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N L Harrison

Publications and source records attributed to N L Harrison.

At least 37 records · Page 2Linked to original sources
PubMed1999

General anaesthetic actions on ligand-gated ion channels.

The molecular mechanisms of general anaesthetics have remained largely obscure since their introduction into clinical practice just over 150 years ago. This review describes the actions of general anaesthetics on mammalian neurotransmitter-gated ion channels. As a result of research during the last several decades, ligand-gated ion channels have emerged as promising molecular targets for the central nervous system effects of general anaesthetics. The last 10 years have witnessed an explosion of studies of anaesthetic modulation of recombinant ligand-gated ion channels, including recent studies which utilize chimeric and mutated receptors to identify regions of ligand-gated ion channels important for the actions of general anaesthetics. Exciting future directions include structural biology and gene-targeting approaches to further the understanding of general anaesthetic molecular mechanisms.

Anesthetics, General↗
PubMed1999

Normal electrophysiological and behavioral responses to ethanol in mice lacking the long splice variant of the gamma2 subunit of the gamma-aminobutyrate type A receptor.

The gamma subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABA(A)-Rs. The long splice variant of the gamma2 subunit (gamma2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABA(A)-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes gamma2L from the short splice variant (gamma2S). Mice homozygous for this exon deletion (gamma2L-/-) are viable and indistinguishable from wild-type (gamma2L+/+) mice. No gamma2L mRNA was detected in these mice, nor could gamma2L-containing GABA(A)-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of gamma2 subunit protein to be not significantly changed, suggesting that gamma2S replaces gamma2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20-200 mM) observed in neurons from gamma2L+/+ or gamma2L-/- mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that gamma2L is not required for ethanol's modulatory action at the GABA(A)-R or whole animal behavioral effects.

Alternative Splicing↗
PubMed1999

Subunit mutations affect ethanol actions on GABA(A) receptors expressed in Xenopus oocytes.

1. Mutations of specific amino acids were introduced in transmembrane domains (TM) of GABA(A) receptor alpha2, beta1 and gamma2L subunits. The effects of these mutations on the action of ethanol were studied using the Xenopus oocyte expression system and two-electrode voltage-clamp recording techniques. 2. Mutant alpha2 subunits containing S270I (TM2) or A291W (TM3) made the receptor more sensitive to GABA, as compared to wild-type alpha2beta1gamma2L receptor. The mutation S265I (TM2) of beta1 and S280I (TM2) or S30IW (TM3) in gamma2L subunits did not alter apparent affinity of the receptor for GABA. M286W (TM3) in the beta1 subunit resulted in a receptor that was tonically open. 3. Using an EC5 concentration of GABA, the function of the wild-type receptor with alpha2beta1gamma2L subunits was potentiated by ethanol (50-200 mM). The mutations in TM2 or TM3 of the alpha2 subunit diminished the potentiation by ethanol. The action of ethanol was also eliminated with a mutation in the TM2 site of the beta1 subunit. Ethanol produced significant inhibition of GABA responses in receptors containing the combination of alpha2 and beta1 TM2 mutants with a wild-type gamma2L subunit. A small but significant reduction in the potentiation by ethanol was observed with gamma2L TM2 and/or TM3 mutants. 4. From these results, we suggest that in heteromeric GABA(A) receptors composed of the alpha, beta and gamma subunits, ethanol may bind in a cavity formed by TM2 and TM3, and that binding to the alpha or beta subunit may be more critical than the gamma subunit.

Amino Acid Sequence↗
PubMed1999

Agonist gating and isoflurane potentiation in the human gamma-aminobutyric acid type A receptor determined by the volume of a second transmembrane domain residue.

