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Keywords:

  • ethanol sites of action;
  • pressure;
  • ion channels;
  • two-electrode voltage clamp;
  • Xenopus oocytes

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

The current study used an ethanol antagonist, increased atmospheric pressure, to test the hypothesis that ethanol acts on multiple sites in glycine receptors (GlyRs). The effects of 12 times normal atmospheric pressure of helium-oxygen gas (pressure) on ethanol-induced potentiation of GlyR function in Xenopus oocytes expressing human α1, α2 or the mutant α1(A52S) GlyRs were measured using two-electrode voltage clamp. Pressure reversibly antagonized potentiation of glycine in α1 GlyR by 40–200 mm ethanol, but did not antagonize 10 and 25 mm ethanol in the same oocytes. In contrast, pressure did not significantly affect potentiation of glycine by 25–100 mm ethanol in α2 GlyRs, nor did pressure alter ethanol response in the A52S mutant. Pressure did not affect baseline receptor function or response to glycine in the absence of ethanol. These findings provide the first direct evidence for multiple sites of ethanol action in GlyRs. The sites can be differentiated on the basis of ethanol concentration, subunit and structural composition and sensitivities to pressure antagonism of ethanol. Parallel studies with butanol support this conclusion. The mutant α1(A52S) GlyR findings suggest that increased attention should be focused on the amino terminus as a potential target for ethanol action.

Abbreviations used
ATA

normal atmospheric pressure

CNS

central nervous system

GlyR

glycine receptors

heliox

helium-oxygen gas

LGIC

ligand-gated ion channels

Glycine is a major inhibitory neurotransmitter in the mammalian central nervous system (CNS). Its predominant effects are in the spinal cord and brain stem through the activation of a ligand-gated receptor linked to an integral chloride channel (Rajendra et al. 1997). This strychnine-sensitive glycine receptor (GlyR) is also found in other areas of the brain such as the hippocampus, cerebral cortex and cerebellum (Betz 1991; Rajendra et al. 1997). Thus, glycine is likely to play a significant role as a neurotransmitter in many brain regions. GlyRs belong to a superfamily of ligand-gated ion channels (LGICs), known as Cys-loop receptors (Ortells and Lunt 1995; Karlin 2002) whose members include γ-aminobutyric acid type-A (GABAA), nicotinic acetylcholine and 5-hydroxytryptamine3, all of which assemble to form ion channels with a pentameric structure (Schofield et al. 1987). Two types of GlyR subunits have been cloned (α1 –α4 and β) and the pentamer is formed from α subunits alone or α and β subunits (Betz 1991; Rajendra et al. 1997). The α1 and α2 GlyR subunits are the most abundant α subunits found in the CNS (Malosio et al. 1991) with various α subtypes showing distinct pharmacological properties and differing developmental expression (Malosio et al. 1991; Rajendra et al. 1997; Eggers et al. 2000).

Previous studies indicate a role for GlyRs in the action of ethanol. Ethanol positively modulates GlyR function measured in synaptoneurosomes of whole-rat brain (Engblom and Åkerman 1991), embryonic spinal neurons of mouse and chick (Celentano et al. 1988; Aguayo and Pancetti 1994), freshly dissociated rat neurons (Ye et al. 2001; Jiang and Ye 2003) and brain slice preparations (Eggers et al. 2000). Recent transgenic studies in mice also add to the evidence that GlyRs participate in the action of ethanol in mammals (Findlay et al. 2002). In addition, behaviorally relevant concentrations of ethanol reliably and robustly potentiate human recombinant α1 and α2 GlyRs expressed in Xenopus oocytes measured electrophysiologically (Mascia et al. 1996a; Mascia et al. 1996b; Mihic et al. 1997; Yamakura and Harris 2000; Davies et al. 2003).

Interestingly, earlier work reported that strychnine-sensitive GlyRs displayed at least two distinct sensitivities to ethanol in embryonic spinal neurons (Aguayo and Pancetti 1994). In one population of cells, low concentrations of ethanol (1–10 mm) potentiated glycine currents, whereas in a second population of cells, these low concentrations of ethanol did not potentiate glycine currents. In contrast, higher concentrations of ethanol (100–450 mm) potentiated glycine in all of the neurons tested. Later studies, which tested the effects of ethanol on recombinant GlyRs expressed in Xenopus oocytes, reported differences in ethanol sensitivity between α1 and α2 GlyRs with α1 GlyRs showing greater sensitivity to ethanol in the 5–50 mm range than did the α2 GlyRs (Mascia et al. 1996b). There was no significant difference in the effects of ethanol between α1 and α2 GlyRs when tested at concentrations above 50 mm. Thus, the differences in ethanol sensitivity identified in embryonic spinal neurons may be attributed to different populations of cells expressing primarily α1 or α2 glycine subunits (Mascia et al. 1996b; Eggers et al. 2000). Moreover, making a point mutation in α1 GlyR of alanine to serine at amino acid 52 (A52S) in the amino-terminal region of the receptor reduced the sensitivity of the receptor to ethanol and yielded a receptor that was similar to α2 GlyR in regards to overall ethanol sensitivity (Mascia et al. 1996b). The authors proposed two explanations for the findings including the possibility that ethanol has multiple sites of action on GlyRs and that the mutation eliminated one of the sites, but not others.

Traditionally, the mechanism and site of drug action can be studied using the appropriate receptor agonists and antagonists. This pharmacological approach is based on the concept that a selective antagonist can differentiate sites of drug action. However, the physical-chemical mechanism of action as well as the low affinities of ethanol limit the utility of traditional pharmacological receptor ligands as tools for investigating ethanol's sites of action (Eckenhoff and Johansson 1997; Davies et al. 2003).

