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The caging of specific residues of proteins is a powerful tool. This discussion attempts to alert the reader to the considerations that must be made in preparing and analyzing a caged protein through nonsense suppression. Although the suppression methodology is conceptually straightforward, it not possible to provide a failsafe "cook book" method for using caged unnaturals. We have emphasized the preparation of caged receptors expressed in Xenopus oocytes, but these approaches can clearly be adapted to many other systems.

The Kir4.1 gene (KCNJ10) encodes an inwardly rectifying K(+) channel subunit abundantly expressed in the CNS. Its expression in the mammalian inner ear has been suggested but its function in vivo in the inner ear is unknown. Because diverse human hereditary deafness syndromes are associated with mutations in K(+) channels, we examined auditory function and inner ear structure in mice with a genetically inactivated Kir4.1 K(+) channel subunit. Startle response experiments suggest that Kir4.1-/- mice are profoundly deaf, whereas Kir4.1+/- mice react like wild-type mice to acoustic stimuli. In Kir4.1-/- mice, the Reissner membrane is collapsed, the tectorial membrane is swollen, and type I hair cells and spiral ganglion neurons as well as their central processes degenerate over the first postnatal weeks. In the vestibular ganglia, neuronal cell death with apoptotic features is also observed. Immunostaining reveals that Kir4.1 is strongly expressed in stria vascularis of wild-type but not Kir4.1-/- mice. Within the spiral ganglion, Kir4.1 labeling was detected on satellite cells surrounding spiral ganglion neurons and axons. We conclude that Kir4.1 is crucial for normal development of the cochlea and hearing, via two distinct aspects of extracellular K(+) homeostasis: (1). in stria vascularis, Kir4.1 helps to generate the cochlear endolymph; and (2). in spiral and vestibular ganglia, Kir4.1 in surrounding glial cells helps to support the spiral and vestibular ganglion neurons and their projecting axons.

The caging of specific residues of proteins is a powerful tool. This discussion attempts to alert the reader to the considerations that must be made in preparing and analyzing a caged protein through nonsense suppression. Although the suppression methodology is conceptually straightforward, it not possible to provide a failsafe “cook book” method for using caged unnaturals. We have emphasized the preparation of caged receptors expressed in Xenopus oocytes, but these approaches can clearly be adapted to many other systems.

Glutamate-gated chloride (GluCl) channels from invertebrates can be activated by ivermectin (IVM) to produce electrical silencing in mammalian neurons. To improve this GluCl/IVM strategy, we sought to mutate the Caenorhabditis elegans GluCl channels so that they become insensitive to glutamate but retain their sensitivity to IVM. Based on structure-function studies of nicotinic acetylcholine receptor superfamily members, we tested in oocytes 19 point mutants at 16 residues in the beta-subunit likely to be involved in the response to glutamate. Y182F reduces the glutamate response by greater than six-fold, with little change to IVM responses, when coexpressed with wild-type (WT) GluCl alpha. For GluCl alphabeta(Y182F), the EC(50) and Hill coefficient for glutamate are similar to those of WT, indicating that the mutant decreases the efficacy of glutamate, but not the potency. Also, fluorescent proteins (enhanced green fluorescent protein, enhanced yellow fluorescent protein, enhanced cyan fluorescent protein; XFP) were inserted into the M3-M4 loop of the GluCl alpha, beta and beta(Y182F). We found no significant functional difference between these XFP-tagged receptors and WT receptors. The modified GluCl channel, without glutamate sensitivity but with a fluorescent tag, may be more useful in GluCl silencing strategies.

Selectively reducing the excitability of specific neurons will (1) allow for the creation of animal models of human neurological disorders and (2) provide insight into the global function of specific sets of neurons. We focus on a combined genetic and pharmacological approach to silence neurons electrically. We express invertebrate ivermectin (IVM)-sensitive chloride channels (Caenorhabditis elegans GluCl alpha and beta) with a Sindbis virus and then activate these channels with IVM to produce inhibition via a Cl- conductance. We constructed a three-cistron Sindbis virus that expresses the alpha and beta subunits of a glutamate-gated chloride channel (GluCl) along with the green fluorescent protein (EGFP) marker. Expression of the C. elegans channel does not affect the normal spike activity or GABA/glutamate postsynaptic currents of cultured embryonic day 18 hippocampal neurons. At concentrations as low as 5 nm, IVM activates a Cl- current large enough to silence infected neurons effectively. This conductance reverses in 8 hr. These low concentrations of IVM do not potentiate GABA responses. Comparable results are observed with plasmid transfection of yellow fluorescent protein-tagged (EYFP) GluCl alpha and cyan fluorescent protein-tagged (ECFP) GluCl beta. The present study provides an in vitro model mimicking conditions that can be obtained in transgenic mice and in viral-mediated gene therapy. These experiments demonstrate the feasibility of using invertebrate ligand-activated Cl- channels as an approach to modulate excitability.

A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation-pi interaction between serotonin and Trp183 of the serotonin channel 5-HT(3A)R is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation-pi interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine.

ATP-gated P2X channels are the simplest of the three families of transmitter-gated ion channels. Some P2X channels display a time- and activation-dependent change in permeability as they undergo the transition from the relatively Na(+)-selective I(1) state to the I(2) state, which is also permeable to organic cations. We report that the previously reported permeability change of rat P2X(2) (rP2X(2)) channels does not occur at mouse P2X(2) (mP2X(2)) channels expressed in oocytes. Domain swaps, species chimeras, and point mutations were employed to determine that two specific amino acid residues in the cytosolic tail domain govern this difference in behavior between the two orthologous channels. The change in pore diameter was characterized using reversal potential measurements and excluded field theory for several organic ions; both rP2X(2) and mP2X(2) channels have a pore diameter of approximately 11 A in the I(1) state, but the transition to the I(2) state increases the rP2X(2) diameter by at least 3 A. The I(1) to I(2) transition occurs with a rate constant of approximately 0.5 s(-1). The data focus attention on specific residues of P2X(2) channel cytoplasmic domains as determinants of permeation in a state-specific manner.