Gamma-aminobutyric acid type A (GABA(A) )receptors are targets for allosteric modulation by general anesthetics. Mutation of Ser270 within the second transmembrane domain of the GABA(A) receptor alpha subunit can ablate the modulation of the receptor by the anesthetic ether isoflurane. To investigate further the function of this critical amino acid residue, we made multiple amino acid substitutions at Ser270 and analyzed the concentration-dependent gating by GABA and regulation by isoflurane in each mutant receptor. There is a strong negative correlation between the EC(50) for GABA and the molecular volume of the amino acid residue at position 270. Replacement of Ser by large residues such as His and Trp produced a shift of the GABA concentration-response curve to the left, whereas replacement of Ser with Gly had the opposite effect. There also was a strong negative association between the molecular volume of the amino acid residue at 270 and the degree of enhancement of submaximal GABA responses by isoflurane. These results indicate the significance of the amino acid at position alpha270 in gating of the GABA(A) receptor. In addition, the data on isoflurane are consistent with the existence of a cavity of finite size in the region of alpha270 that may be filled by the anesthetic molecule or by the side chain of a larger residue at alpha270. The introduction of isoflurane, or of a large residue, into this cavity may stabilize the open state of the GABA(A) receptor relative to the closed state.

Anesthetics, Inhalation↗
PubMed1998

Mutations of gamma-aminobutyric acid and glycine receptors change alcohol cutoff: evidence for an alcohol receptor?

Alcohols in the homologous series of n-alcohols increase in central nervous system depressant potency with increasing chain length until a "cutoff" is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the alpha subunit. We now demonstrate that these residues in the glycine alpha1 and the gamma-aminobutyric acid rho1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine alpha1 receptor, whereas mutation of Ile-307 and/or Trp-328 in the gamma-aminobutyric acid rho1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.

Alcohols↗
PubMed1998

Neurosteroids act on the GABA(A) receptor at sites on the N-terminal side of the middle of TM2.

Two sets of chimeras between alphaxalone-sensitive GABA(A) receptor alpha2 or beta1 subunits and the alphaxalone-insensitive glycine receptor alpha1 subunit were constructed to determine the structural domains important for the modulatory actions of neuroactive steroids. These data suggest that the site of action for neurosteroids on GABA(A) receptors is not the same as that for volatile anesthetics and ethanol, but is on the N-terminal side of the middle of TM2.

Anesthetics↗
PubMed1998

Enhancement of glycine receptor function by ethanol is inversely correlated with molecular volume at position alpha267.

Glycine and gamma-aminobutyric acid (GABA)A receptors are members of the "superfamily" of ion channels, and are sensitive to allosteric modulation by n-alcohols such as ethanol and butanol. We recently demonstrated that the mutation of Ser-267 to Ile in the alpha1 subunit abolished ethanol regulation of glycine receptors (Gly-R). In the present study, a pair of chimeric receptors was studied, in which a 45-amino acid domain comprising transmembrane domains 2 and 3 was exchanged between the Gly-Ralpha1 and gamma-aminobutyric acid rho1 subunits. Detailed pharmacologic analysis of these chimeras confirmed that this domain of the Gly-R confers enhancement of receptor function by ethanol and butanol. An extensive series of mutations at Ser-267 in the Gly-Ralpha1 subunit was also prepared, and the resulting homomeric receptors were expressed and tested for sensitivity to glycine, and allosteric modulation by alcohols. All of the mutant receptors expressed successfully in Xenopus oocytes. Mutation of Ser-267 to small amino acid residues such as Gly or Ala produced receptors in which glycine responses were potentiated by ethanol. As we have reported previously, the mutant Gly-Ralpha1 (Ser-267 --> Ile) was completely insensitive to ethanol; mutation of Ser-267 to Val had a similar effect. Mutation of Ser-267 to large residues such as His, Cys, or Tyr resulted in inhibition of Gly-R function by ethanol. These results demonstrate that the size of the amino acid residue at position alpha267 plays a crucial role in determining the functional consequences of allosteric modulation of the Gly-R by alcohols.

Animals↗
PubMed1998

A deficit of functional GABA(A) receptors in neurons of beta 3 subunit knockout mice.

Mice whose gamma-aminobutyric acid type A (GABA(A)) beta3 subunit gene is inactivated ('beta3 knockout mice') have been previously shown to have epilepsy, hypersensitive behavior, cleft palate, and a high incidence of neonatal mortality. In this study, we analyze whole-cell responses to GABA in neurons from beta3+/+, beta3+/- and beta3-/- mice. We demonstrate markedly decreased responses to GABA in both hippocampal and dorsal root ganglion neurons isolated from beta3-/- mice without major differences in the GABA concentration-response curves. We also utilize the subunit selective pharmacology of Zn2+ and the anticonvulsant drug loreclezole to help infer the presence of beta2 and gamma subunits in the GABA(A) receptors remaining in neurons from beta3-/- mice.

