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PubMed2000

The Croonian Lecture 2000. Nicotinic acetylcholine receptor and the structural basis of fast synaptic transmission.

Communication in the nervous system takes place at chemical and electrical synapses, where neurotransmitter-gated ion channels, such as the nicotinic acetylcholine (ACh) receptor, and gap junction channels control propagation of electrical signals from one cell to the next. Newly developed electron crystallographic methods have revealed the structures of these channels trapped in open as well as closed states, suggesting how they work. The ACh receptor has large vestibules extending from the membrane which shape the ACh-binding pockets and facilitate selective transport of cations across a narrow membrane-spanning pore. When ACh enters the pockets it triggers a concerted conformational change that opens the pore by destabilizing a gate in the middle of the membrane made by a ring of pore-lining alpha-helical segmets. The alternative 'open' configuration of pore-lining segments reshapes the lumen and creates new surfaces, allowing the ions to pass through. The gap junction channel uses a similar structural mechanism, involving coordinated rearrangements of alpha-helical segments in the plane of the membrane, to open its pore.

Acetylcholine↗
PubMed1997

Cable properties of dendrites in hippocampal neurons of the rat mapped by a voltage-sensitive dye.

Dendrites of pyramidal neurons from embryonic rat hippocampus are investigated in culture using a voltage-sensitive fluorescent dye. The electrical response to somatic stimulation is observed as a time-resolved map with a resolution of 0.9 microm at a time constant of 0.4 ms without signal averaging. The data are interpreted in terms of a tapering cable with Hodgkin-Huxley parametrization. The spread of short hyperpolarizing transients is damped by capacitive shunting. The invasion of an action potential is boosted by voltage-gated conductances of a low density. No irregularity is observed at a bifurcation. The passive cable parameters of internal resistance and membrane resistance at resting voltage are Ri = 300 omega cm and Rm = 40 (k)omega cm2 respectively, at a maximum sodium conductance of approximately 4.4 mS/cm2. The electrotonic length constant and the dynamic length constant at 1 kHz are 580 and 90 microm respectively. These results are compatible with electrophysiological data of dendrites in slices of adult hippocampus and with optical data of narrow processes of leech neurons in culture. The functional implications of boosting an action potential by voltage-gated channels of low density are considered.

Action Potentials↗
PubMed1992

Effects of a benz[e]indene on gamma-aminobutyric acid-gated chloride currents in cultured postnatal rat hippocampal neurons.

Benz[e]indenes (BIs) are tricyclic molecules that can be envisioned as steroids without an A-ring. Because certain steroids are known to alter gamma-aminobutyric acid (GABA) responses in central neurons, we examined the effects of a substituted BI resembling 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP) on GABA-gated chloride currents in cultured postnatal rat hippocampal neurons. The compound, BI-1, reversibly potentiated GABA currents at concentrations of > 10 nM, with an EC50 value of 0.2 microM. BI-1 increased the apparent affinity of GABA for its receptor, decreasing the GABA EC50 from 9 microM to 3 microM. BI-1 had no effect on the shape of the GABA current-voltage relationship and did not alter the GABA reversal potential. The effects of BI-1 were not altered by benzodiazepine or picrotoxin site antagonists. At concentrations up to 10 microM, where maximal effects on GABA currents were seen, BI-1 did not directly activate a membrane current. This contrasts with the effects of 3 alpha-OH-DHP, which activated chloride currents at concentrations that were subsaturating for GABA potentiation. These results suggest that the BIs may be useful for determining the mechanisms by which steroids potentiate GABA responses and directly gate chloride channels.

Animals↗
PubMed1996

Oxidant stress activates a non-selective cation channel responsible for membrane depolarization in calf vascular endothelial cells.

