MolPharm xPharm- The Comprehensive Pharmacology Reference

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thompson, S.A.
Right arrow Articles by Wafford, K.A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thompson, S.A.
Right arrow Articles by Wafford, K.A.

Vol. 55, Issue 6, 993-999, June 1999

Residues in Transmembrane Domains I and II Determine gamma -Aminobutyric Acid Type AA Receptor Subtype-Selective Antagonism by Furosemide

S.A. Thompson, S.A. Arden, G. Marshall, P.B. Wingrove, P.J. Whiting, and K.A. Wafford

Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, United Kingdom

    Summary
Top
Summary
Introduction
Materials and Methods
Results and Discussion
References

GABAA receptors in cerebellar granule cells are unique in expressing a subtype containing the alpha 6 subunit. This receptor subtype has high affinity for GABA and produces a degree of tonic inhibition on cerebellar granule cells, modulating the firing of these cells via spillover of GABA from GABAergic synapses. This receptor subtype also has selective affinity for the diuretic furosemide over receptors containing other alpha -subunits. Furosemide exhibits approximately 100-fold selectivity for alpha 6-containing receptors over alpha 1-containing receptors. By making alpha 1/alpha 6 chimeras we have identified a transmembrane region (209-279) responsible for the high furosemide sensitivity of alpha 6beta 3gamma 2s receptors. Within the alpha 1 transmembrane region, a single amino acid was identified that when mutated from threonine to isoleucine, increased furosemide sensitivity by 20-fold. We demonstrate the beta -subunit selectivity of furosemide to be due to asparagine 265 in the beta 2 and beta 3 transmembrane-domain II similar to that observed with potentiation by the anticonvulsant loreclezole. We also show that Ile in transmembrane-domain I accounts for the increased GABA sensitivity observed at alpha 6beta 3gamma 2s compared with alpha 1beta 3gamma 2s receptors, but did not affect direct activation by pentobarbital or potentiation by the benzodiazepine flunitrazepam. Location of these residues within transmembrane domains leads to speculation that they may be involved in the channel-gating mechanism conferring increased receptor activation by GABA, in addition to conferring furosemide sensitivity.

    Introduction
Top
Summary
Introduction
Materials and Methods
Results and Discussion
References

In the mammalian brain, inhibitory neurotransmission is mainly mediated via activation of GABAA receptors, which belong to a superfamily of ligand-gated ion channels. The mammalian GABAA receptor gene family consists of a number of subunit polypeptides (alpha 1-6, beta 1-3, gamma 1-2, delta , and epsilon ) that are thought to coassemble as pentamers (Whiting et al., 1995; Sieghart, 1995). Native GABAA receptor subtypes most likely consist of alpha  and beta  subunits together with a gamma , delta , or epsilon  subunit. The binding of GABA to the receptor complex results in the opening of an anion channel through which chloride ions flow. In addition to the GABA binding site, a number of allosteric sites have been identified on the receptor, which can modulate GABAergic activity. These include the benzodiazepines and anesthetics, which potentiate GABAergic responses, and antagonists such as picrotoxin and zinc, which act in a noncompetitive manner (Macdonald and Olsen, 1994).

Another compound identified as a noncompetitive antagonist at GABAA receptors is the diuretic compound furosemide. This blocker of the Na+/2Cl-/K+ cotransporter, has also been shown to be receptor subtype-selective, eliciting approximately 100-fold greater sensitivity for alpha 6beta 2gamma 2s receptors than for alpha 1beta 2gamma 2s receptors (Korpi et al., 1995), as well as selectivity for alpha 6beta 3gamma 2s over alpha 6beta 1gamma 2s. The aim of this study was to identify the amino acids within the alpha 6 subunit and beta 3 subunit that are responsible for conferring high affinity for this antagonist, using chimeric receptors and point mutations.

    Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results and Discussion
References

Cloning of human GABAA receptor subunit cDNAs (alpha 1-alpha 6, beta 3, and gamma 2s) has been described previously (Hadingham et al., 1993a,b, 1996; Wafford et al., 1996). Chimeric and point-mutated cDNAs were generated by standard techniques as described previously (Wingrove et al., 1994). Mutations were confirmed by DNA sequencing.

Expression of Human GABAA Receptors in Xenopus Oocytes. Adult female Xenopus laevis were anesthetized by immersion in a 0.4% solution of 3-aminobenzoic acid ethylester for 30 to 45 min (or until unresponsive). Ovary tissue was removed via a small abdominal incision and stage V and VI oocytes were isolated with fine forceps. After mild collagenase treatment to remove follicle cells (Type IA, 0.5 mg ml-1 for 6 min), the oocyte nuclei were directly injected with 10 to 20 nl of injection buffer (88 mM NaCl, 1 mM KCl, 15 mM HEPES, at pH 7, filtered through nitro-cellulose) containing different combinations of human GABAA subunit cDNAs (20 ng µl-1) engineered into the expression vector pCDM8 or pcDNAI/Amp. After incubation for 24 to 72 h, oocytes were placed in a 50 µl bath and perfused at 4 to 6 ml/min-1 with modified Barth's medium consisting of 88 mM NaCl, 1 mM KCl, 10 mM HEPES, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.91 mM CaCl2, 2.4 mM NaHCO3, at pH 7.5. Cells were impaled with two 1 to 3 MOmega electrodes containing 2 M KCl and voltage-clamped at -70 mV.

In all experiments drugs were applied in the perfusate until the peak of the response was observed. Noncumulative concentration-response curves to GABA and pentobarbital were constructed with an interval of 3 min between each agonist application.

Inhibition curves to furosemide (0.3-3000 µM) were constructed using a GABA EC50 concentration. Furosemide was preapplied for 30 s before addition of the GABA EC50 concentration and furosemide. The effects of flunitrazepam were examined on control GABA EC20 responses with a preapplication time of 30 s. A minimum of two Xenopus toads were used for each data set.

Whole Cell Patch-Clamp of Human Embryonic Kidney (HEK) 293 Cells Transiently Transfected with Human GABAA Receptors. Experiments were performed on HEK 293 cells transiently transfected with human cDNA combinations alpha 1beta 3gamma 2s, alpha 6beta 3gamma 2s, alpha 1T230Ibeta 3gamma 2s, and alpha 6I228Tbeta 3gamma 2s (6 µg of cDNA total per coverslip) using calcium phosphate precipitation (Chen and Okayama, 1988) as described previously (Hadingham et al., 1993a). Glass coverslips containing the cells in a monolayer culture were transferred to a perspex chamber on the stage of Nikon Diaphot inverted microscope. Cells were continuously perfused with a solution containing 124 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM KH2PO4, 25 mM NaHCO3, 11 mM D-glucose, at pH 7.2, and observed using phase-contrast optics. Patch-pipettes were pulled with an approximate tip diameter of 2 µm and a resistance of 4 MOmega with borosilicate glass and filled with 130 mM CsCl, 10 mM HEPES, 10 mM EGTA, 3 mM Mg+-ATP, pH adjusted to 7.3 with CsOH. Cells were patch-clamped in whole-cell mode using an Axopatch-200B patch-clamp amplifier (Axon Inst., Foster City, CA). Drug solutions were applied by a double-barreled pipette assembly, controlled by a stepping motor attached to a Prior manipulator, enabling rapid equilibration around the cell. Increasing GABA concentrations were applied for 5-s pulses with a 30-s interval between applications.

Analysis. Curves were fitted using a nonlinear square-fitting program to the equation f(x) = BMAX/[1 + (EC50/x)n] where x is the drug concentration, EC50 is the concentration of drug eliciting a half-maximal response and n is the Hill coefficient. EC50 and IC50 values are shown as mean (95% CL), n = 3 or more, and differences between means were evaluated by Student's t test and considered significant if P < .05.

