Browsing by Subject "Single channel"
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Item Activation and allosteric modulation of the [alpha]1 glycine receptor(2010-05) Welsh, Brian Thomas; Mihic, S. John; Aldrich, Richard W.; Dopico, Alejandro M.; Harris, Robert A.; Zakon, Harold H.The glycine receptor (GlyR) is a ligand-gated ion channel and member of the nicotinic acetylcholine receptor superfamily. Glycine and the partial agonist taurine are both believed to be the endogenous ligands of the receptor. Partial agonists have lower efficacies than full agonists, eliciting submaximal responses even at saturating concentrations. Recent evidence suggests that efficacy at these receptors is determined by conformational changes that occur early in the process of receptor activation. We previously identified a mutation of the aspartate-97 residue to arginine (D97R), which produces a spontaneously active mutant with behavior that mimics the effects of saturating glycine concentrations on wildtype (WT) GlyR. This D97 residue is hypothesized to form an electrostatic interaction with arginine-119 on an adjacent subunit to stabilize a closed channel closed state. We found that the disruption of this bond converts taurine into a full agonist and greatly increases the efficacies of other [beta]-amino acid partial agonists. Our findings suggest that the determination of efficacy in the GlyR involves the disruption of an inter-subunit electrostatic interaction soon after binding. We next investigated whether the taurine efficacy could be enhanced by ethanol, a well-studied positive allosteric modulator of receptor function. Whole-cell recordings of WT GlyRs demonstrated that alcohol could potentiate the effect of low concentrations of taurine, but did not increase the efficacy of a saturating concentration. Therefore we sought to understand the mechanism by which alcohol enhances the GlyR, because ethanol's actions at inhibitory receptors in the brain are thought to produce many of the physiological effects associated with its use. We examined the effects of 3 [mu]M glycine ± 50 or 200 mM ethanol on outside-out patches expressing WT [alpha]1 GlyR, to determine the effects of alcohol at the single-channel level. Alcohol enhanced GlyR function in a very specific manner. It had minimal effects on open and closed dwell times. Instead, ethanol potentiated GlyR function almost exclusively by increasing burst durations and increasing the number of channel openings per burst, without affecting the percentage of open time within bursts. Kinetic modeling suggests that ethanol increases burst durations by decreasing the rate of glycine unbinding.Item Critical elements contributing to the control of glycine receptor activation and allosteric modulation(2010-12) Todorovic, Jelena, 1981-; Mihic, S. John; Richburg, John H.; Aldrich, Richard W.; Duvauchelle, Christine L.; Pierce-Shimomura, Jonathan T.Glycine receptors (GlyRs) are ligand-gated ion channels (LGICs) that, along with other members of the cys-loop superfamily of receptors, mediate a considerable portion of fast neurotransmission in the central nervous system (CNS). GlyRs are pentameric channels, organized quasi-symmetrically around an ion-conducting pore. Opening of the integral ion pore depends on ligand binding and transduction of this binding signal to the channel gate. Research presented in this dissertation describes a number of critical electrostatic interactions that play a role in conserving the closed-state stability of the receptor in the absence of ligand, ensuring that receptor activation occurs only upon neurotransmitter binding. These amino acids, aspartic acid at position 97 (D97), lysine 116 (K116), arginine 119 (R119) and arginine R131 (R131) are charged residues that interact with one another through electrostatic attraction. When D97 is replaced with any other amino acid this destabilizes the closed state of the channel and causes spontaneous GlyR channel opening. I show that restoration of this electrostatic interaction in GlyR bearing double mutations in which the charges are swapped (D97R/R119E and D97R/R131D) markedly decreases this spontaneous current. In addition, I investigate how these residues that interact at the interfaces between receptor subunits affect the efficacies of GlyR partial agonists. My work shows that the partial agonist taurine is converted into a full agonist at both D97R and R131D receptors. Furthermore, I analyzed the structure of the more extracellular part of the transmembrane (TM) 2 segment that lines the ion channel pore, showing that it is unlikely that this fragment (stretching from T13’ to S18’) is constrained in a true alpha helical conformation. From this work, using disulfide trapping and whole cell electrophysiology, I conclude that a significant level of flexibility characterizes this part of the TM2 domain. This segment includes residue S267, previously shown to be significant for alcohol and anesthetic actions, as well as residue Q266 that, when mutated, produces a hyperekplexia-like phenotype. The range of movement of residues in this region may therefore play an important role not only in channel gating but also in how modulators of GlyR function exert their actions.Item The relationship between glycine receptor agonist efficacy and allosteric modulation(2014-05) Kirson, Dean; Mihic, S. JohnThe glycine receptor (GlyR) is a ligand-gated ion channel member of the cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Both glycine and the partial agonist taurine act as endogenous ligands of the GlyR. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse, such as ethanol. As a partial agonist, taurine has a decreased efficacy relative to glycine, resulting in a decreased maximum response. We investigated the effects of ethanol, anesthetics, inhalants, and zinc to determine if these allosteric modulators could increase the efficacy of the taurine-activated GlyR. Whole cell recordings of wild type GlyR revealed that each of the allosteric modulators potentiated currents generated by saturating concentrations of taurine but not glycine, implying an increase in efficacy. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of these allosteric modulators with zinc to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by another allosteric modulator, indicating no synergistic effects on efficacy. We next investigated the actions of ethanol and isoflurane on the taurine-activated GlyR at the single channel level, finding that both allosteric modulators stabilized the channel open state, increasing the efficacy of the taurine-activated GlyR. We previously identified a mutation in the ligand-binding domain of the GlyR (D97R) that produces spontaneously activating channels, on which taurine has increased efficacy. We identified a residue, R131, as a possible binding partner of D97 in forming an electrostatic interaction that holds the channel in the closed state. We found that disruption of this interaction results in greatly increased taurine efficacy, indicating that efficacy for partial agonists may be determined by agonist ability to break this bond early in the activation process following binding. Thus we find differential mechanisms of allosteric modulation and efficacy determinations for the GlyR when activated by taurine vs. glycine.Item The relationship between glycine receptor agonist efficacy and allosteric modulation(2014-05) Kirson, Dean; Mihic, S. JohnThe glycine receptor (GlyR) is a ligand-gated ion channel member of the cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Both glycine and the partial agonist taurine act as endogenous ligands of the GlyR. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse, such as ethanol. As a partial agonist, taurine has a decreased efficacy relative to glycine, resulting in a decreased maximum response. We investigated the effects of ethanol, anesthetics, inhalants, and zinc to determine if these allosteric modulators could increase the efficacy of the taurine-activated GlyR. Whole cell recordings of wild type GlyR revealed that each of the allosteric modulators potentiated currents generated by saturating concentrations of taurine but not glycine, implying an increase in efficacy. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of these allosteric modulators with zinc to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by another allosteric modulator, indicating no synergistic effects on efficacy. We next investigated the actions of ethanol and isoflurane on the taurine-activated GlyR at the single channel level, finding that both allosteric modulators stabilized the channel open state, increasing the efficacy of the taurine-activated GlyR. We previously identified a mutation in the ligand-binding domain of the GlyR (D97R) that produces spontaneously activating channels, on which taurine has increased efficacy. We identified a residue, R131, as a possible binding partner of D97 in forming an electrostatic interaction that holds the channel in the closed state. We found that disruption of this interaction results in greatly increased taurine efficacy, indicating that efficacy for partial agonists may be determined by agonist ability to break this bond early in the activation process following binding. Thus we find differential mechanisms of allosteric modulation and efficacy determinations for the GlyR when activated by taurine vs. glycine.