Prior studies have shown that exposure to 12 times normal atmospheric pressure (ATA) of helium-oxygen gas (heliox) directly antagonizes the behavioral and biochemical actions of ethanol (Alkana and Malcolm 1981; Alkana et al. 1992; Bejanian et al. 1993; Davies and Alkana 1998, 2001). More recent two-electrode voltage clamp studies demonstrated that pressure antagonized ethanol potentiation (50–200 mm) of α1 GlyR function in a direct, reversible, concentration and pressure dependent manner (Davies et al. 2003). This work also showed that pressure did not antagonize allosteric modulation of α1 GlyR function by Zn2+. Taken together, these findings indicate that pressure is a direct, ethanol antagonist with the selectivity and other properties necessary for it to be used to help differentiate the sites and mechanisms of ethanol action within GlyRs (Davies et al. 2003).

The present study uses increased atmospheric pressure as an alternative to a traditional pharmacological antagonist to test the hypothesis that ethanol acts at multiple sites in GlyRs. We tested the effects of pressure on ethanol potentiation of glycine-activated currents in Xenopus oocytes expressing wildtype α1, wildtype α2, or mutant α1(A52S) homomeric GlyRs. The results suggest multiple sites of action for ethanol that can be differentiated on the basis of ethanol concentration, subunit and structural composition and sensitivities to pressure antagonism of ethanol. Parallel studies with butanol support this conclusion. This is the first direct evidence for multiple sites of action for ethanol in GlyRs.

Atmospheric conditions

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

The 1 ATA air condition (control) was achieved by testing in a hyperbaric chamber with the lid removed or by using compressed air with the chamber sealed. Pilot studies determined that there was no difference between these two methods. To achieve the 12 ATA heliox condition, the chamber was purged with heliox and then pressurized to the experimental ATA at the rate of 1 ATA/min using premixed certified compressed gases. The heliox mixture consisted of 1.7% oxygen and 98.3% helium resulting in a 0.2 ATA partial pressure for oxygen at 12 ATA. This provides normal oxygenation and avoids complications from higher oxygen partial pressures. In addition, we replace the nitrogen in our gas mixture with helium to avoid the depressant effect of nitrogen at increased atmospheric pressure (Alkana and Malcolm 1982a).

We modeled our hyperbaric electrophysiology studies after our behavioral and biochemical work, which utilized hyperbaric helium-oxygen (heliox) gas to achieve pressurization. We did not include a 1 ATA heliox control in the present study based on prior behavioral, biochemical and electrophysiological studies that found no significant difference between 1 ATA air and 1 ATA heliox (Davies et al. 1999, 2003). Previous work indicates that ethanol antagonism by 12 ATA heliox results from the action of pressure per se, not from the pharmacological effects of helium. This work demonstrated that exposure to 1 ATA heliox does not antagonize the effects of ethanol (Malcolm and Alkana 1982; Davies et al. 1999) or other general anesthetics (Wann and MacDonald 1988) and that exposure to hydrostatic pressure antagonized general anesthetics in frogs (Dodson et al. 1985). Further evidence that heliox does not mediate antagonism came from studies in which exposure to 4 or 6 ATA oxygen antagonized the behavioral effects of ethanol in mice. Moreover, the antagonism by oxygen could not be explained by increased oxygen partial pressure or oxygen-induced changes in ethanol pharmacokinetics (Alkana and Malcolm 1982a).

Expression in oocytes

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

Xenopus oocytes were isolated (Davies et al. 2003) and injected with either human α1, α2 or the mutant α1(A52S) cDNAs 1.5 ng per 30 nL cloned into the mammalian expression vector pCIS2 or pBKCMV (Mascia et al. 1996b; Davies et al. 2003). Mutagenesis of alanine to serine was performed as previously described (Ryan et al. 1994). After injection, oocytes were stored individually in incubation medium (MBS supplemented with 2 mm sodium pyruvate, 0.5 mm theophylline, 10 000 U/L penicillin, 10 mg/L streptomycin and 50 mg/L gentamycin) in Petri dishes (VWR, San Dimas, CA). All solutions were sterilized by passage through 0.22 µm filters. Oocytes, stored at 18°C, usually expressed GlyRs the day after injection. Oocytes were used in electrophysiological recordings for 1–5 days after cDNA injection.

Hyperbaric two-electrode whole-cell voltage clamp recording

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

Two-electrode voltage-clamp recording was performed using techniques similar to those previously reported (Davies et al. 2003). Briefly, electrodes pulled (P-30, Sutter Instruments, Novato, CA, USA) from borosilicate glass [1.2 mm thick-walled filamented glass capillaries (WPI, Sarasota, FL, USA)] were back-filled with 3 m KCl to yield resistances of 0.5–3 MΩ. All electrophysiological recordings were conducted within a specially designed hyperbaric chamber that contains a vibration resistant platform that supports an oocyte bath, two micro positioners (WPI or Narishige International USA, Inc. East Meadow, NY, USA) and bath clamp (Davies et al. 2003). Oocytes were perfused in a 100 µL oocyte bath with MBS ± drugs via a custom high pressure drug delivery system (Alcott Chromatography, Norcross, GA, USA or Shimadzu, Columbia, MD, USA) at 2 mL/min using 1/16 OD high pressure PEEK tubing (Upchurch Scientific, Oak Harbor, WA, USA). Oocytes were voltage clamped at a membrane potential of − 70 mV using a Warner Instruments Model OC-725C (Hamden, CT, USA) oocyte clamp. Individual oocytes were tested at both control and experimental atmospheric conditions and checked for normal function during pressurization and depressurization. Note that the order of testing control and atmospheric conditions was counterbalanced to minimize the effects of testing order and to determine if pressure effects were reversible.