The hyperpolarization-activated transient current of mammalian 5-hydroxytryptamine transporters (SERT) expressed in Xenopus oocytes was studied. Human (h) and rat (r) SERT transient currents are blocked by HEPES with changes in the waveform kinetics, and the blockade of hSERT has use-dependent properties. HEPES also changes the time course of the prepriming step, especially for hSERT. Transient currents at hSERT and rSERT are also blocked by spermine and spermidine in the mM range, and by fluoxetine, cocaine, QX-314, and QX-222 in the μM range. These pharmacological and kinetic properties of transient current blockade emphasize the similarities between the transient current and phenomena at ion channels.

The nonsense codon suppression technique was used to incorporate o-nitrobenzyl cysteine or o-nitrobenzyl tyrosine (caged Cys or Tyr) into the 9' position of the M2 transmembrane segment of the gamma-subunit of the muscle nicotinic ACh receptor expressed in Xenopus oocytes. The caged amino acids replaced an endogenous Leu residue that has been implicated in channel gating. ACh-induced current increased substantially after ultraviolet (UV) irradiation to remove the caging group. This represents the first successful incorporation of caged Cys into a protein in vivo and the first incorporation of caged amino acids within a transmembrane segment of a membrane protein. The bulky nitrobenzyl group does not prevent the synthesis, assembly, or trafficking of the ACh receptor. When side chains were decaged using 1-ms UV light flashes, the channels with caged Cys or caged Tyr responded with strikingly different kinetics. The increase in current upon photolysis of caged Cys was too rapid for resolution by the voltage-clamp circuit [time constant (tau)10 ms], whereas the increase in current upon photolysis of caged Tyr was dominated by a phase with tau approximately 500 ms. Apparently, the presence of a bulky o-nitrobenzyl Tyr residue distorts the receptor into an abnormal conformation. Upon release of the caging group, the receptor relaxes, with tau approximately 500 ms, into a conformation that allows the channel to open. Tyr at the 9' position of the gamma-subunit greatly increases the ability of ACh to block the channel by binding within the channel pore. This is manifested in two ways. 1) A "rebound," or increase in current, occurs upon removal of ACh from the bathing medium; and 2) at ACh concentrations400 microM, inward currents are decreased through the mutated channel. The ability to incorporate caged amino acids into proteins should have widespread utility.

cAMP induces a protein-synthesis-dependent late phase of long-term potentiation (LTP) at CA3–CA1 synapses in acute hippocampal slices. Herein we report cAMP-mediated LTP and long-term depression (LTD) at monosynaptic CA3–CA1 cell pairs in organotypic hippocampal slice cultures. After bath application of the membrane-permeable cAMP analog adenosine 3′,5′-cyclic monophosphorothioate, Sp isomer (Sp-cAMPS), synaptic transmission was enhanced for at least 2 h. Consistent with previous findings, the late phase of LTP requires activation of cAMP-dependent protein kinase A and protein synthesis. There is also an early phase of LTP induced by cAMP; the early phase depends on protein kinase A but, in contrast to the later phase, does not require protein synthesis. In addition, the cAMP-induced LTP is associated with a reduction of paired-pulse facilitation, suggesting that presynaptic modification may be involved. Furthermore, we found that Sp-cAMPS induced LTD in slices pretreated with picrotoxin, a γ-aminobutyric acid type A (GABA A ) receptor antagonist. This form of LTD depends on protein synthesis and protein phosphatase(s) and is accompanied by an increased ratio of failed synaptic transmission. These results suggest that GABA A receptors can modulate the effect of cAMP on synaptic transmission and thus determine the direction of synaptic plasticity.

The integral membrane proteins of neurons and other excitable cells are generally resistant to high-resolution structural tools. In this thesis we present our efforts to probe the structure of the agonist-binding site of the nicotinic acetylcholine receptor (nAChR) using the tethered agonist approach, which combines chemical synthesis, the nonsense suppression methodology for unnatural amino acid incorporation and electrophysiology. In Chapter 2, we present the results of incorporating a series of tethered quaternary ammonium derivatives of tyrosine into the nAChR using the in vivo nonsense suppression methodology for incorporating unnatural amino acids site-specifically. At three sites, a constitutively active receptor results, but the pattern of activation as a function of chain length is different. At position alpha149, there is a clear preference for a three-carbon tether, while at position alpha93 tethers of 2-5 carbons are comparably effective. At position gamma55/delta57, all tethers except the shortest one can activate the receptor. Based on these and other data, a model for the binding site of the receptor can be developed by analogy to the acetylcholine esterase crystal structure. In Chapter 3, we report evidence that the N-terminal extracellular domain of nAChR is closely related to acetylcholine binding protein (AChBP), whose crystal structure was solved in May 2001. Based on the model obtained from docking acetylcholine into the structure of AChBP, we designed and incorporated a new tethered agonist, lysyl-carbamylcholine. Incorporation of this tethered agonist at several positions produced constitutively active receptors, with significant activity seen at alpha192, alpha193, and gamma119/delta121. These results demonstrated that the loop E residue gamma119/delta121 on the complementary subunit is very near the agonist-binding site. We also investigated the role of an intersubunit hydrogen bond, which was seen in the crystal structure of AChBP. Incorporation of tryptophan analogs that abolish the hydrogen bonding abilities slowed the desensitization of the receptor, which implied that this hydrogen bond might play a key role in the allosteric transitions of desensitization. In Chapter 4, we describe our efforts to prepare a short tethered agonist and the results of incorporating it into nAChR at alpha198 by chemical modification of cysteine mutants introduced by nonsense suppression methodology. Methanethiosulphonate ethyltrimethylammonium (MTSET) modification resulted in constitutive activity, which suggested the closeness of alpha198 to the agonist-binding site. In Chapter 5, methods in molecular biology, electrophysiology and molecular docking, and the synthesis of amino acids and dinucleotide dCA-amino acids are summarized.

Quantitative aspects of synaptic transmission can be studied by inserting green fluorescent protein (GFP) moieties into the genes encoding membrane proteins. To provide calibrations for measurements on synapses expressing such proteins, we developed methods to quantify histidine-tagged GFP molecules (His6-GFP) bound to Ni-NTA moieties on transparent beads (80‐120 mm diameter) over a density range comprising nearly four orders of magnitude (to 30 000 GFP:mm 2 ). The procedures employ commonly available Hg lamps, fluorescent microscopes, and CCD cameras. Two independent routes are employed: (1) single-molecule fluorescence measurements are made at the lowest GFP densities, providing an absolute calibration for macroscopic signals at higher GFP densities; (2) known numbers of His6-GFP molecules are coupled quantitatively to the beads. Each of the two independent routes provides linear data over the measured density range, and the two independent methods agree with root mean square (rms) deviation of 11‐21% over this range. These satisfactory results are obtained on two separate microscope systems. The data can be corrected for bleaching rates, which are linear with light intensity and become appreciable at intensities\ 1W :cm 2 . If a suitable GFP-tagged protein can be chosen and incorporated into a ‘knock-in’ animal, the density of the protein can be measured with an absolute accuracy on the order of 20%. © 2001 Elsevier Science B.V. All rights reserved.

