Henry A. Lester

The inward rectifier potassium channel family.

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+.

Cis – trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel

5-Hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2–M3), can link binding to gating through a cis–trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis–trans energy gap of the proline analogue and the activation of the channel, suggesting that cis–trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2–M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2–M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.

Neuronal P2X transmitter-gated cation channels change their ion selectivity in seconds.

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.

A rat brain na+ channel α subunit with novel gating properties

Abstract We have constructed a full-length rat brain Na + channel α subunit cDNA that differs from the previously reported a subunit of Noda et al. at 6 amino acid positions. Transcription of the cDNA in vitro and injection into Xenopus oocytes resulted in the synthesis of functional Na + channels. Although the single-channel conductance of the channels resulting from cloned cDNA was the same as that of channels resulting from injection of rat brain RNA, we observed two significant differences in the gating properties of the channels. The Na + currents from cloned cDNA displayed much slower macroscopic inactivation compared with those from rat brain mRNA. In addition, the current-voltage relationship for currents from cloned cDNA was shifted 20–25 mV in the depolarizing direction compared with currents from rat brain RNA. Coinjection of low MW rat brain RNA restored normal inactivation of the channels indicating the presence of a component, either a structural subunit of the channel complex or a modifying enzyme, necessary for normal gating of the channel.

Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes

Voltage-clamp analysis was applied to study the currents associated with the uptake of extracellular gamma-aminobutyric acid (GABA) by the cloned transporter GAT1 expressed at high efficiency in Xenopus oocytes. Steady-state GABA currents were increased at higher extracellular [GABA], [Na+], and [Cl-] and at more negative potentials. The Hill coefficient for Na+ exceeded unity, suggesting the involvement of two Na+ ions. In the absence of GABA, voltage jumps produced transient currents that behaved like capacitive charge movements; these were suppressed by the uptake inhibitor SKF-89976A, were shifted to more negative potentials at lower external [Na+] and [Cl-], and had an effective valence of 1.1 elementary charge. A turnover rate per transporter of 6-13/s at maximal [GABA] (-80 mV, 96 mM NaCl, 22 degrees C) is given both by the kinetics of voltage jump relaxations and by the ratio between the maximal GABA currents and the charge movements. These quantitative data are necessary for evaluating the roles of GAT1 in synaptic function.

International Union of Pharmacology. XLI. Compendium of Voltage-Gated Ion Channels: Potassium Channels

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

Nicotine binding to brain receptors requires a strong cation–π interaction

Nicotine addiction begins with high-affinity binding of nicotine to acetylcholine (ACh) receptors in the brain. The end result is over 4,000,000 smoking-related deaths annually worldwide and the largest source of preventable mortality in developed countries. Stress reduction, pleasure, improved cognition and other central nervous system effects are strongly associated with smoking. However, if nicotine activated ACh receptors found in muscle as potently as it does brain ACh receptors, smoking would cause intolerable and perhaps fatal muscle contractions. Despite extensive pharmacological, functional and structural studies of ACh receptors, the basis for the differential action of nicotine on brain compared with muscle ACh receptors has not been determined. Here we show that at the α4β2 brain receptors thought to underlie nicotine addiction, the high affinity for nicotine is the result of a strong cation–π interaction to a specific aromatic amino acid of the receptor, TrpB. In contrast, the low affinity for nicotine at the muscle-type ACh receptor is largely due to the fact that this key interaction is absent, even though the immediate binding site residues, including the key amino acid TrpB, are identical in the brain and muscle receptors. At the same time a hydrogen bond from nicotine to the backbone carbonyl of TrpB is enhanced in the neuronal receptor relative to the muscle type. A point mutation near TrpB that differentiates α4β2 and muscle-type receptors seems to influence the shape of the binding site, allowing nicotine to interact more strongly with TrpB in the neuronal receptor. ACh receptors are established therapeutic targets for Alzheimer’s disease, schizophrenia, Parkinson’s disease, smoking cessation, pain, attention-deficit hyperactivity disorder, epilepsy, autism and depression. Along with solving a chemical mystery in nicotine addiction, our results provide guidance for efforts to develop drugs that target specific types of nicotinic receptors.

Conducting states of a mammalian serotonin transporter

We have studied permeation at a cloned rat 5-HT transporter expressed in Xenopus oocytes. [3H]5-HT uptake and [125I]RTI-55 binding yield a turnover rate of approximately 1/s that does not depend on membrane potential. However, in voltage-clamp experiments, three distinct currents results from 5-HT transporter expression. First, a steady-state, voltage-dependent transport-associated current is induced by 5-HT application. Second, a transient inward current is activated by voltage jumps to high negative potentials in the absence of 5-HT and is blocked by 5-HT itself. Third, a small leakage current is observed in the absence of 5-HT. All the observed currents are blocked by inhibitors of 5-HT uptake but are differentially affected by Na+, Li+, K+, Ba2+, Cs+, Cl-, and amiloride. The conducting states of the 5-HT transporter may reflect the existence of a permeation pathway similar to that of ionic channels.

RGS9 modulates dopamine signaling in the basal ganglia.

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. We examined a role for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduced locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice showed heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerated the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increased RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness.

Chronic Nicotine Cell Specifically Upregulates Functional α4* Nicotinic Receptors: Basis for Both Tolerance in Midbrain and Enhanced Long-Term Potentiation in Perforant Path

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose α4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased α4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change α4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional α4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases α4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated α4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional α4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.