Studies in our laboratory (Davies et al. 2003) and studies conducted elsewhere (Mascia et al. 1996a; Mascia et al. 1996b) found that ethanol potentiation of GlyR function is more robust and reliable when tested in the presence of low concentrations of glycine (typically EC2-10). Moreover, the action of ethanol is difficult to measure when tested in the presence of higher glycine levels (e.g. EC50) because the degree of potentiation by ethanol is smaller and the receptors are more prone to desensitization (Mascia et al. 1996b). For these reasons we elected to use low ECs of glycine for the present studies. Except where noted, the concentration of glycine producing 2% of a maximal effect (EC2) was determined for each oocyte and was used in studies that tested the effects of ethanol. Previous studies report that glycine is more potent at α1 (EC50 = 155 ± 19 µm) than α2 (EC50 = 257 ± 14 µm) receptors (Mascia et al. 1996b). Utilizing a set effective concentration of agonist (e.g. EC2) with each oocyte allowed comparison of drug effects across oocytes and across receptor subtypes while minimizing influence by differences in levels of receptor expression (Mascia et al. 1996a; Mascia et al. 1996b). The effects of drugs were measured for 30 s unless otherwise noted. When testing drug-potentiating effects, the oocytes were preincubated with drug for 60 s prior to coapplication of drug and glycine. Washout periods (5–15 min) were allowed between drug applications to ensure complete resensitization of receptors (Mascia et al. 1996a; Mascia et al. 1996b). A chart recorder (Barnstead/Thermolyne, Dubuque, IA, USA) continuously plotted the clamped currents. The peak currents were measured and used in data analysis. All experiments were performed at room temperature (20–23°C).

Data analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

Results are presented as percentage potentiation of control glycine current responses. Glycine-activated control responses were measured before and after each drug application to take into account possible shifts in the control currents as the recordings proceeded. All results are expressed as mean ± SEM. Data were obtained from oocytes from at least two different frogs. The n refers to the number of different oocytes tested. Non-linear regression analysis of the concentration-response curves and statistical analyzes were performed using Prism (GraphPAD Software, San Diego, CA, USA). Statistical significance was defined as p < 0.05.

Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

We began testing the hypothesis that ethanol acts by multiple sites and/or mechanisms in GlyR by extending our initial studies in strychnine-sensitive α1 GlyR expressed in Xenopus oocytes (Davies et al. 2003) to include a full spectrum of ethanol concentrations. The rationale for this experiment derived from earlier work, which found that 12 ATA heliox antagonized the effects of 50–200 mm ethanol, combined with prior studies elsewhere suggesting that lower ethanol concentrations may act on different populations of receptors or on a different location on these receptors (Aguayo and Pancetti 1994; Mascia et al. 1996b). In agreement with our previous investigations, we found that 10–200 mm ethanol induced a concentration-dependent potentiation of EC2 glycine in α1 GlyR. Ethanol in the absence of glycine did not measurably alter resting membrane currents (Fig. 1a). As reported previously (Davies et al. 2003), exposure to 12 ATA heliox significantly antagonized potentiation of glycine response by 50–200 mm ethanol (Fig. 1). In agreement with expectations of a competitive antagonist, the degree of pressure antagonism significantly decreased as the concentration of ethanol increased from 50 to 200 mm (Fig. 1b) and reversed when the oocytes were returned to 1 ATA air (Fig. 1a). Pressure did not measurably alter resting membrane currents (Fig. 1a), nor did pressure significantly affect glycine-activated currents in the absence of ethanol (Fig. 1a,b), thus supporting a direct mechanism of pressure antagonism of ethanol. Further, EC2 concentrations of glycine elicited similar currents for all applications of glycine across time regardless of atmospheric conditions, which indicates that the antagonism of ethanol by pressure cannot be attributed to receptor desensitization or rundown (Fig. 1a).

image

Figure 1. Pressure significantly antagonizes the effects of high but not low ethanol concentrations in α1 GlyRs. (a) Representative sequential tracing (from left to right) for data from a single oocyte in 1 ATA air (effects of glycine [Gly] alone, Gly + 25 mm ethanol [EtOH], Gly alone, Gly + 200 mm EtOH, Gly alone) and then in 12 ATA heliox (effects of Gly alone, Gly + 25 mm EtOH, Gly alone, Gly + 200 mm EtOH, Gly alone) showing typical responses observed under these experimental conditions. The data were the same for oocytes tested in the reverse order. Ethanol (25 mm) potentiation of GlyR function was not antagonized by pressure whereas ethanol (200 mm) potentiation of GlyR function was antagonized by pressure. The slanted double vertical lines represent the transition from 1 ATA air to 12 ATA heliox. The vertical scale bar represents 50 nA and the horizontal scale bar represents 48 s. The horizontal bars above the tracing indicate time of glycine (lower) and ethanol (upper) applications. The dashed horizontal line represents initial glycine EC2 response. (b) Depicts results from all concentrations of ethanol (10–200 mm) tested at both atmospheric conditions. Oocytes were tested in both ascending and descending pressure conditions. Two-way anova revealed significant main effects of atmospheric condition [F1,94 = 26.79, p < 0.001] and drug condition [F6,94 = 35.60, p < 0.001] and a significant interaction between main effects [F6,94 = 5.09, p < 0.001]. Glycine EC2 was determined for each oocyte and ranged from 13 to 60 µm glycine (30.4 ± 2 µm). Glycine was applied for 30 s. Ethanol was applied for 1 min, and then coapplied with glycine for 30 s. Washout time between drug applications was at least 5 min. Glycine-activated currents completely recovered after ethanol washout. (Inset) Expanded scale displaying the effects of lower concentrations of ethanol (10–50 mm) tested at both atmospheric conditions.

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In contrast to the results for 50–200 mm ethanol, the degree of pressure antagonism was less at 40 mm than at 50 mm ethanol. More remarkably, pressure did not significantly affect potentiation of glycine response by 25 and 10 mm ethanol (Fig. 1b inset) in the same oocytes in which there was antagonism of ethanol at higher concentrations (Fig. 1). Pressure antagonized ethanol at 40–200 mm regardless of whether the higher concentrations were tested before or after the low ethanol concentrations.