Animals↗
PubMed1998

Propofol and other intravenous anesthetics have sites of action on the gamma-aminobutyric acid type A receptor distinct from that for isoflurane.

Both volatile and intravenous general anesthetics allosterically enhance gamma-aminobutyric acid (GABA)-evoked chloride currents at the GABA type A (GABAA) receptor. Recent work has revealed that two specific amino acid residues within transmembrane domain (TM)2 and TM3 are necessary for positive modulation of GABAA and glycine receptors by the volatile anesthetic enflurane. We now report that mutation of these residues within either GABAA alpha2 (S270 or A291) or beta1 (S265 or M286) subunits resulted in receptors that retain normal or near-normal gating by GABA but are insensitive to clinically relevant concentrations of another inhaled anesthetic, isoflurane. To determine whether receptor modulation by intravenous general anesthetics also was affected by these point mutations, we examined the effects of propofol, etomidate, the barbiturate methohexital, and the steroid alphaxalone on wild-type and mutant GABAA receptors expressed in human embryonic kidney 293 cells. In most cases, these mutations had little or no effect on the actions of these intravenous anesthetics. However, a point mutation in the beta1 subunit (M286W) abolished potentiation of GABA by propofol but did not alter direct activation of the receptor by high concentrations of propofol. These data indicate that the receptor structural requirements for positive modulation by volatile and intravenous general anesthetics may be quite distinct.

Anesthetics, Inhalation↗
PubMed1998

Trichloroethanol modulation of recombinant GABAA, glycine and GABA rho 1 receptors.

The actions of 2,2,2,-trichloroethanol were studied on agonist-activated Cl- currents in gamma-aminobutyric acid type A (GABAA), glycine and GABA rho 1 receptors by use of the whole-cell patch-clamp technique. Recombinant wild-type and mutant receptor subunits were transiently expressed in human embryonic kidney (HEK) 293 cells. Trichloroethanol enhanced currents elicited by submaximal (EC20) agonist concentrations at GABAA alpha 2 beta 1 receptors and glycine alpha 1 homomeric receptors in a reversible, concentration-dependent manner. Trichloroethanol, at concentrations of < or = 2 mM, did not significantly alter the magnitude of submaximal GABA currents at GABA rho 1 receptors, whereas higher concentrations inhibited submaximal GABA currents. Recent work has identified residues within putative transmembrane domains 2 and 3 as critical for positive modulation of GABAA and glycine receptors by n-alkanols and volatile ether anesthetics. Submaximal glycine currents at receptors containing either of two specific mutations within the glycine receptor alpha 1 subunit (S267I and A288W) were not enhanced by low concentrations of trichloroethanol and were inhibited by higher concentrations of trichloroethanol. In the GABAA alpha 2 beta 1 receptor, a specific mutation within transmembrane domain 3 of the beta 1 subunit (M286W) also abolished positive modulation by trichloroethanol. Mutations within the GABAA alpha 2 receptor subunit did not alter positive modulation by TCEt, whereas such mutations ablate positive modulation by n-alkanols and volatile anesthetics. In summary, trichloroethanol modulation of GABAA, glycine and GABA rho 1 receptors shares some, but not all, features in common with the requirements for modulation by n-alkanols and volatile anesthetics.

Amino Acid Sequence↗
PubMed1997

Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors.

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABA(A) and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.