We cloned the inebriated homologue MasIne from Manduca sexta and expressed it in Xenopus laevis oocytes. MasIne is homologous to neurotransmitter transporters but no transport was observed with a number of putative substrates. Oocytes expressing MasIne respond to hyperosmotic stimulation by releasing intracellular Ca2+, as revealed by activation of the endogenous Ca2+-activated Cl− current. This Ca2+ release requires the N-terminal 108 amino acid residues of MasIne and occurs via the inositol trisphosphate pathway. Fusion of the N terminus to the rat γ-aminobutyric acid transporter (rGAT1) also renders rGAT1 responsive to hyperosmotic stimulation. Immunohistochemical analyses show that MasIne and Drosophila Ine have similar tissue distribution patterns, suggesting functional identity. Inebriated is expressed in tissues and cells actively involved in K+ transport, which suggests that it may have a role in ion transport, particularly of K+. We propose that stimulation of MasIne releases intracellular Ca2+ in native tissues, activating Ca2+-dependent K+ channels, and leading to K+ transport.

Lentiviral vectors were constructed to express the weakly rectifying kidney K+ channel ROMK1 (Kir1.1), either fused to enhanced green fluorescent protein (EGFP) or as a bicistronic message (ROMK1-CITE-EGFP). The channel was stably expressed in cultured rat hippocampal neurons. Infected cells were maintained for 2–4 wk without decrease in expression level or evidence of viral toxicity, although 15.4 mM external KCl was required to prevent apoptosis of neurons expressing functional ROMK1. No other trophic agents tested could prevent cell death, which was probably caused by K+loss. This cell death did not occur in glia, which were able to support ROMK1 expression indefinitely. Functional ROMK1, quantified as the nonnative inward current at −144 mV in 5.4 mM external K+blockable by 500 μM Ba2+, ranged from 1 to 40 pA/pF. Infected neurons exhibited a Ba2+-induced depolarization of 7 ± 2 mV relative to matched EGFP-infected controls, as well as a 30% decrease in input resistance and a shift in action potential threshold of 2.6 ± 0.5 mV. This led to a shift in the relation between injected current and firing frequency, without changes in spike shape, size, or timing. This shift, which quantifies silencing as a function of ROMK1 expression, was predicted from Hodgkin-Huxley models. No cellular compensatory mechanisms in response to expression of ROMK1 were identified, making ROMK1 potentially useful for transgenic studies of silencing and neurodegeneration, although its lethality in normal K+ has implications for the use of K+ channels in gene therapy.

Recording and analysis of neuronal patch-clamp data involve many assumptions about membrane properties and cell morphology. Some of these assumptions introduce large errors or oversimplifications into the results. In particular, dendritic branching with high intracellular resistance leads to difficulty with capacitance calculation and transient subtraction, and may significantly distort measured currents. A two-compartment model, presented in detail here, provides a simple method of reducing many of these problems for the relatively simple case of cultured neurons studied with whole-cell patch electrodes. Some passive membrane properties may be accurately calculated, and the results may be used to correct recorded currents for resulting series resistance, intracellular resistance, and capacitive transient errors. The model may be tailored to particular cell types or experimental conditions. Programs to implement the algorithms are available from http://www.its.caltech.edu/ approximately nadeau/Rscomp.html.

The rat gamma-aminobutyric acid transporter GAT1 expressed in Xenopus oocytes was labeled at Cys74, and at one or more other sites, by tetramethylrhodamine-5-maleimide, without significantly altering GAT1 function. Voltage-jump relaxation analysis showed that fluorescence increased slightly and monotonically with hyperpolarization; the fluorescence at -140 mV was approximately 0. 8% greater than at +60 mV. The time course of the fluorescence relaxations was mostly described by a single exponential with voltage-dependent but history-independent time constants ranging from approximately 20 ms at +60 mV to approximately 150 ms at -140 mV. The fluorescence did not saturate at the most negative potentials tested, and the midpoint of the fluorescence-voltage relation was at least 50 mV more negative than the midpoint of the charge-voltage relation previously identified with Na(+) binding to GAT1. The presence of gamma-aminobutyric acid did not noticeably affect the fluorescence waveforms. The fluorescence signal depended on Na(+) concentration with a Hill coefficient approaching 2. Increasing Cl(-) concentration modestly increased and accelerated the fluorescence relaxations for hyperpolarizing jumps. The fluorescence change was blocked by the GAT1 inhibitor, NO-711. For the W68L mutant of GAT1, the fluorescence relaxations occurred only during jumps to high positive potentials, in agreement with previous suggestions that this mutant is trapped in one conformational state except at these potentials. These observations suggest that the fluorescence signals monitor a novel state of GAT1, intermediate between the E*(out) and E(out) states of Hilgemann, D.W., and C.-C. Lu (1999. J. Gen. Physiol. 114:459-476). Therefore, the study provides verification that conformational changes occur during GAT1 function.

Many ion channels and receptors display striking phenotypes for gainof-function mutations but milder phenotypes for null mutations. Gain of molecular function can have several mechanistic bases: selectivity changes, gating changes including constitutive activation and slowed inactivation, elimination of a subunit that enhances inactivation, decreased drug sensitivity, changes in regulation or trafficking of the channel, or induction of apoptosis. Decreased firing frequency can occur via increased function of K+or Cl−channels. Channel mutants also cause gain-of-function syndromes at the cellular and circuit level; of these syndromes, the cardiac long-QT syndromes are explained in a more straightforward way than are the epilepsies. G protein– coupled receptors are also affected by activating mutations.