The unexpected insensitivity to pressure antagonism of lower ethanol concentrations, combined with the sensitivity to pressure antagonism of higher ethanol concentrations, is consistent with the notion that ethanol acts via at least two different sites in α1 GlyRs. These sites would correspond to one site that is pressure antagonism insensitive and another site, which has a higher ethanol sensitivity threshold that is pressure antagonism sensitive.

Pressure does not antagonize the effects of ethanol in α2 GlyRs

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

Recombinant studies have shown that homomeric α1 GlyRs are more sensitive to ethanol than are homomeric α2 GlyRs (Mascia et al. 1996b). The difference in ethanol sensitivity shown in the aforementioned recombinant studies is also present for native heteromeric channels (Eggers et al. 2000). Based in part on these reported differences, we extended our investigation to α2 GlyRs to determine whether these subunit-dependent differences in ethanol sensitivity might reflect the action of ethanol on different sites that can be differentiated on the basis of their sensitivity to pressure antagonism of ethanol.

Oocytes expressing human homomeric α2 GlyRs were voltage-clamped and tested with glycine±ethanol under both control and pressure conditions. As a positive control, other oocytes, isolated from the same frogs at the same time expressing α1 GlyRs were also tested with glycine ± 100 mm ethanol under both control and pressure conditions. Ethanol (25–100 mm) potentiated responses of an EC2 concentration of glycine in α2 GlyRs in a concentration dependent manner under control (1 ATA air) atmospheric conditions (Fig. 2b). Ethanol, when tested in the absence of glycine, did not measurably alter the resting membrane currents of the oocytes (Fig. 2a). In addition, α1 GlyRs were more sensitive to the effects of ethanol than α2 GlyRs (e.g. 100 mm ethanol: α1 GlyRs SEM = 66.2 ± 3.0%; α2 GlyRs SEM = 45.9 ± 4.5%; p < 0.05). These initial findings, conducted at control atmospheric conditions, are consistent with findings reported by others (Mascia et al. 1996b; Eggers et al. 2000).

image

Figure 2. Ethanol potentiation of α2 GlyR function is not antagonized by pressure. (a) Sequential tracings (from left to right) from a single oocyte from data summarized in the lower panel. The oocyte was tested in 1 ATA air (effects of Gly alone, Gly + 100 mm EtOH, Gly alone), in 12 ATA heliox (effects of Gly alone, Gly + 100 mm EtOH, Gly alone) and then in 1 ATA Air (effects of Gly alone, Gly + 100 mm EtOH, Gly alone). The horizontal bars above each tracing indicate time of glycine EC2 and 100 mm ethanol applications. The vertical scale bars represent 50 nA and the horizontal scale represents 48 s. (b) Depicts results from all concentrations of ethanol (25–100 mm) in α2 GlyRs tested at both atmospheric conditions. Two-way anova revealed a significant main effect of drug condition [F2,11 = 11.94, p < 0.01] but no significant effect of atmospheric condition on glycine-activated chloride currents. There was no significant interaction between main effects of drug and atmospheric condition. Glycine EC2 was determined for each oocyte and ranged from 40 to 120 µm glycine (69.5 ± 6 µm). The points that are connected by the dashed line on the graph are taken from Fig. 1 (α1 GlyRs) to allow for comparison of ethanol sensitivity when tested in α1 and α2 GlyRs. See Fig. 1 legend for other figure parameters. Data are presented as mean ± SEM of 4–5 oocytes.

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When these same oocytes were tested under hyperbaric conditions, we found that exposure to 12 ATA heliox did not significantly affect potentiation by either low (25 mm) or high (50–100 mm) ethanol concentrations on α2 GlyRs (Fig. 2b). In contrast, pressure did significantly antagonize ethanol potentiation of 100 mm ethanol in α1 GlyRs expressed in the same batches of oocytes used for the α2GlyR study [t(4) = 5.099, p < 0.01]. Because α1 and α2 GlyRs were expressed in oocytes isolated at the same time and from the same frogs, the inability of pressure to antagonize ethanol in α2 GlyRs can not be explained by differences in batches of oocytes. Furthermore, the differences in sensitivity to pressure antagonism were present regardless of whether pressure was tested before or after the control condition indicating that the results cannot be due to receptor rundown or the development of tolerance to ethanol. Consistent with our initial study, pressure did not measurably influence α1 GlyR or α2 GlyR baseline function (Fig. 2a), nor did pressure significantly affect responses to glycine in the absence of ethanol in either receptor (Fig. 2a,b), further supporting a direct mechanism of pressure antagonism in α1 GlyRs. The insensitivity to pressure antagonism of all concentrations of ethanol in α2 GlyRs suggests that α2 receptors have a pressure antagonism insensitive ethanol site, and lack the pressure antagonism sensitive ethanol site found in α1 GlyRs.

Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

To test the generality of the pressure/ethanol findings in GlyRs, we also tested the effects of pressure vs. the n-alcohol, butanol. Previous studies demonstrated that glycine-induced currents of homomeric α1 and α2 GlyRs are potentiated by n-alcohols and that the potencies of these alcohols in potentiating glycine receptor function increases as the carbon chain length increases up to decanol (Mascia et al. 1996a). The results from this and other previous work suggest that the α subunits of strychnine-sensitive glycine receptors contain common sites of action for n-alcohols (Mascia et al. 1996a). The current study tested the hypothesis that differences in sensitivity to pressure antagonism of butanol in α1 and α2 GlyRs would parallel findings with ethanol.

