We describe a maximum likelihood method for direct estimation of rate constants from macroscopic ion channel data for kinetic models of arbitrary size and topology. The number of channels in the preparation, and the mean and standard deviation of the unitary current can be estimated, and a priori constraints can be imposed on rate constants. The method allows for arbitrary stimulation protocols, including stimuli with finite rise time, trains of ligand or voltage steps, and global fitting across different experimental conditions. The initial state occupancies can be optimized from the fit kinetics. Utilizing arbitrary stimulation protocols and using the mean and the variance of the current reduce or eliminate problems of model identifiability (Kienker, 1989). The algorithm is faster than a recent method that uses the full autocovariance matrix (Celentano and Hawkes, 2004), in part due to the analytical calculation of the likelihood gradients. We tested the method with simulated data and with real macroscopic currents from acetylcholine receptors, elicited in response to brief pulses of carbachol. Given appropriate stimulation protocols, our method chose a reasonable model size and topology.

GABA A receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABA A 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 GABA A 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.

Neuroactive steroids have some of their most potent actions by augmenting the function of GABA A receptors. Endogenous steroid actions on GABA A receptors may underlie important effects on mood and behavior. Exogenous neuroactive steroids have potential as anesthetics, anticonvulsants, and neuroprotectants. We have taken multiple approaches to understand more completely the interaction of neuroactive steroids with GABA A receptors. We have developed many novel steroid analogues in this effort. Recent work has resulted in synthesis of new enantiomer analogue pairs, novel ligands that probe various properties of the steroid pharmacophore, fluorescent neuroactive steroid analogues, and photoaffinity labels. Using these tools, combined with receptor binding and electrophysiological assays, we have begun to untangle the complexity of steroid actions at this important class of ligand-gated ion channel.

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 y-aminobutyric acid A (GABA A ) receptor. We analysed the actions of PS on single-channel currents from recombinant GABA A receptors formed from a1, b2 and y2L 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 a1 subunit had a major effect on the rate of block. Mutation of the residue a1 V256S reduced the rate of block by 30-fold. A mutation at the homologous position of the b2 subunit (b2 A252S) had no effect, nor did a complementary mutation in the y2L subunit (y2L 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.

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 ␣1Asn407/Tyr410 in the M4 transmembrane domain and residue ␣1Gln241 in the M1 domain. We examined the role of residues in the ␣1 subunit M1 domain in the modulation of the rat ␣1␤2␥2L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the ␣1Q241W or ␣1Q241L mutations were insensitive to (3␣,5␣)-3-hydroxypregnan-20-one (3␣5␣P), albeit with different underlying mechanisms. The ␣1Q241S mutant was potentiated by 3␣5␣P, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the ␣1Q241L mutation had no effect on channel potentiation by (3␣,5␣)-3-hydroxymethylpregnan-20-one, but mutation of the neighboring residue, ␣1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5␣-pregnan-20-one) potentiated the wild-type receptor but not the ␣1Q241L mutant. The findings are consistent with a model in which the ␣1Ser240 and ␣1Gln241 residues shape the surface to which steroid molecules bind.Potentiating neurosteroids are among the most efficacious modulators of the mammalian GABA-A receptor having potential applications as anxiolytics, anticonvulsants, sedatives, and anesthetics. Recent work has given significant insights into the functional and structural mechanisms of steroid actions. Potentiating steroids [e.g., (3␣,5␣)-3-hydroxypregnan-20-one (3␣5␣P) and (3␣,5␤)-3-hydroxypregnan-20-one (3␣5␤P)] act on the GABA-A receptor by modifying the channel open and closed times, leading to an increase in the open probability of the channel, enhanced macroscopic peak current, and a slower current decay when exposure to agonist is terminated. The putative steroid binding site is located in the membrane-spanning regions of the ␣ subunit of the receptor, extending from the ␣1Gln241 residue in the M1 membrane-spanning region to the residues ␣1Asn407 and ␣1Tyr410 in the M4 domain (Hosie et al., 2006). Mutations that reduce the H-bonding ability of these residues reduce receptor potentiation by both 5␣-and 5␤-reduced steroids. It was proposed that a common interaction site mediates the effects of the two classes of steroids, the ␣1Gln241 residue acting as an H-bond acceptor to the 3␣-hydroxyl group of the steroid molecule and the ␣1Asn407/Tyr410 residues interacting with the ketone group in the side chain on the D ring of steroids (Hosie et al., 2006). Subsequent studies showed that mutations that disrupt channel potentiation by steroids also affect modulation by a tricyclic benz[e]indene neurosteroid analog (Li et al., 2006), enantiomers of natural steroids (Li et al., 2007a), and the marine cembranoid eupalmerin acetate (Li et al., 2008), suggesting that the site may function as a common interaction site for a number of GABA-A receptor modul...