Drugs Used. gamma -Aminobutyric acid (Sigma Chemical Co., St. Louis, MO) was prepared as a 1 M stock solution in modified Barth's medium. Concentrated stock solutions of furosemide (1 M) and flunitrazepam (10 mM) (both obtained from Sigma) were freshly prepared in 100% dimethyl sulfoxide. Pentobarbital was obtained from Rhône Mérieux (Harlow, UK) as a concentrate in alcohol (Sagatal for injection containing 60 mg ml-1 pentobarbitone sodium). The concentrates were diluted into buffer and the maximal final vehicle concentration was 0.3% v/v for dimethyl sulfoxide and 0.4% v/v for the alcohol. No effects on GABA currents were observed with either vehicle.

    Results and Discussion
Top
Summary
Introduction
Materials and Methods
Results and Discussion
References

As has previously been reported (Korpi et al., 1995; Wafford et al., 1996) furosemide displays a greater sensitivity for alpha 6beta 3gamma 2s receptors [IC50 = 12.1 (11.4, 12.9) µM] compared with alpha 1-5beta 3gamma 2s receptors [IC50 values ranging from 234.9 (212.3, 260) µM for alpha 4beta 3gamma 2s to >= 3 mM for alpha 2beta 3gamma 2s] (Fig. 1). In addition, the gamma -subunit appears not to be required for furosemide antagonism, as alpha 6beta 3 receptors are also highly sensitive to block by furosemide, with an IC50 of 14.4 (9.5, 21.9) µM (data not shown).


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1.   Concentration-inhibition curves for furosemide versus a GABA EC50 response on Xenopus oocytes expressing human GABAA receptors composed of alpha 1beta 3gamma 2s (open circle ), alpha 2beta 3gamma 2s (black-down-triangle ), alpha 3beta 3gamma 2s (black-diamond ), alpha 4beta 3gamma 2s (), alpha 5beta 3gamma 2s (black-square), and alpha 6beta 3gamma 2s () Data represents the mean ± S.E.M. of at least three individual cells.

A number of chimeras (C1-C5) were made encompassing different regions of the alpha 1 and alpha 6 subunits (Fig. 2A) and expressed with human beta 3 and gamma 2 subunits in Xenopus oocytes. Chimeras 1 and 3 both displayed alpha 1-like furosemide sensitivity (C1 IC50 = 1.38 (1.32, 1.45) mM and C3 IC50 = 0.98 (0.87, 1.10) mM). The furosemide sensitivity for chimera 2 was not significantly different from alpha 6beta 3gamma 2s receptors [17.1 (14.9, 19.7) µM compared with 12.1 (11.4, 12.9) µM] whereas chimeras 4 and 5 displayed intermediate sensitivity [78.6 (55.8, 110.6) µM and 56.3 (37.6, 84.3) µM respectively; Fig. 2B] (Table 1a). These results suggest that there are at least two amino acids responsible for the high furosemide sensitivity of alpha 6-containing receptors, the first being located within a region between amino acids 131 and 160 and the second between 209 and 279 (Fig. 3).


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 2.   A, schematic diagram of the five alpha 1/alpha 6 chimeras generated for this study. Numbering is according to mature alpha 1 polypeptide and for each chimera the first and last amino acid of the alpha 6 sequence are numbered. The transmembrane domains are illustrated. Shaded area represents alpha 6 sequence and clear areas represent alpha 1 sequence. B, concentration-inhibition curves for furosemide versus a GABA EC50 response on Xenopus oocytes expressing human GABAA receptors composed of alpha 1beta 3gamma 2s (open circle ), alpha 6beta 3gamma 2s (), and the chimeras C1 (), C2 (black-triangle), C3 (black-down-triangle ), C4 (black-diamond ), and C5 (black-square) expressed with beta 3gamma 2s. Data represents the mean ± S.E.M. of at least four individual cells.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1
Summary of furosemide IC50 values and GABA EC50 values determined for the different chimeras and point mutants in this study, expressed using (a) Xenopus oocytes or (b) HEK cells. Data represents mean (±95% confidence limits) of n >=  3, n.d. (not determined)



View larger version (59K):
[in this window]
[in a new window]
  		Fig. 3.   Alignment of human GABAA alpha 1 and alpha 6 subunits. Numbering is according to mature alpha 1 polypeptide. The figure shows the four putative transmembrane domains and the two regions containing the amino acids responsible for the high sensitivity at alpha 6-containing receptors identified from the alpha 1/6 chimeras. Within the first region shaded in yellow (131-160) there are seven amino acid differences between alpha 1 and alpha 6 whereas the second region shaded in blue (209-279) has 12.