Alanine↗
PubMed1997

Mice devoid of gamma-aminobutyrate type A receptor beta3 subunit have epilepsy, cleft palate, and hypersensitive behavior.

gamma-Aminobutyric acid type A receptors (GABA(A)-Rs) mediate the bulk of rapid inhibitory synaptic transmission in the central nervous system. The beta3 subunit is an essential component of the GABA(A)-R in many brain regions, especially during development, and is implicated in several pathophysiologic processes. We examined mice harboring a beta3 gene inactivated by gene targeting. GABA(A)-R density is approximately halved in brain of beta3-deficient mice, and GABA(A)-R function is severely impaired. Most beta3-deficient mice die as neonates; some neonatal mortality, but not all, is accompanied by cleft palate. beta3-deficient mice that survive are runted until weaning but achieve normal body size by adulthood, although with reduced life span. These mice are fertile but mothers fail to nurture offspring. Brain morphology is grossly normal, but a number of behaviors are abnormal, consistent with the widespread location of the beta3 subunit. The mice are very hyperactive and hyperresponsive to human contact and other sensory stimuli, and often run continuously in tight circles. When held by the tail, they hold all paws in like a ball, which is frequently a sign of neurological impairment. They have difficulty swimming, walking on grids, and fall off platforms and rotarods, although they do not have a jerky gait. beta3-deficient mice display frequent myoclonus and occasional epileptic seizures, documented by electroencephalographic recording. Hyperactivity, lack of coordination, and seizures are consistent with reduced presynaptic inhibition in spinal cord and impaired inhibition in higher cortical centers and/or pleiotropic developmental defects.

Angelman Syndrome↗
PubMed1997

Activation and deactivation rates of recombinant GABA(A) receptor channels are dependent on alpha-subunit isoform.

The role of subunit composition in determining intrinsic maximum activation and deactivation kinetics of GABA(A) receptor channels is unknown. We used rapid ligand application (100-micros solution exchange) to examine the effects of alpha-subunit composition on GABA-evoked activation and deactivation rates. HEK 293 cells were transfected with human cDNAs encoding alpha1beta1gamma2- or alpha2beta1gamma2-subunits. Channel kinetics were similar across different transfections of the same subunits and reproducible across several GABA applications in the same patch. Current rise to peak was at least twice as fast for alpha2beta1gamma2 receptors than for alpha1beta1gamma2 receptors (reflected in 10-90% rise times of 0.5 versus 1.0 ms, respectively), and deactivation was six to seven times slower (long time constants of 208 ms versus 31 ms) after saturating GABA applications. Thus alpha-subunit composition determined activation and deactivation kinetics of GABA(A) receptor channels and is therefore likely to influence the kinetics and efficacy of inhibitory postsynaptic currents.

Cells, Cultured↗
PubMed1997

Alpha subunit isoform influences GABA(A) receptor modulation by propofol.

We have investigated the role of the alpha subunit in the modulation of gamma-aminobutyric acid type A (GABA(A)) receptors by the general anesthetic propofol, using whole-cell patch clamp recordings made from distinct stable fibroblast cell lines which expressed only alpha1beta3gamma2 or alpha6beta3gamma2 GABA(A) receptors. At clinically relevant anesthetic concentrations, propofol potentiated submaximal GABA currents in alpha1beta3gamma2 receptors to a far greater degree than those in alpha6beta3gamma2 receptors. The alpha subunit influenced the efficacy of propofol for modulation, but not its potency. In contrast, direct gating of the ion channel by propofol, in the absence of GABA, was significantly larger in the alpha6 than the alpha1 containing receptors. The potentiation of submaximal GABA by trichloroethanol, and the potentiation and direct gating by methohexital was also studied, and showed the same relative trends as propofol.

Anesthetics, Intravenous↗
PubMed1996

Chimeric GABAA/glycine receptors: expression and barbiturate pharmacology.

