probably due to channel block. The results from single‐channel experiments were confirmed by macroscopic recordings from HEK cells in the presence of GABA or pentobarbital.

Mutations of the GABA-A receptor α1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids

Neuroactive steroids are among the most efficacious modulators of the mammalian GABA-A receptor. Previous work has proposed that receptor potentiation is mediated by steroid interactions with a site defined by the residues α1Asn407/Tyr410 in the M4 transmembrane domain and residue α1Gln241 in the M1 domain. We examined the role of residues in the α1 subunit M1 domain in the modulation of the rat α1β2γ2L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the α1Q241W or α1Q241L mutations were insensitive to (3α,5α)-3-hydroxypregnan-20-one (3α5αP), albeit with different underlying mechanisms. The α1Q241S mutant was potentiated by 3α5αP, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the α1Q241L mutation had no effect on channel potentiation by (3α,5α)-3-hydroxymethyl-pregnan-20-one, but mutation of the neighboring residue, α1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5α-pregnan-20-one) potentiated the wild-type receptor but not the α1Q241L mutant. The findings are consistent with a model in which the α1Ser240 and α1Gln241 residues shape the surface to which steroid molecules bind.

Differential regulation of GABA release and neuronal excitability mediated by neuropeptide Y1 and Y2 receptors in rat thalamic neurons

1 Neuropeptide Y (NPY) produced inhibitory effects on neurons of the thalamic reticular nucleus (RT; n= 18) and adjacent ventral basal complex (VB; n= 22), which included hyperpolarization (∼4 mV), a reduction in rebound and regular spikes and an increased membrane conductance. These effects were mediated predominantly via NPY1 receptor activation of G‐protein‐activated, inwardly rectifying K+ (GIRK) channels. 2 NPY reduced the frequency of spontaneous GABAA receptor‐mediated inhibitory postsynaptic currents (sIPSCs) in RT (by 60 ± 7 %, n= 14) and VB neurons (by 25 ± 11 %, n= 16), but had no effect on the kinetic properties of sIPSCs. After removal of the RT nucleus, the inhibitory effects of NPY on sIPSCs in VB neurons remained (29 ± 7 %, n= 5). The synaptic effects were mediated via NPY2 receptors. 3 NPY inhibited the frequency of miniature IPSCs (mIPSCs) in RT and VB neurons (by 63 ± 7 %, n= 5, and 37 ± 8 %, n= 10, respectively) in the presence of tetrodotoxin (TTX) (1 μM) but not TTX (1 μM) and Cd2+ (200 μM). 4 NPY inhibited evoked IPSCs in both RT (by 18 ± 3 %, n= 6) and VB (by 5 ± 4 %, n= 6) neurons without change in short‐term synaptic plasticity. 5 We conclude that NPY1 and NPY2 receptors are functionally segregated in the thalamus: NPY1 receptors are predominantly expressed at the somata and dendrites and directly reduce the excitability of neurons in both the RT and VB nuclei by activating GIRK channels. NPY2 receptors are located at recurrent (RT) and feed‐forward GABAergic terminals (VB) and downregulate GABA release via inhibition of Ca2+ influx from voltage‐gated Ca2+ channels.

INORGANIC, MONOVALENT CATIONS COMPETE WITH AGONISTS FOR THE TRANSMITTER BINDING SITE OF NICOTINIC ACETYLCHOLINE RECEPTORS

The properties of adult mouse recombinant nicotinic acetylcholine receptors activated by acetylcholine (ACh+) or tetramethylammonium (TMA+) were examined at the single-channel level. The midpoint of the dose-response curve depended on the type of monovalent cation present in the extracellular solution. The shifts in the midpoint were apparent with both inward and outward currents, suggesting that the salient interaction is with the extracellular domain of the receptor. Kinetic modeling was used to estimate the rate constants for agonist binding and channel gating in both wild-type and mutant receptors exposed to Na+, K+, or Cs+. The results indicate that in adult receptors, the two binding sites have the same equilibrium dissociation constant for agonists. The agonist association rate constant was influenced by the ionic composition of the extracellular solution whereas the rate constants for agonist dissociation, channel opening, and channel closing were not. In low-ionic-strength solutions the apparent association rate constant increased in a manner that suggests that inorganic cations are competitive inhibitors of ACh+ binding. There was no evidence of an electrostatic potential at the transmitter binding site. The equilibrium dissociation constants for inorganic ions (Na+, 151 mM; K+, 92 mM; Cs+, 38 mM) and agonists (TMA+, 0.5 mM) indicate that the transmitter binding site is hydrophobic. Under physiological conditions, about half of the binding sites in resting receptors are occupied by Na+.

Activation and block of recombinant GABAA receptors by pentobarbitone: a single‐channel study

Recombinant GABAA receptors (α1β2γ2L) were transiently expressed in HEK 293 cells. We have investigated activation and block of these receptors by pentobarbitone (PB) using cell‐attached single‐channel patch clamp. Clusters of single‐channel activity elicited by 500 μM PB were analysed to estimate rate constants for agonist binding and channel gating. The minimal model able to describe the kinetic data involved two sequential binding steps, followed by channel opening. The estimated channel opening rate constant is ∼1500 s−1, and the estimated equilibrium dissociation constants for the binding steps involved in activation are ∼2 mM. Our results show a dose‐dependent block of receptors at millimolar concentrations of PB that results in reduced open interval durations. The reduction in mean open time is linearly proportional to PB concentration, indicating that block can be produced by binding of a single PB molecule. Addition of millimolar concentrations of PB in the presence of GABA also produces a reduction of open channel lifetime in addition to a progressive increase in the closed interval durations within a cluster. The data suggest that the receptor contains two or more blocking sites while occupancy of only one of the sites is sufficient for channel block. Neither the blocking rate constant nor return rate from the blocked state(s) is affected by pH (ionization status of the PB molecule) demonstrating that both neutral and anionic forms of PB cause channel block.