Our initial butanol study tested the effects of pressure on butanol-induced potentiation of α1 GlyR function (Fig. 3). We elected to use an EC10 in this study rather than the EC2 that we used in our previous studies because we felt that this would allow us to further test the effects of pressure vs. glycine at a second, low glycine EC value. In agreement with previous work, pilot studies determined that glycine EC10 caused minimal receptor desensitization. Oocytes expressing α1 GlyR were voltage-clamped and tested with EC10 glycine±butanol (0.5–5 mm) under both 1 ATA control and 12 ATA heliox conditions. We tested butanol concentrations that yielded degrees of potentiation functionally equivalent to the potentiation by key ethanol concentrations in the prior study (e.g. 0.5 mm butanol potentiated α1 GlyR function by about 20%, which was similar to potentiation by 10 and 25 mm ethanol). Low (10 mm) and high (100 mm) concentrations of ethanol were also tested in this first butanol study to ensure that the low and high concentrations of butanol selected yielded functional outcomes that were similar to the functional outcomes of low and high ethanol concentrations. Ethanol and butanol were tested in the same oocytes.

image

Figure 3. Pressure significantly antagonizes the effects of high but not low butanol concentrations in α1 GlyRs. (a) Representative sequential tracing (from left to right) for data from a single oocyte in 1 ATA air (effects of Gly alone, Gly + 0.5 mm butanol [BuOH], Gly alone, Gly + 1.0 mm BuOH, Gly alone, Gly + 100 mm EtOH, Gly alone) and then in 12 ATA heliox (effects of Gly alone, Gly + 0.5 mm butanol [BuOH], Gly alone, Gly + 1.0 mm BuOH, Gly alone, Gly + 100 mm EtOH, Gly alone) showing typical responses observed under these experimental conditions. Oocytes were tested in both ascending and descending pressure conditions and the data were the same when oocytes were tested in the reverse order. Butanol (0.5 mm) potentiation of GlyR function was not antagonized by pressure whereas higher butanol (1 mm) and ethanol (100 mm) concentrations that potentiated GlyR function were antagonized by pressure. The slanted double vertical lines represent the transition from 1 ATA air to 12 ATA heliox. The vertical scale bar represents 100 nA and the horizontal scale bar represents 48 s. The horizontal bars above the tracing indicate time of glycine (lower) and butanol (upper) or ethanol (middle) applications. The dashed horizontal line represents initial glycine EC10 response. (b) Depicts results from all concentrations of butanol (0.5–5 mm) in α1 GlyRs tested at both atmospheric conditions. Two-way anova revealed a significant main effects of atmospheric condition [F1,13 = 52.30, p < 0.0001] and drug condition [F4,13 = 17.66, p < 0.0001] and a significant interaction between main effects [F4,13 = 16.28, p < 0.0001]. (c) Depicts results from all concentrations of ethanol (10 and 100 mm) in α1 GlyRs tested at both atmospheric conditions. Butanol and ethanol were tested in the same oocytes. Glycine EC10 was determined for each oocyte and ranged from 40 to 75 µm glycine (57.4 ± 3 µm). Glycine was applied for 30 s. Alcohols were applied for 1 min, then coapplied with glycine for 30 s. Washout time between drug applications was at least 5 min. Glycine-activated currents completely recovered after alcohol washout. Values represent the currents induced by butanol and ethanol and are expressed as mean percent ± SEM of control glycine response as measured in 4–5 oocytes. (*p < 0.05, compared with respective 1 ATA Air control by Bonferroni Multiple Comparisons [butanol] and unpaired t-tests [ethanol]).

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In agreement with our ethanol studies (Fig. 1), pressure directly antagonized butanol potentiation of GlyR function at the higher functional concentrations tested (1–5 mm), but did not antagonize potentiation by the lower functional butanol concentration (0.5 mm) (Fig. 3b). Butanol did not measurably alter the resting membrane currents of the oocytes when tested in the absence of glycine supporting a modulatory role for butanol (Fig. 3a). Moreover, in agreement with previous studies pressure did not significantly affect responses to glycine alone (Fig. 3a,b). The butanol concentration-dependent differences in sensitivity to pressure antagonism parallel those seen with ethanol and suggest that butanol also acts via two different sites in α1 GlyR that appear to correspond to the sites for ethanol.

Consistent with our initial ethanol study (Fig. 1), pressure directly antagonized ethanol potentiation of GlyR function at the higher functional concentration tested (100 mm), but did not antagonize potentiation by the lower functional ethanol concentration (10 mm) (Fig. 3c). These later findings with ethanol suggest that the ethanol concentration dependent differences in sensitivity to pressure antagonism in α1GlyRs are not due to an unexpected phenomenon based on the glycine EC value tested.

Pressure does not antagonize the effects of butanol in α2 GlyRs

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

We next tested the effects of pressure vs. butanol potentiation of glycine-activated currents in α2 GlyRs. Oocytes expressing human homomeric α2 GlyRs were voltage-clamped and tested with glycine±butanol under both control and pressure conditions. Butanol (0.5–5 mm) potentiated responses of an EC10 concentration of glycine in α2 GlyRs in a concentration dependent manner under control (1 ATA air) atmospheric conditions (Fig. 4). Butanol, when tested in the absence of glycine, did not measurably alter the resting membrane currents of the oocytes (Data not shown).

image

Figure 4. Butanol potentiation of α2 GlyR function is not antagonized by pressure. The figure depicts results from three concentrations of butanol (0.5–5 mm) in α2 GlyRs tested at both 1 ATA air and 12 ATA heliox. Two-way anova revealed a significant main effect of drug condition [F2,9 = 9.10, p < 0.01] but no significant effect of atmospheric condition on glycine-activated chloride currents. There was no significant interaction between main effects of drug and atmospheric condition. Glycine EC10 was determined for each oocyte and ranged from 90 to 210 µm glycine (135.0 ± 17 µm). The points that are connected by the dashed line on the graph are taken from Fig. 3 (α1 GlyRs) to allow for comparison of butanol sensitivity when tested in α1 and α2 GlyRs. See Fig. 1 legend for other figure parameters. Data are presented as mean ± SEM of 4 oocytes.