G protein-activated K+ channel (GIRK) subunits possess a conserved extracellular integrin-binding motif (RGD) and bind directly to beta1 integrins. We expressed GIRK1/GIRK4 channels labeled with green fluorescent protein in fibroblast cell lines expressing or lacking beta1 integrins. Neither plasma membrane localization nor agonist-evoked GIRK currents were affected by the absence of beta1 integrins or by incubation with externally applied RGD-containing peptide. Mutation of the aspartate (D) of RGD impaired currents, GIRK glycosylation, and membrane localization, but the interaction with beta1 integrins remained intact. Thus, beta1 integrins are not essential for functional GIRK expression; and the GIRK-integrin interactions involve structural elements other than the RGD motif.

Regulators of G protein signaling (RGS) stimulate the GTPase activity of G protein Galpha subunits and probably play additional roles. Some RGS proteins contain a Ggamma subunit-like (GGL) domain, which mediates a specific interaction with Gbeta 5. The role of such interactions in RGS function is unclear. RGS proteins can accelerate the kinetics of coupling of G protein-coupled receptors to G-protein-gated inwardly rectifying K+ (GIRK) channels. Therefore, we coupled m2-muscarinic acetylcholine receptors to GIRK channels in Xenopus oocytes to evaluate the effect of Gbeta 5 on RGS function. Co-expression of either RGS7 or RGS9 modestly accelerated GIRK channel kinetics. When Gbeta 5 was co-expressed with either RGS7 or RGS9, the acceleration of GIRK channel kinetics was strongly increased over that produced by RGS7 or RGS9 alone. RGS function was not enhanced by co-expression of Gbeta 1, and co-expression of Gbeta 5 alone had no effect on GIRK channel kinetics. Gbeta 5 did not modulate the function either of RGS4, an RGS protein that lacks a GGL domain, or of a functional RGS7 construct in which the GGL domain was omitted. Enhancement of RGS7 function by Gbeta 5 was not a consequence of an increase in the amount of plasma membrane or cytosolic RGS7 protein.

We demonstrate that it is possible to observe single fluorescent molecules using a standard fluorescence microscope with mercury lamp excitation and an inexpensive cooled charge-coupled device (CCD) camera. With this equipment, we have been able to observe single molecules of tetramethyl-rhodamine, rhodamine 6G, fluorescein isothiocyanate and green fluorescent protein. Immobilized molecules were observed both in air and in aqueous solution.

The CNS abundantly expresses P2X receptor channels for ATP; of these the most widespread in the brain is the P2X(4) channel. We show that ivermectin (IVM) is a specific positive allosteric effector of heterologously expressed P2X(4) and possibly of heteromeric P2X(4)/P2X(6) channels, but not of P2X(2), P2X(3), P2X(2)/P2X(3,) or P2X(7) channels. In the submicromolar range (EC(50,) approximately 250 nM) the action of IVM was rapid and reversible, resulting in increased amplitude and slowed deactivation of P2X(4) channel currents evoked by ATP. IVM also markedly increased the potency of ATP and that of the normally low-potency agonist alpha, beta-methylene-ATP in a use- and voltage-independent manner without changing the ion selectivity of P2X(4) channels. Therefore, IVM evokes a potent pharmacological gain-of-function phenotype that is specific for P2X(4) channels. We also tested whether IVM could modulate endogenously expressed P2X channels in the adult trigeminal mesencephalic nucleus and hippocampal CA1 neurons. Surprisingly, IVM produced no significant effect on the fast ATP-evoked inward currents in either type of neuron, despite the fact that IVM modulated P2X(4) channels heterologously expressed in embryonic hippocampal neurons. These results suggest that homomeric P2X(4) channels are not the primary subtype of P2X receptor in the adult trigeminal mesencephalic nucleus and in hippocampal CA1 neurons.

Fast synaptic transmission depends on the selective ionic permeability of transmitter-gated ion channels. Here we show changes in the ion selectivity of neuronal P2X transmitter-gated cation channels as a function of time (on the order of seconds) and previous ATP exposure. Heterologously expressed P2X2, P2X2/P2X3 and P2X4 channels as well as native neuronal P2X channels possess various combinations of mono- or biphasic responses and permeability changes, measured by NMDG+ and fluorescent dye. Furthermore, in P2X4 receptors, this ability to alter ion selectivity can be increased or decreased by altering an amino-acid residue thought to line the ion permeation pathway, identifying a region that governs this activity-dependent change.

Heteromultimeric G protein-activated inward rectifier K+ (GIRK) channels, abundant in heart and brain, help to determine the cellular membrane potential as well as the frequency and duration of electrical impulses. The sequence arginine-glycine-aspartate (RGD), located extracellularly between the first membrane-spanning region and the pore, is conserved among all identified GIRK subunits but is not found in the extracellular domain of any other cloned K+ channels. Many integrins, which, like channels, are integral membrane proteins, recognize this RGD sequence on other proteins, usually in the extracellular matrix. We therefore asked whether GIRK activity might be regulated by direct interaction with integrin. Here, we present evidence that mutation of the RGD site to RGE, particularly on the GIRK4 subunit, decreases or abolishes GIRK current. Furthermore, wild-type channels can be co-immunoprecipitated with integrin. The total cellular amount of expressed mutant GIRK channel protein is the same as the wild-type protein; however, the amount of mutant channel protein that localizes to the plasma membrane is decreased relative to wild-type, most likely accounting for the diminished GIRK current detected. GIRK channels appear to bind directly to integrin and to require this interaction for proper GIRK channel membrane localization and function.

The rat and human serotonin transporters (rSERT and hSERT, respectively) were expressed inXenopusoocytes and studied using site-directed mutagenesis, electrophysiological recordings, and [3H]5-HT uptake measurements. rSERT, but not hSERT, displayed increased transport-associated current at low pH. Chimeras and point mutations showed that, of the 52 nonidentical residues, a single residue at position 490 (threonine in rSERT and lysine in hSERT) governs this difference. Furthermore, potentiation required the glutamate residue at position 493. Cysteine substitution and alkylation experiments showed that residue 493 is extracellular. Cysteine at 493 increased, whereas aspartate decreased, the net charge movement per transported 5-HT molecule. The mutations at this region did not significantly affect other aspects of SERT function, including agonist-independent leakage current, voltage-dependent transient current, and H+current. This region may therefore be part of an external gate required for rSERT function. The data and analyses show that, in the absence of detailed structural information, a gate–lumen–gate scheme is useful for interpreting results from mutations that alter functional properties of neurotransmitter transporters.