GABAA and glycine receptors are close relatives in the "gene superfamily" of ligand-gated ion channels, but have distinctly different pharmacology. For example, barbiturates have two effects on GABAA receptors (GABAA-R): at low micromolar concentrations (2-5 microM), the anesthetic barbiturate methohexital potentiates submaximal chloride current responses to GABA; at higher concentrations (20-50 microM), the barbiturate causes direct gating of the channel in the absence of agonist. Neither of these barbiturate effects is seen on the glycine receptor (Gly-R). In order to study the structural parts of the GABAA-R involved in this barbiturate pharmacology, two unique restriction sites were introduced into the cDNAs encoding the alpha 2 and beta 1 subunits of the human GABAA-R and the alpha 1 subunit of the human gly-R. The first site ('X') corresponded to the C-terminal end of the third transmembrane domain (M3) in each subunit and enabled exchange of C-terminal fragment of approximately 100 amino acids (which includes the large 'cytoplasmic loop' and M4 segment) between GABAA-R and Gly-R subunits. The second site ('S') was approximately 30 amino acids 3'- from the N-terminal end of each subunit and enabled exchange of a small N-terminal fragment between GABAA-R and Gly-R subunits. Several chimeric receptor subunit cDNAs were constructed and the resulting receptors tested for their ability to respond to GABA and glycine and for sensitivity to the barbiturate methohextial (MTX). The results show that neither the large C-terminal fragment nor the smaller N-terminal fragment is associated with the enhancement or direct activation of the GABAA-R by MTX. These results demonstrate the viability of chimeric GABAA/Gly-R and suggest that the method will be suitable for further investigation of the molecular basis of the barbiturate pharmacology of the GABA-R.

Anesthetics, General↗
PubMed1996

Inhibition of a fast inwardly rectifying potassium conductance by barbiturates.

Whole cell voltage clamp recordings were used to study the effects of two barbiturates, methohexital and pentobarbital, on inwardly rectifying K+ currents in the plasma membrane of a rat basophilic granulocyte cell line (RBL-1). Inwardly rectifying K+ currents are responsible for maintaining the resting membrane potential in a variety of cell types including skeletal and cardiac muscle, neurons, glia, blood cells, and endothelial cells. RBL-1 cells are unusual because the inward rectifier is the only apparent voltage-dependent current in these cells. Steps to command potentials between + 80 and -120 mV evoked only this strongly rectifying, rapidly developing current at membrane potentials more hyperpolarized than the reversal potential for K' ions. Extracellular Cs+ (10 mM) and Ba2+ (100 microM and 1 mM) blocked this current in a reversible and voltage-dependent manner. The voltage threshold for activation of the inwardly rectifying K+ current is dependent on the extracellular K+ concentration as predicted by the Nernst equation. Methohexital and pentobarbital reversibly inhibited the current in a concentration-dependent fashion with 50% inhibitory concentration (IC50) values of 145 microM and 218 microM respectively. The Hill slopes for both of these effects were approximately 1. The inhibition was not voltage dependent. These results indicate that fast inwardly rectifying K+ channels are potential molecular targets for barbiturates and could explain some of the diverse clinical effects of these drugs.

Adjuvants, Anesthesia↗
PubMed1995

The effects of four general anesthetics on intracellular [Ca2+] in cultured rat hippocampal neurons.

It has been suggested that general anesthesia might arise as a consequence of increased cytoplasmic free ionized calcium concentration ([Ca2+]i). The effect of increased [Ca2+]i might be to activate K+ channels or to modulate other ion channels important for the control of excitability, such as the GABAA receptor. A direct test of this hypothesis has not been reported. Microfluorimetry with the calcium-sensitive dye fura-2 was used to study the effects of four anesthetic agents on the regulation of intracellular free Ca2+ in hippocampal neurons cultured from the embryonic rat hippocampus. Basal intracellular free ionized calcium concentration [Ca2+]i in the neurons was 50-100 nM. Depolarization of the neurons with 50 mM K+ resulted in the elevation of [Ca2+]i to 200-800 nM, with subsequent recovery of [Ca2+]i over several minutes. The volatile anesthetics halothane, enflurane and isoflurane did not alter basal [Ca2+]i, even above clinically relevant concentrations; however, they did inhibit elevation of [Ca2+]i by high K+ stimulation. The intravenous anesthetic methohexital caused small increases in basal [Ca2+]i at concentrations > or = 50 microM; methohexital (5-50 microM) also inhibited elevations of [Ca2+]i induced by high K+. The evidence presented here suggests that the anesthetics studied do not produce their actions via sustained or transient increases in [Ca2+]i. However, all of the anesthetics studied appear to possess inhibitory effects on hippocampal voltage-dependent Ca2+ channels, in addition to their previously described effects at GABAA receptors.

Anesthetics↗