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When these same oocytes were tested under hyperbaric conditions, we found that exposure to 12 ATA heliox did not significantly affect potentiation by either low or high butanol concentrations on α2 GlyRs (Fig. 4). The insensitivity to pressure antagonism of all concentrations of butanol in α2 GlyRs suggests that α2 receptors have a pressure antagonism insensitive butanol site, and lack the pressure antagonism sensitive butanol site found in α1 GlyRs. The results parallel the findings with ethanol in α2 GlyRs and further support the hypothesis that short-chain n-alcohols act on similar targets in GlyRs. In addition, the findings provide evidence that α1 and α2 GlyRs differ in sensitivity to pressure antagonism of ethanol and butanol.

Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

The amino acid sequence identity between α1GlyR and α2GlyR is high, indicating that differences in function arise from relatively minor differences in their structures. Therefore, the differences in sensitivity to pressure antagonism of ethanol between α1 and α2 GlyR indicates that one or more of the limited number of amino acid residues that differ between these two homomeric GlyRs play a critical role in determining whether the receptor is sensitive or insensitive to pressure antagonism.

The mutant α1(A52S) GlyR is similar to the wildtype α2 GlyR in regards to overall ethanol sensitivity (i.e. less sensitive to ethanol) (Mascia et al. 1996b). Thus, the replacement of the hydrophobic amino acid alanine with the polar, uncharged amino acid serine appears to participate in the reduced ethanol sensitivity of the mutant α1 GlyR. Interestingly, sequence comparisons between α1 and α2 GlyRs indicates that a threonine residue (polar, uncharged) resides at the postition equivalent to alanine at position 52 in the α2 GlyR. Based on these findings and the lack of pressure antagonism in α2 GlyRs, we reasoned that the mutant α1(A52S) GlyR may also be less sensitive to pressure antagonism. If true, this finding would provide a new line of evidence that this site is an initial target for ethanol.

To test this notion, oocytes expressing the mutant α1(A52S) GlyRs were voltage-clamped and tested with glycine ± 100 mm ethanol under both control and pressure conditions. We elected to test 100 mm ethanol because this concentration produced a degree of potentiation that was functionally equivalent to the potentiation by ethanol in wildtype α1 GlyRs that were antagonized by pressure. We found that exposure to 12 ATA heliox did not significantly affect potentiation by 100 mm ethanol on mutant α1(A52S) GlyRs (Fig. 5). This finding indicates that the single point mutation in a1GlyR (A52S) that caused the receptor to behave like the α2 GlyR in regards to sensitivity to ethanol (Mascia et al. 1996b) also caused the mutant receptor to behave like the pressure antagonism insensitive α2 GlyR.

image

Figure 5. Ethanol potentiation of mutant α1(A52S) GlyR function is not antagonized by pressure. The figure depicts results from 100 mm ethanol in the mutant (A52S) α1 GlyRs tested at both 1 ATA air and 12 ATA heliox. Unpaired t-tests determined that there was no significant effect of atmospheric condition on ethanol potentiation of glycine-activated chloride currents (t(8) = 1.030, p = 0.33). Glycine EC2 was determined for each oocyte and ranged from 70 to 180 µm glycine (110.0 ± 19 µm). See Fig. 1 legend for other figure parameters. Data are presented as mean ± SEM of 5 oocytes.

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Glycine concentration response experiment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

Glycine concentration-response curves were determined at 1 ATA air conditions in Xenopus oocytes expressing either homomeric α1, α2 or the mutant α1(A52S) GlyR subunit (Fig. 6). In agreement with previous studies in oocytes (Saul et al. 1994; Mascia et al. 1996b) and HEK 293 cells (Ryan et al. 1994), the glycine EC50 was significantly lower in the α1 receptors (120.2 ± 6 µm) than in the α2 (238.2 ± 28 µm) or the mutant α1(A52S) receptors (286.1 ± 12 µm) [F (2,11) = 22.7, p < 0.01]. Hill coefficients were 1.98 ± 0.2 for α1, 2.25 ± 0.1 for α2, and 2.85 ± 0.1 for α1(A52S). These differences in EC50 are consistent with the contention that the amino acid residue at the 52 position influences glycine activation (Ryan et al. 1994; Saul et al. 1994; Absalom et al. 2003).

image

Figure 6. Concentration-response curves for glycine (10–1000 µm) activated Cl- currents in Xenopus oocytes expressing α1, α2, or the mutant α1(A52S) glycine receptor subunits. Glycine induced Cl currents were normalized to the maximal current activated by a saturating concentration of glycine (1 mm) when tested under 1 ATA air conditions. As determined from the curve fits, the glycine EC50 values for α1, α2 and α1(A52S) were 120.2 ± 6, 238.2 ± 28, and 286.1 ± 12 µm, respectively. Hill coefficients were 1.98 ± 0.2 for α1, 2.25 ± 0.1 for α2, and 2.85 ± 0.1 for α1(A52S). Glycine was applied for 30 s. Washout time was at least 5 min after application of glycine. Each data point represents the means ± SEM from 4 different oocytes.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