We have developed a method to analyze the relative contributions of pre- and postsynaptic actions of a particular gene product in neurons in culture and potentially in slices using adenovirus-mediated gene transfer. A recombinant virus directed the expression of both a GFP reporter protein and TrkB.T1, a C-terminal truncated dominant negative TrkB neurotrophin receptor. When expressed in the presynaptic cell at synapses between embryonic hippocampal neurons in culture, the dominant negative TrkB.T1 inhibited two forms of synaptic potentiation induced by the neurotrophin brain-derived neurotrophic factor (BDNF): (i) greater evoked synaptic transmission and (ii) higher frequency of spontaneous miniature synaptic currents. These inhibition effects are not seen if the transgene is expressed only in the postsynaptic cell. We conclude that BDNF-TrkB signal transduction in the presynaptic terminal leads to both types of potentiation and is therefore the primary cause of synaptic enhancement by BDNF in these neurons.

Electrophysiological measurements add new dimensions to the study of neurotransmitter transporters. (1) One can perform measurements with high temporal resolution (however, uptake of radioactive substrate is limited in that it cannot resolve events that occur within 1 sec, which is greater than the time of a single transport cycle). (2) Electrophysiology provides information about partial steps in transport cycles, including the fact that ion binding and dissociation at transporters can generate currents, which provides new insights about ion- transporter interaction. (3) Electrophysiology provides information about single transporter molecules, from patch-clamp recordings of single-channel activity of neurotransmitter transporters. At present, little is known about the molecular mechanisms that underlie transport. Electrophysiological measurements of ion binding and permeation contribute to the analysis of mutations that affect transport.13,21 Electrophysiology may help to identify amino acids and domains in neurotransmitter transporters that participate in specific ways in the transport process, such as ion neurotransmitter binding, permeation pathways, voltage sensors, and gates. In combination with spectroscopic measurements,25 it may also be possible to identify the actual conformational changes of the proteins that enable substrate translocation.

Publisher Summary Recombinant (and thus nonreplicating) adenovirus is at present the gene transfer vector of choice for introducing genes of interest into postmitotic neurons in vitro, in slice cultures, and in vivo. The per-cell efficiency can be close to 100%. By controlling the multiplicity of infection and by choice of a suitable promoter, the level of expression can be controlled. Expression can persist for weeks under favorable conditions. Cytotoxic effects are sometimes a problem, but usually are not severe. Adenovirions enter the cells by a complex pathway, usually by initial attachment to an integrin, followed by endocytosis into endosomes. The viral DNA is transported to the nucleus where it exists as a linear molecule of approximately 36-40 kb, with a covalently attached protein at each end. This protein presumably contributes to nuclease resistance of the DNA and thus to the persistence of expression. In proliferating cells, the DNA is diluted during cell division. Transgenic mice, either deleted in (i.e., “knockouts”) or overexpressing a gene of interest, are an attractive tool for studying effects of genes in neurobiology. The recombinant adenovirus system can be established in a somewhat shorter period of time than can a transgenic mouse. If an introduced gene has a lethal embryonic phenotype, the adenovirus system is much more convenient for studying the adult phenotype. On the other hand, the volume of tissue in vivo that can be infected with adenovirus is limited by the number of injections and volume per injection that an animal will tolerate, because the virus does not spread appreciably through the extracellular space. Therefore, for a nonsecreted molecule such as a receptor or an ion channel, the broad region of expression achieved with transgenic mice cannot be duplicated by adenovirus.

Publisher Summary This chapter has adapted the nonsense codon suppression method for the incorporation of unnatural amino acids into membrane proteins in a Xenopus oocyte expression system. Combining this method with electrophysiologic analysis allows it to probe structure-function relationships in ion channels and receptors in ways not possible with conventional mutagenesis. In the absence of atomic-scale, structural data for membrane proteins, these techniques can provide detailed structural information. In the nonsense codon suppression method, Xenopus oocytes are coinjected with two RNAs: (1) mRNA transcribed in vitro from a mutated cDNA containing a TAG nonsense (stop) codon at the position of interest and (2) a suppressor tRNA containing the corresponding anticodon, CUA, and chemically acylated at the end with an amino acid. During protein synthesis, the aminoacylated suppressor tRNA directs the incorporation of the amino acid into the desired position of the protein. Because the amino acid is appended synthetically, it is not limited to the natural, and many unnatural amino acids have been incorporated into various proteins using this method. This chapter describes suppressor tRNA design and synthesis, chemical acylation of the suppressor tRNA, relevant organic synthesis methods, and optimization of mRNA and suppressor tRNA for Xenopus oocyte expression.

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K + channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the “signature” Cys128–Cys142 disulfide loop of the nAChR α subunit.

Most working proteins, including metabolic enzymes, transcription regulators, and membrane receptors, transporters, and ion channels, share the property of allosteric coupling. The term 'allosteric' means that these proteins mediate indirect interactions between sites that are physically separated on the protein. In the example of ligand-gated ion channels, the binding of a suitable ligand elicits local conformational changes at the binding site, which are coupled to further conformational changes in regions distant from the binding site. The physical motions finally arrive at the site of biological activity: the ion-permeating pore. The conformational changes that lead from the ligand binding to the actual opening of the pore comprise 'gating'. In 1956, del Castillo and Katz suggested that the competition between different ligands at nicotinic acetylcholine receptors (nAChRs) could be explained by formation of an intermediate, ligand-bound, yet inactive state of the receptor, which separates the active state of the receptor from the initial binding of the ligand (del Castillo & Katz, 1957). This 'binding-then-gating', two-step model went beyond the then-prevailing drug-receptor model that assumes a single bimolecular binding reaction, and paralleled Stephenson's conceptual dichotomy of 'affinity' and 'efficacy' (Stephenson, 1956). In 1965 Monod, Wyman and Changeux presented a simple allosteric model (the MWC model) (Monod et al. 1965) that explained the cooperative binding of oxygen to haemoglobin; it was adopted as an important paradigm for ligand-gated channels soon after its initial formulation (Changeux et al. 1967; Karlin, 1967; Colquhoun, 1973).