1. In vascular endothelial cells, oxidant stress increases cell Na+ content and inhibits the agonist-stimulated influx of external Ca2+. Further, oxidant stress increases uptake of Ca2+ into otherwise quiescent endothelial cells. To determine the mechanism responsible for altered Na+ and Ca2+ homeostasis, the present study examined the effect of oxidant stress on ionic current and channel activity in calf pulmonary artery endothelial cells. 2. Voltage-clamped control cells had a zero-current potential of -60 mV. Incubation of cells with the oxidant tert-butylhydroperoxide (tBuOOH; 0.4 mM, 1 h) caused depolarization to -4 mV and activation of ionic current equally selective for Na+ and K+. 3. Cell-attached membrane patches made on tBuOOH-treated cells contained ion channels that had a bidirectional conductance of 30 pS and that were not present in patches from control cells. Inside-out patches excised from oxidant-treated cells showed the channel to be equally selective for Na+ and K+ and to allow inward Ca2+ current. 4. Oxidant-activated channels were observed to display two gating modalities that were further evident during analysis of single-channel open probability. Neither modality was significantly affected by altering internal [Ca2+] (1 microM-10 nM). 5. Activation of non-selective channels provides a possible mechanism by which oxidants may increase endothelial cell Na+ content. Channel permeability to Ca2+ may account in part for the elevation of cytosolic free [Ca2+] that occurs in oxidant-treated cells. 6. Channel activation is associated with membrane depolarization, a mechanism that may contribute to oxidant inhibition of the agonist-stimulated Ca2+ influx pathway.

Animals↗
PubMed2006

Measurement of conformational changes accompanying desensitization in an ionotropic glutamate receptor.

The canonical conformational states occupied by most ligand-gated ion channels, and many cell-surface receptors, are the resting, activated, and desensitized states. While the resting and activated states of multiple receptors are well characterized, elaboration of the structural properties of the desensitized state, a state that is by definition inactive, has proven difficult. Here we use electrical, chemical, and crystallographic experiments on the AMPA-sensitive GluR2 receptor, defining the conformational rearrangements of the agonist binding cores that occur upon desensitization of this ligand-gated ion channel. These studies demonstrate that desensitization involves the rupture of an extensive interface between domain 1 of 2-fold related glutamate-binding core subunits, compensating for the ca. 21 degrees of domain closure induced by glutamate binding. The rupture of the domain 1 interface allows the ion channel to close and thereby provides a simple explanation to the long-standing question of how agonist binding is decoupled from ion channel gating upon receptor desensitization.

Animals↗
PubMed1996

Activation kinetics of glutamate receptor channels from wild-type Drosophila muscle.

Outside-out patches from wild-type Drosophila larval muscle were exposed briefly to L-Glutamate (Glu) using a piezo-driven application system. Glu in concentrations of 0.1 to 30 mM was applied and the responses to repeated applications of a given concentration were averaged. The peak current, î, and the current rise time, tr, from 0.1 î to 0.9 î were determined from the averages. Half-maximum activation of the channels was reached with approximately 2 mM Glu. î increased proportional to the power n = 3. 5 to n = 5.8 (average of four experiments, n = 4.4) for Glu concentrations between 0.3 and 0.5 mM. tr increased from approximately 0.2 ms at 10 mM Glu to a value of approximately 3.5 ms at 0.2 mM Glu. A linear reaction scheme with five binding steps preceding the channel-opening conformational change is proposed as the kinetic mechanism of channel activation and investigated in computer simulations. A set of rate constants assuming the same affinity for each binding site is found to describe the data better than one assuming positive cooperativity. The results are very similar to those for Glu-gated channels of crayfish and locust muscle, which is evidence for a common kinetic mechanism of these channels.

Animals↗
PubMed1993

Molluscan ligand-gated ion-channel receptors.

In this chapter we introduce the reader to the structures of the different types of ligand-gated ion-channel receptor, and the numerous receptor subtypes that have recently been revealed to exist, in both invertebrate and vertebrate species, by the application of molecular biological methods. We then review some of the data in support of the existence, in molluscs, of such receptor/channel complexes for gamma-aminobutyric acid, glutamate and acetylcholine. Finally, recent results from our laboratory on the cloning and expression of complementary DNAs, from the pond-snail Lymnaea stagnalis, that encode GABA(A) and glutamate receptor subunits will be described.

Amino Acid Sequence↗
PubMed2001

DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature.

Several DEG/ENaC cation channel subunits are expressed in the tongue and in cutaneous sensory neurons, where they are postulated to function as receptors for salt and sour taste and for touch. Because these tissues are exposed to large temperature variations, we examined how temperature affects DEG/ENaC channel function. We found that cold temperature markedly increased the constitutively active Na(+) currents generated by epithelial Na(+) channels (ENaC). Half-maximal stimulation occurred at 25 degrees C. Cold temperature did not induce current from other DEG/ENaC family members (BNC1, ASIC, and DRASIC). However, when these channels were activated by acid, cold temperature potentiated the currents by slowing the rate of desensitization. Potentiation was abolished by a "Deg" mutation that alters channel gating. Temperature changes in the physiologic range had prominent effects on current in cells heterologously expressing acid-gated DEG/ENaC channels, as well as in dorsal root ganglion sensory neurons. The finding that cold temperature modulates DEG/ENaC channel function may provide a molecular explanation for the widely recognized ability of temperature to modify taste sensation and mechanosensation.