The current study used an ethanol antagonist, increased atmospheric pressure, to test the hypothesis that ethanol acts on multiple sites in GlyRs expressed in Xenopus oocytes. Pressure reversibly antagonized ethanol potentiation of α1 GlyR function at ethanol concentrations from 40 to 200 mm, but did not significantly affect ethanol potentiation in these receptors at lower concentrations of 10 and 25 mm. The results were not due to technical adjustments necessary to conduct studies under pressure or to alterations by pressure of normal GlyR function. These concentration dependent differences in sensitivity of α1 GlyRs to pressure antagonism of ethanol are consistent with the notion that ethanol acts via multiple sites in α1 GlyRs that can be differentiated on the basis of their sensitivities to pressure antagonism. These correspond to a relatively low ethanol threshold (≤ 10 mm ethanol) pressure-antagonism insensitive site and a relatively high ethanol threshold (≤ 40 mm ethanol) pressure-antagonism sensitive site. Subsequent studies found that pressure did not significantly alter potentiation of glycine-activated currents by 25–100 mm ethanol in α2 GlyRs. The insensitivity to pressure antagonism at these concentrations in α2 GlyRs is consistent with the notion that α2 receptors have a pressure-antagonism insensitive ethanol site, but lack the pressure-antagonism sensitive ethanol site present on α1 GlyRs. Similar concentration-dependent and subunit-dependent differences in sensitivity to pressure antagonism were found with butanol indicating that the pressure/ethanol findings generalize to other short chain n-alcohols in GlyRs. Taken together, these findings provide the first direct evidence for multiple sites of action for ethanol and other n-chain alcohols in GlyRs.

The notion that there may be more than one site of action for ethanol on GlyRs is a new concept that has received little attention. As discussed in the Introduction, prior studies found concentration dependent differences in ethanol sensitivity of neurons and recombinant GlyRs that were consistent with the hypothesis that ethanol acts on multiple sites in GlyRs. First, Aguayo and Pancetti (1994) found populations of embryonic spinal cord neurons that had at least two distinct sensitivities to ethanol potentiation of glycine responses with one population responding to ethanol concentrations from 1 to 10 mm and a resistant population with an ethanol threshold of 100 mm. Second, recombinant studies in Xenopus oocytes found that α1 GlyR were more sensitive to ethanol than α2 GlyR at ethanol concentrations from 5 to 50 mm (Mascia et al. 1996b; Mihic et al. 1997). These previous findings in neurons and oocytes parallel the concentration-dependent differences in sensitivity to pressure antagonism of ethanol in α1 and α2 GlyR reported in the current study. It is noteworthy that native receptors typically are heteromeric combinations of α and β subunits (Rajendra et al. 1997) and that other work suggests both the addition of a β subunit and the expression system utilized can alter GlyR sensitivity to ethanol (Valenzuela et al. 1998; McCool et al. 2003). Hence, the present studies in homomeric α1 GlyR must be extended before conclusions can be drawn. Nonetheless, the present results with pressure, taken in the context of prior work, draw a link from GlyR in the recombinant model to neurons and suggest that ethanol has multiple sites of action on GlyR in the mammalian nervous system.

The present experiments do not distinguish between actual multiple sites and multiple substates of a single binding cavity. Such an internal cavity was resolved by cryoelectron microscopy between the four alpha helices that form the transmembrane domain of the nicotinic acetylcholine receptor (Mizazawa et al. 2003). An example of an alcohol binding cavity that is in dynamic motion and could accommodate multiple substates is found in the structure of LUSH (Kruse et al. 2003). Such a water-filled cavity, in which the alcohol ligand participates with water in a complex hydrogen-bonding network (Trudell 2004), could exhibit multiple substates whose populations change as a function of applied pressure. Understanding the subtle energetic changes that make the sites in GlyR α2 subunits insensitive to pressure is a goal of ongoing studies.

The rationale for using pressure in the present studies was derived from earlier work, which found that exposure to 50–300 times normal ATA could reverse anesthetic effects of ethanol and other agents (Johnson et al. 1942; Lever et al. 1971; Trudell et al. 1973; Halsey and Wardley-Smith 1975; Miller and Wilson 1978; Galla and Trudell 1980). However, exposure to these high pressures caused non-specific excitation that complicated interpretation of the findings and thus limited the use of high pressure as a tool for investigating the mechanism of action of ethanol (for review see Wann and MacDonald 1988). Subsequent experiments determined that the non-specific excitatory effects attributed to pressures above 50 ATA could be eliminated by using lower levels of hyperbaric exposure. This work used pressures up to 12 ATA heliox to antagonize behavioral and biochemical effects of ethanol (Alkana and Malcolm 1981; Alkana et al. 1992; Bejanian et al. 1993; Davies and Alkana 1998, 2001; Davies et al. 1999). The antagonism occurred without measurable changes in baseline behavior, CNS or receptor excitability (Bejanian et al. 1993; Davies et al. 1994; Syapin et al. 1996; Davies et al. 1999) or other changes (Malcolm and Alkana 1982; Alkana and Malcolm 1982a; Alkana and Malcolm 1982b) that could explain the antagonism through indirect mechanisms.

The present work, taken with other recent electrophysiological studies in oocytes, provides compelling evidence supporting a direct mechanism of pressure antagonism of ethanol in GlyRs. This earlier work, which focused on the effects of pressure vs. higher concentrations of ethanol (50–200 mm) in α1 GlyRs, demonstrated that neither the pressurization procedure nor exposure to 12 ATA heliox per se affects: (1) baseline GlyR function; (2) Hill slope or maximal current response (Emax) to glycine; (3) glycine EC50 or (4) the effects of the GlyR antagonist strychnine (Davies et al. 2003). In addition, pressure antagonized ethanol regardless of the order in which pressure was tested, thus eliminating the possibility that pressure antagonism can be attributed to the development of tolerance with repeated ethanol applications or to receptor desensitization or receptor rundown (Davies et al. 2003). Moreover, in the present study, sensitivity to pressure antagonism in GlyR was both ethanol concentration dependent in the same oocytes and receptor subunit and structure dependent across oocytes. This pattern of differences in sensitivity to pressure antagonism in α1 and α2 GlyRs, taken together, is incompatible with a mechanism of pressure antagonism that reflects general alterations in receptor function, post-translational processes or other biochemical systems. Collectively, these behavioral, biochemical and electrophysiological studies provide several lines of evidence supporting the hypothesis that pressure is a direct antagonist of the effects of ethanol. Therefore, analogous to a traditional pharmacological antagonist, pressure acts by blocking or offsetting the initial action of ethanol, and thus, can be used to identify the initial pressure-sensitive targets for ethanol.