Recent evidence indicates that several members of the Na+-coupled transporter family are regulated, and this regulation in part occurs by redistribution of transporters between intracellular locations and the plasma membrane. We elucidate components of this process for both wild-type and mutant GABA transporters (GAT1) expressed inXenopusoocytes using a combination of uptake assays, immunoblots, and electrophysiological measurements of membrane capacitance, transport-associated currents, and GAT1-specific charge movements. At low GAT1 expression levels, activators of protein kinase C (PKC) induce redistribution of GAT1 from intracellular vesicles to the plasma membrane; at higher GAT1 expression levels, activators of PKC fail to induce this redistribution. However, coinjection of total rat brain mRNA with GAT1 permits PKC-mediated modulation at high transporter expression levels. This effect of brain mRNA on modulation is mimicked by coinjection of syntaxin 1a mRNA and is eliminated by injecting synaptophysin or syntaxin antisense oligonucleotides. Additionally, botulinum toxins, which inactivate proteins involved in vesicle release and recycling, reduce basal GAT1 expression and prevent PKC-induced translocation. Mutant GAT1 proteins, in which most or all of a leucine heptad repeat sequence was removed, display altered basal distribution and lack susceptibility to modulation by PKC, delineating one region of GAT1 necessary for its targeting. Thus, functional regulation of GAT1 in oocytes occurs via components common to transporters and to trafficking in both neural and non-neural cells, and suggests a relationship between factors that control neurotransmitter secretion and the components necessary for neurotransmitter uptake.

The rat serotonin transporter expressed inXenopusoocytes displays an inward current in the absence of 5-HT when external pH is lowered to 6.5 or below. The new current differs from the leakage current described previously in two ways. (1) It is ∼10-fold larger at pH 5 than the leakage current at pH 7.5 and reaches 1000 H+/sec per transporter at extremes of voltage and pH with no signs of saturation. (2) It is selective for H+by reversal potential measurements. Similar H+-induced currents are also observed in several other ion-coupled transporters, including the GABA transporter, the dopamine transporter, and the Na+/glucose transporter. The high conductance and high selectivity of the H+-induced current suggest that protons may be conducted via a hydrogen-bonded chain (a “proton-wire mechanism”) formed at least partially by side chains within the transporter. In addition, pH affects other conducting states of rat serotonin transporter. Acidic pH potentiates the 5-HT-induced, transport-associated current and inhibits the hyperpolarization-activated transient current. The dose–response relationships for these two effects suggest that two H+binding sites, with pKavalues close to 5.1 and close to 6.3, govern the potentiation of the 5-HT-induced current and the inhibition of the transient current, respectively. These results are important for developing structure-function models that explain permeation properties of neurotransmitter transporters.

Cyclic nucleotide-gated (cng) channels are important components of signaling systems mediating sensory transduction. In vertebrate photoreceptors, light activates a signaling cascade that causes a decrease in intracellular cGMP concentrations, closing retinal cng channels. Signal transduction in olfactory receptor neurons is believed to proceed via G-protein-mediated elevation of intracellular cAMP in response to odorant binding by 7-helix receptors. cAMP opens the olfactory cng channel, which is highly permeable to Ca2+. Here we demonstrate byin situhybridization and immunohistochemistry with subunit-specific antibodies that both subunits of the heteromeric rat olfactory cng channel are also widely expressed in the brain. Expression of the retinal rod cng channel, however, can be detected only in the eye. In the adult hippocampus, the olfactory cng channel is expressed on cell bodies and processes of CA1 and CA3 neurons. In cultured embryonic hippocampal neurons, the channel is localized to a subset of growth cones and processes. We recorded conductances with the electrophysiological characteristics of the heteromeric olfactory cng channel in excised inside-out patches from these cultured neurons. We also show that Ca2+influx into hippocampal neurons in response to cyclic nucleotide elevation can be detected using fura-2 imaging. Cyclic nucleotide elevation has been implicated in several mechanisms of synaptic plasticity in the hippocampus, and these mechanisms also require elevation of intracellular Ca2+. Our results suggest that the “olfactory” cng channel could regulate synaptic efficacy in brain neurons by modulating Ca2+levels in response to changes in cyclic nucleotide concentrations.

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K + channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by G βγ subunits, but instead shows constitutive activation, and (ii) is no longer a K + -selective channel but conducts Na + as well. The present experiments on weaver GIRK2 ( wv GIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na + concentration. In particular, manipulations that decrease intracellular Na + produce a component of Na + -permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na + influx through the wv GIRK2 channel, leading to additional Na + activation. We also show that the wv GIRK2 channel is permeable to Ca 2+ , providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wv GIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K + channels.

Expression of G protein alpha subunits of the Gq family with various G protein-coupled receptors induces activation of an inositol 1,4,5-trisphosphate (IP3)/Ca2+-mediated Cl- conductance in Xenopus oocytes. Our present data show that two members of this family, the human Galpha 16 subunit and the murine homologue Galpha 15, can induce both activation and inhibition of these agonist-induced currents. Although extremely low amounts (10-50 pg) of injected Galpha 16 subunit cRNA cause modest (~2-fold) enhancement of ligand-induced Cl- currents in oocytes co-injected with thyrotropin-releasing hormone (TRH) receptor cRNA 48 h postinjection, larger Galpha 16 and Galpha 15 cRNA injections cause >10-fold inhibition of TRH or 5HT2c receptor responses. The inhibition is analyzed in this study. The inhibited currents are recovered if various Gbeta gamma subunit combinations are also expressed with the Galpha subunits. The constitutively active mutant, Galpha 16Q212L, also causes a strong attenuation of the ligand-induced Cl- currents, but this inhibition is not recovered by co-expression of Gbeta gamma subunits. These results indicate that the free Galpha subunit is responsible for the inhibitory signal. Although expression of TRH receptor alone produces maximum responses approximately 48 h after injection, co-expression of TRH receptor with Galpha 16 results in enhanced responses 6-12 h postinjection, followed by complete attenuation at 36 h. Furthermore, injection of Galpha 16 cRNA alone at comparable levels gives rise to spontaneous Cl- currents within 6-12 h postinjection, suggesting that the early spontaneous activation underlies the later suppression. Expression of other G protein alpha subunits of the Gq family, at cRNA levels considerably higher than effective for Galpha 16, produces both analogous spontaneous Cl- currents and, later, inhibition of ligand-induced Cl- currents. Experiments with direct injection of IP3 and of Ca2+ suggest that this inhibition is consistent with the down-regulation of IP3 receptors. These data indicate that both enhancement and inhibition of signaling through G protein-coupled receptors can be mediated by the expression level and/or activity of an individual G protein.