Acid Sensing Ion Channels↗
PubMed2003

Adenophostin A and imipramine are two activators of the olfactory inositol 1,4,5-trisphosphate-gated channel in fish olfatory cilia.

Binding of an odorant to its receptor activates the cAMP-dependent pathway, and also leads to inositol 1,4,5-trisphosphate (InsP(3)) production. This induces opening of a plasma membrane channel in olfactory receptor cells (ORCs). We investigated single-channel properties of this channel in the presence of a phospholipase C (PLC) activator (imipramine) and of a potent activator of the InsP(3)/Ca(2+) release channel (adenophostin A) by reconstituting carp olfactory cilia into planar lipid bilayers. In the presence of 53 mM barium as a charge carrier, the addition of 50 microM imipramine induced a current of 1.53+/-0.05 pA at 0 mV. There were two different mean open times (6.0+/-0.6 ms and 49.6+/-6.4 ms). The I/ V curve displayed a slope conductance of 50+/-2 pS. Channel activity was transient and was blocked by neomycin (50 microM). These observations suggest that imipramine may activate the olfactory InsP(3)-gated channel through PLC. Using the same ionic conditions, the application of 0.5 microM adenophostin A triggered a current of 1.47+/-0.04 pA at 0 mV. The I/ V curve displayed a slope conductance of 60+/-2 pS. This channel showed only a single mean open time (15.0+/-0.3 ms) and was strongly inhibited by ruthenium red (30 microM) and heparin (10 microg/mL). These results indicate that adenophostin A and imipramine may act on the ciliary InsP(3)-gated channel and are potentially valuable pharmacological tools for studying olfactory transduction mechanisms.

Adenosine↗
PubMed1999

Controversial issues in vertebrate olfactory transduction.

A number of controversial issues in olfactory transduction are discussed including the matter of multiple transduction pathways, with a new experiment proposed. Evidence is reviewed concerning the fact that cyclic AMP is the only pathway mediating olfactory transduction. Two knockout mice have been produced: a knockout for a cyclic nucleotide-gated channel and a G(olf) knockout. The results obtained with both mice are consistent with cyclic AMP being the only second messenger. The evidence for gaseous second channel messengers is also reviewed. Slow gating kinetics of the cyclic nucleotide-gated channel and the detection of single-odorant molecules are reviewed. A new phenomenon in which odorants can block odorant responses is discussed.

Animals↗
PubMed1990

Voltage dependence of NMDA-activated macroscopic conductances predicted by single-channel kinetics.

The conductance activated in many mammalian CNS neurons by the glutamate analog NMDA is inhibited at hyperpolarized potentials by extracellular magnesium. Whole-cell recordings from hippocampal neurons in culture were used to determine the voltage dependence of the NMDA conductance in the presence of extracellular magnesium concentrations from 1 microM to 10 mM. The conductance-voltage data are well fitted by a gating function derived from rate constants determined in an earlier study of the kinetic behavior of single channels activated by NMDA. The results are consistent with the assumption that magnesium inhibits current through the NMDA-activated channel by directly blocking the ion pore. In addition, another voltage-dependent blocking or flicker-producing mechanism has to be invoked to account for the behavior of the conductance at both the single-channel and whole-cell level, especially at low concentrations of extracellular magnesium.

Animals↗
PubMed1990

Ion channels and B cell mitogenesis.

Given the presence of ionic channels at the membrane of lymphocytes, we have analyzed the effect of various channels blockers on B lymphocytes activation. TEA and 4-AP, two K+ channels blockers, quinine, a blocker of Ca2(+)-activated K+ channels, nickel and verapamil, two Ca2+ channels blockers, all inhibited LPS-induced B cell proliferation. However, these drugs neither inhibited the induction of Ia and Fc gamma RII expression nor cell enlargement and early RNA synthesis, indicating that the entry of B lymphocytes into G1 phase was not affected. In contrast, both late RNA synthesis and the induction of the TfR, which occur while the cell progress through G1, were inhibited by these blockers. These data show that TEA, quinine and verapamil block B lymphocyte activation during the G1 phase, probably between G1A and G1B. To question whether these effects were due to the block of voltage-activated K+ channels, we compared the ability of TEA, quinine, verapamil, 4-AP and nickel to block proliferation and K+ channels. A striking correlation was found for all the drugs but less for 4-AP. Moreover, TMA, a TEA analog unable to block K+ currents, did not affect B cell proliferation. Taken together, our data suggests that functional voltage-gated K+ channels are required at a precise stage of the G1 phase of the B cell cycle.