In this context, the present studies suggest molecular structures on which ethanol acts in GlyRs. We found qualitative differences in sensitivity to pressure antagonism of ethanol between α1 GlyR (pressure-antagonism sensitive) and α2 GlyR (pressure-antagonism insensitive). The amino acid sequence identity between α1 and α2 GlyRs is high (approximately 79%) (Rajendra et al. 1997). Thus, the differences in sensitivity to pressure antagonism of ethanol between α1 and α2 GlyRs indicate that one or more of the limited number of amino acid residues that differ between these receptors play a critical role in determining the sensitivity of the site to ethanol and to pressure antagonism of ethanol.

This conclusion is supported by our initial findings indicating that a single point mutation at position 52 (A52S) in the terminal region of the α1GlyR converts the pressure antagonism sensitive α1GlyR to be pressure antagonism insensitive. This hyperbaric finding also complements prior studies, which found that the same mutation also affects ethanol sensitivity (Mascia et al. 1996b). These findings support the notion that structural differences that affect sensitivity to pressure antagonism play a role in determining sensitivity of the site to ethanol. Together, this work indicates that the region at and around A52 is a site for both pressure antagonism and ethanol action. This correspondence at one site adds new support for A52 as an important target for ethanol.

Sequence alignment of a portion of the amino terminal regions from human α1GlyR and α2GlyR subunits indicates that the α2GlyR subunit contains a threonine residue at a position equivalent to alanine 52 of the α1 wild-type. Since both serine and threonine are polar residues, one could speculate that in the amino terminus region of the glycine receptor that side chain differences may play a role in ethanol sensitivity and sensitivity to pressure antagonism.

Previous studies have shown that the alanine-to-serine exchange at position 52 in the α1 GlyR subunit is responsible for the spasmodic phenotype, an inherited startle syndrome, in mice (Ryan et al. 1994; Saul et al. 1994). This A52S substitution in α1 GlyR is characterized by normal glycine binding properties, with an incerease in the glycine EC50 (Ryan et al. 1994; Saul et al. 1994; Mascia et al. 1996b). In agreement with these reports, we also found an increase in the EC50 for glycine in the α1(A52S) receptor compared with the α1 wild-type. Taken together, these findings are consistent with the notion that A52S defines part of a site involved in receptor activation rather than receptor binding (Saul et al. 1994; Ryan et al. 1994; Absalom et al. 2003).

If this notion that the A52 region is involved in glycine activation of α1GlyRs is valid, then the present findings point to the A52 region as a site of ethanol action and suggest that ethanol may act through the A52 region by potentiating glycine-induced activation and that pressure antagonizes ethanol by blocking ethanol potentiation of glycine activation in the A52 region. This scenario would explain the lack of direct effects of ethanol on the receptor, could provide an explanation for why the mutant α1 GlyR and wildtype α2 GlyR are less sensitive to ethanol than α1 GlyR and would be consistent with findings suggesting that ethanol binds to the water accessible S267 region (Mascia et al. 2000) with ethanol transduction (i.e. increase in glycine activation) occurring in the A52 region. This scheme for ethanol action in α1GlyR might generalize to other LGICs.

In conclusion, the present findings with pressure indicate that there are multiple sites of action for ethanol in GlyR. The sites can be differentiated on the bases of their sensitivities to pressure antagonism in conjunction with their threshold sensitivities to ethanol and their subunit composition. This evidence from hyperbaric studies that there are multiple sites or mechanisms of action for ethanol fit and help to explain the findings of prior work which found concentration dependent differences in the sensitivities to ethanol potentiation of GlyR function in different neuronal populations. The hyperbaric studies are also consistent with prior findings suggesting that there are targets for ethanol at both the amino-terminus and transmembrane regions of GlyR (Mascia et al. 1996b; Mihic et al. 1997) and suggest that increased attention should be focused on the amino terminus as a potential site of glycine activation and target for ethanol transduction. Collectively, this work provides several lines of evidence supporting multiple sites of action for ethanol in GlyR.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References

The authors thank Dr Peter Schofield for providing the α1 and α2 GlyR cDNA used in this study and Ashutosh Kulkarni for technical assistance. We also thank Dr R. Adron Harris for his early encouragement in developing the hyperbaric oocyte technique and helpful comments and suggestions during the course of this investigation. This work was supported in part by: United States Public Health Service Research Grants AA03972 (R.L.A), AA013890 (D.L.D) and AA11525 (S.J.M) (National Institute on Alcohol Abuse and Alcoholism – NIAAA) and NIGMS RO1 G64371 (J.R.T), the National Science Foundation IBN-9818422 (R.L.A), the Alcohol Beverage Medical Research Foundation (R.L.A).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Atmospheric conditions
  6. Expression in oocytes
  7. Hyperbaric two-electrode whole-cell voltage clamp recording
  8. Data analysis
  9. Results
  10. Ethanol experiments
  11. Pressure selectively antagonizes the effects of high but not low concentrations of ethanol in α1 GlyRs
  12. Pressure does not antagonize the effects of ethanol in α2 GlyRs
  13. Butanol experiments
  14. Pressure antagonizes the effects of high but not low concentrations of butanol in α1 GlyRs
  15. Pressure does not antagonize the effects of butanol in α2 GlyRs
  16. Mutant α1(A52S) GlyR experiment
  17. Pressure does not antagonize the effects of ethanol in mutant α1(A52S) GlyRs
  18. Glycine concentration response experiment
  19. Discussion
  20. Acknowledgments
  21. References
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