We investigated the stoichiometry of the heteromultimeric G protein-coupled inward-recitfier K^+ channel (GIRK) formed from GIRK1 and GIRK4 subunits. Multimeric GIRK constructs with several concatenated channel subunits were expressed in Xenopus oocytes. Coexpression of various trimeric constructs with different monomers clearly showed that the functional channel has stoichiometry (GIRK1)_2(GIRK4)_2. Efforts to establish a preferred arrangement of subunits around the channel pore suggest that more than one arrangement may be viable.

1. The structural determinants of a G protein-activated inwardly rectifying potassium channel, GIRK1 (KIR3.1), involved in voltage- and time-dependent gating properties were investigated by heterologous expression of chimeric constructs and point mutants in Xenopus oocytes. 2. Chimeras between GIRK1 and the weakly rectifying potassium channel, ROMK1 (KIR1.1), indicate that residues in the putative transmembrane segments TM1 and TM2 affect the steep inward rectification of GIRK1, while residues in the main pore-forming domain, the P-region segment, are critical for the manifestation of GIRK1 time-dependent activation. 3. Phenylalanine 137 in the P-region of GIRK1 is unique; in ROMK1, as in other inward rectifiers, there is a serine residue at this position. Mutation of the phenylalanine 137 to serine leads to expression of currents with nearly time-independent activation. 4. An acidic residue (aspartate) in TM2 partially controls the time- and voltage-dependent gating in IRK1 (KIR2.1). Mutation of the equivalent aspartate 173 to glutamine in GIRK1 did not abolish the time-dependent activation but did decrease the degree of inward rectification. 5. These results reveal an important role for the P-region in controlling the time-dependent gating of an inwardly rectifying potassium channel and suggest a close relationship between permeation and gating in this family of K+ channels.

The direct interaction of recombinant G beta 1 gamma 2 proteins with the carboxyl terminal domain of a G protein-gated inward rectifier K channel subunit, Kir3.4 (GIRK4), was measured in real time using biosensor chip technology. The carboxyl terminus of Kir3.4 (a.a. 186-419) was expressed in bacteria as a glutathione-S-transferase (GST) fusion protein, GST-Kir3. 4ct. GST-Kir3.4ct was immobilized to the surface of a biosensor chip by high affinity binding of the GST domain to a covalently attached anti-GST antibody. The association and dissociation rates of G beta 1 gamma 2 dimers with the immobilized Kir3.4ct domain were temporally resolved as a change in refractive index detected by surface plasmon resonance. Specific binding of G beta 1 gamma 2 dimers to Kir3.4ct was characterized by a dissociation rate (kd) of approximately 0.003 s-1. Association kinetics were dominated by a concentration-independent component (time constant approximately 50 s) which complicates models of binding and may indicate conformational changes during binding of G beta 1 gamma 2 to Kir3.4ct. The estimated equilibrium dissociation binding constant (Kd) was approximately 800 nM. These studies demonstrate that G beta gamma dimers interact directly with the Kir3.4 channel subunit, and suggest interesting details in the interaction with the major cytosolic carboxyl terminal domain. The slow G beta 1 gamma 2 dissociation rate measured on the sensor chip is similar in magnitude to a slow component of channel deactivation measured electrophysiologically in Xenopus oocytes expressing Kir3.1/3.4 multimeric channels and a G protein-coupled receptor. Biosensor-based experiments such as those described here will complement electrophysiological studies on the molecular basis of G protein interactions with Kir channels and other ion channel proteins.

We have investigated the possible role of selected negatively-charged amino acids of the sodium and chloride-coupled GABA transporter GAT-1 on sodium binding. These residues located adjacent to putative transmembrane domains and which are conserved throughout the large superfamily of neurotransmitter transporters were changed by site-directed mutagenesis. The functional consequences were that one of the residues, glutamate-101, was critical for transport. Its replacement by aspartate left only 1% of the activity, and no activity could be detected when it was replaced by other residues. Expression levels and targeting to the plasma membrane of the mutant transporters appeared normal. Transient sodium currents were not observed in the mutants, and increased sodium concentrations did not affect the percentage of wild type transport of the E101D mutant. It is concluded that residue glutamate-101 is critical for one or more of the conformational changes of GAT-1 during its transport cycle.

In nicotinic acetylcholine receptors (nAChR), as well as glycine, GABAA (gamma-aminobutyric acid), serotonin (5-HT3), and GluCl glutamate receptors, a leucine residue at the approximate midpoint of the M2 transmembrane domain (the 9' position) is conserved across most known subunits. Structural data for the nAChR suggest that the Leu 9' residues occupy a 'kink' in each of the five M2 helices and point into the closed channel; in the opening step, the M2 helices rotate so that Leu 9' side chains no longer occlude the conduction pathway. Mutation of Leu 9' to one of several other residues slows desensitization and increases sensitivity to agonist. We have exploited the alpha 2 beta gamma delta stoichiometry of muscle nAChR to express receptors with ms* = 0 to 5 Leu 9'Ser mutated subunits. Strikingly, each Leu 9'Ser mutation shifts the dose-response relation for ACh to the left by approximately 10-fold; a nAChR with ms* = 4 is 10(4)-fold more sensitive than the wild type. The results suggest that each of the five Leu 9' residues participates independently and symmetrically in a key step in the structural transition between the closed and open states.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

The voltage-, time-, and K(+)-dependent properties of a G protein-activated inwardly rectifying K+ channel (GIRK1/KGA/Kir3.1) cloned from rat atrium were studied in Xenopus oocytes under two-electrode voltage clamp. During maintained G protein activation and in the presence of high external K+ (VK = 0 mV), voltage jumps from VK to negative membrane potentials activated inward GIRK1 K+ currents with three distinct time-resolved current components. GIRK1 current activation consisted of an instantaneous component that was followed by two components with time constants tau f approximately 50 ms and tau s approximately 400 ms. These activation time constants were weakly voltage dependent, increasing approximately twofold with maximal hyperpolarization from VK. Voltage-dependent GIRK1 availability, revealed by tail currents at -80 mV after long prepulses, was greatest at potentials negative to VK and declined to a plateau of approximately half the maximal level at positive voltages. Voltage-dependent GIRK1 availability shifted with VK and was half maximal at VK -20 mV; the equivalent gating charge was approximately 1.6 e-. The voltage-dependent gating parameters of GIRK1 did not significantly differ for G protein activation by three heterologously expressed signaling pathways: m2 muscarinic receptors, serotonin 1A receptors, or G protein beta 1 gamma 2 subunits. Voltage dependence was also unaffected by agonist concentration. These results indicate that the voltage-dependent gating properties of GIRK1 are not due to extrinsic factors such as agonist-receptor interactions and G protein-channel coupling, but instead are analogous to the intrinsic gating behaviors of other inwardly rectifying K+ channels.