Animals↗
PubMed2005

Allosteric control of an ionotropic glutamate receptor with an optical switch.

The precise regulation of protein activity is fundamental to life. The allosteric control of an active site by a remote regulatory binding site is a mechanism of regulation found across protein classes, from enzymes to motors to signaling proteins. We describe a general approach for manipulating allosteric control using synthetic optical switches. Our strategy is exemplified by a ligand-gated ion channel of central importance in neuroscience, the ionotropic glutamate receptor (iGluR). Using structure-based design, we have modified its ubiquitous clamshell-type ligand-binding domain to develop a light-activated channel, which we call LiGluR. An agonist is covalently tethered to the protein through an azobenzene moiety, which functions as the optical switch. The agonist is reversibly presented to the binding site upon photoisomerization, initiating clamshell domain closure and concomitant channel gating. Photoswitching occurs on a millisecond timescale, with channel conductances that reflect the photostationary state of the azobenzene at a given wavelength. Our device has potential uses not only in biology but also in bioelectronics and nanotechnology.

Allosteric Regulation↗
PubMed1991

Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium.

Cyclic nucleotide-gated ionic channels in photoreceptors and olfactory sensory neurons are activated by binding of cGMP or cAMP to a receptor site on the channel polypeptide. By site-directed mutagenesis and functional expression of bovine wild-type and mutant channels in Xenopus oocytes, we have tested the hypothesis that an alanine/threonine difference in the cyclic nucleotide-binding site determines the specificity of ligand binding, as has been proposed for cyclic nucleotide-dependent protein kinases [Weber, I.T., Shabb, J.B. & Corbin, J.D. (1989) Biochemistry 28, 6122-6127]. The wild-type olfactory channel is approximately 25-fold more sensitive to both cAMP and cGMP than the wild-type rod photoreceptor channel, and both channels are 30- to 40-fold more sensitive to cGMP than to cAMP. Substitution of the respective threonine by alanine in the rod photoreceptor and olfactory channels decreases the cGMP sensitivity of channel activation 30-fold but little affects activation by cAMP. Substitution of threonine by serine, an amino acid that also carries a hydroxyl group, even improves cGMP sensitivity of the wild-type channels 2- to 5-fold. We conclude that the hydroxyl group of Thr-560 (rod) and Thr-537 (olfactory) forms an additional hydrogen bond with cGMP, but not cAMP, and thereby provides the structural basis for ligand discrimination in cyclic nucleotide-gated channels.

Alanine↗
PubMed1992

Mouse cortical collecting duct cells show nonselective cation channel activity and express a gene related to the cGMP-gated rod photoreceptor channel.

Apical nonselective cation channels with an average single-channel conductance of 34 +/- 2.3 pS were found in M-1 mouse cortical collecting duct cells. Channel activity is increased by depolarization and abolished by cytoplasmic calcium removal. Cytoplasmic application of 0.1 mM cGMP decreases channel open probability by 27%. cDNAs corresponding to approximately 40% of the coding region of the photoreceptor channel were isolated by the polymerase chain reaction from M-1 cells and a rat kidney cDNA library. The rat kidney-derived sequence differs by a single base, and the M-1-cell-derived sequence differs by only two bases, from the photoreceptor sequence. A second clone from M-1 cells differs by 20 out of 426 bases from the photoreceptor sequence. In all three clones, the deduced amino acid sequence is identical to that of the rat photoreceptor channel. Northern blot analysis of poly(A)+ RNA from M-1 cells reveals the presence of a 3.2-kilobase band hybridizing with a retinal cGMP-gated cation channel probe. The results suggest the expression in M-1 cells of more than one gene coding for nonselective cation channels or channel subunits, one of which is identical to the cGMP-gated cation channel gene of rod photoreceptors.

Animals↗