Recent cloning of a family of genes encoding inwardly rectifying K+ channels has provided the opportunity to explain some venerable problems in membrane biology. An expanding number of novel inwardly rectifying K+ channel clones has revealed multiple channel subfamilies that have specialized roles in cell function. The molecular determinants of inward rectification have been largely elucidated with the discovery of endogenous polyamines that act as voltage-dependent intracellular channel blockers, and with the identification of a critical site in the channel that mediates high-affinity block by both polyamines and Mg2+.

We describe a highly disabling congenital myasthenic syndrome (CMS) associated with rapidly decaying, low-amplitude synaptic currents, and trace its cause to a valine to leucine mutation in the signature cystine loop (cys-loop) of the AChR α subunit. The recently solved crystal structure of an ACh-binding protein places the cys-loop at the junction between the extracellular ligand-binding and transmembrane domains where it may couple agonist binding to channel gating. We therefore analyzed the kinetics of ACh-induced single-channel currents to identify elementary steps in the receptor activation mechanism altered by the αV132L mutation. The analysis reveals that αV132L markedly impairs ACh binding to receptors in the resting closed state, decreasing binding affinity for the second binding step 30-fold, but attenuates gating efficiency only about twofold. By contrast, mutation of the equivalent valine residue in the δ subunit impairs channel gating approximately fourfold with little effect on ACh binding, while corresponding mutations in the β and ε subunits are without effect. The unique functional contribution of the α subunit cys-loop likely owes to its direct connection via a β strand to αW149 at the center of the ligand-binding domain. The overall findings reveal functional asymmetry between cys-loops of the different AChR subunits in contributing to ACh binding and channel gating.

Drug-receptor binding interactions of four agonists, ACh, nicotine, and the smoking cessation compounds varenicline (Chantix) and cytisine (Tabex), have been evaluated at both the 2:3 and 3:2 stoichiometries of the α4β2 nicotinic acetylcholine receptor (nAChR). Previous studies have established that unnatural amino acid mutagenesis can probe three key binding interactions at the nAChR: a cation−π interaction, and two hydrogen-bonding interactions to the protein backbone of the receptor. We find that all drugs make a cation−π interaction to TrpB of the receptor. All drugs except ACh, which lacks an N^(+)H group, make a hydrogen bond to a backbone carbonyl, and ACh and nicotine behave similarly in acting as a hydrogen-bond acceptor. However, varenicline is not a hydrogen-bond acceptor to the backbone NH that interacts strongly with the other three compounds considered. In addition, we see interesting variations in hydrogen bonding interactions with cytisine that provide a rationalization for the stoichiometry selectivity seen with this compound.

The glutamate-gated chloride channel (GluCl) is an invertebrate, ligand-gated anion channel of the Cys-loop receptor family. It is activated by the endogenous neurotransmitter L-glutamate and by the antiparasitic drug ivermectin. The crystal structure of the Caenorhabditis elegans GluCl alpha homopentamer (3.3A resolution; Hibbs & Gouaux, 2011) shows the location of the glutamate binding site, the separate ivermectin site, and the highly conserved leucine residue at the 9’ position of the pore-lining M2 transmembrane domain. Mutation of this L9’ residue in other Cys-loop receptors dramatically increases agonist sensitivity. Using whole-cell patch clamp, we found that six of seven mutations at this position (L9's, A, F, I, T, V, but not G) increased the glutamate sensitivity of the heteromeric GluCl channel by factors of 5- to 80-fold. Beta-branched amino acids (Ile, Thr, Val) gave the greatest reductions in EC50. Analysis of side chain properties revealed that helix-destabilizing energy correlated with increased glutamate sensitivity. Except for WT and the L9’F mutation, we were unable to detect glutamate- and/or ivermectin-induced changes in membrane potential using a fluorescent membrane potential assay (FlexStation, Molecular Devices), probably because the spontaneous activity of the mutant channels obscured the fluorescence changes. However, whole-cell patch clamp and fluorescent membrane potential experiments confirmed that the L9’F mutation increased both the glutamate and ivermectin sensitivity of the GluCl channel. Increasing GluCl sensitivity to ivermectin will benefit its use as a neuronal silencing tool.

Neuronal nicotinic acetylcholine receptors (nAChRs) are abundantly expressed in the central nervous system. Neurons in several brain regions co-express alpha5, alpha4 and beta2 subunits. Changes in the receptor composition and stoichiometry of alpha4 and beta2 containing nAChRs are pertinent to disease states. Several genome-wide association and candidate gene studies have identified polymorphisms in the gene for the alpha5 subunit that are linked to an increased risk for nicotine addiction, alcohol addiction and lung cancer. However more information is required about the functional effects of incorporating a normal or mutated alpha5 subunit into the alpha4beta2 receptor pentamer. Here we examine differences in I-V relationships and dose-response relations between alpha4beta2 receptors and alpha4beta2 receptors including alpha5 subunits containing various mutations to the M2 domain expressed in Xenopus oocytes.

An essential function of myelinating oligodendroglia in the mammalian central nervous system is the regulation of extracellular potassium levels by means of a prominent inwardly rectifying K+ current. Cardiac and neuronal K+ inward rectifiers are either activated by hyperpolarizing voltages or controlled by neurotransmitters through the action of receptor-activated G proteins. Neuromodulation of inward rectifiers has not previously been considered as a way to regulate oligodendrocyte function. Here we report the expression of serotonin, somatostatin and muscarinic acetylcholine G protein-coupled receptors in rat brain oligodendrocytes. Activation of these receptors leads to pertussis toxin-sensitive inhibition of inwardly rectifying K+ channels within < 1 s. By contrast, in the heart and in neurons, similar pathways activate an inwardly rectifying conductance. Thus, transmitter-mediated blockade of inward rectifiers appears to be an oligodendrocyte-specific variation of a common motif for convergent signalling pathways. In vivo, expression of this mechanism, which may be dependent on neuron-glia signalling, may have a regulatory role in K+ homeostasis during neuron activity in the central nervous system.