Identification of Isoleucine228 in Transmembrane-Domain (TM) 1. In the region between positions 209 and 279 there are 12 amino acid differences between alpha 1 and alpha 6. Fisher et al. (1997) described a rat alpha 6/alpha 1 chimera with a splice site within TM1 that conferred high furosemide sensitivity and a alpha 1 point mutation (alpha 1L258T) where furosemide sensitivity was unchanged. These results eliminated 5 of the 12 amino acids identified within this region. The remaining seven amino acids were mutated (in groups of 2 or 3) in alpha 1 to the alpha 6 equivalent and the furosemide IC50 determined.

alpha 1V212I,T215V,H216Ybeta 3gamma 2s and alpha 1K220Q,I223Mbeta 3gamma 2s receptors both displayed alpha 1-like furosemide sensitivity [IC50 = 2.12 (1.78, 2.51) mM and 1.15 (0.94, 1.40) mM respectively]. alpha 1V227M,T230Ibeta 3gamma 2s receptors, however, revealed an intermediate sensitivity [IC50 = 51.4 (44.6, 59.2) µM] similar to that of chimeras 4 and 5. Individual point mutations produced IC50 values of 0.7 (0.63, 0.78) mM for alpha 1V227Mbeta 3gamma 2s and 40.9 (34.6, 48.3) µM for alpha 1T230Ibeta 3gamma 2s (Fig. 4), demonstrating a critical role for isoleucine 228 within the alpha 6 subunit in conferring furosemide selectivity. Figure 5 illustrates the effects of furosemide on oocytes expressing wild-type alpha 1beta 3gamma 2s, alpha 6beta 3gamma 2s, and alpha 1T230Ibeta 3gamma 2s receptors. The effects of furosemide were shown to be significantly reduced on the equivalent alpha 6 receptor mutant (alpha 6I228T), producing a 10-fold reduction in furosemide sensitivity with an IC50 of 127.6 (86.3, 188.6) µM (Fig. 6A). In addition, when the same wild-type and equivalent mutants were expressed in HEK cells and studied using whole cell-patch-clamp techniques, similar differences were observed in furosemide sensitivity (Fig. 6B, Table 1b).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 4.   Concentration-inhibition curves for furosemide versus a GABA EC50 response on Xenopus oocytes expressing human GABAA receptors composed of alpha 1beta 3gamma 2s (open circle ), alpha 6beta 3gamma 2s (), and the individual point mutants alpha 1V227Mbeta 3gamma 2s (black-down-triangle ) and alpha 1T230Ibeta 3gamma 2s (). Data represents the mean ± S.E.M. of four individual cells.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 5.   The sensitivity of furosemide is determined in part by the presence of isoleucine at position 228 within the alpha 6 subunit. Representative current recordings illustrate the concentration-dependent inhibition by furosemide (0.3 µM -3 mM) of the inward current evoked by an EC50 concentration of GABA on Xenopus oocytes expressing alpha 6beta 3gamma 2s (A), alpha 1beta 3gamma 2s (B), and alpha 1T230Ibeta 3gamma 2s (C) receptors. Drug application is illustrated by the horizontal bars above the current records.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 6.   Concentration-inhibition curves for furosemide versus a GABA EC50 response on Xenopus oocytes (A) and transiently transfected HEK cells (B) expressing alpha 1beta 3gamma 2s (open circle ), alpha 1T230Ibeta 3gamma 2s (), alpha 6beta 3gamma 2s (), and alpha 6I228Tbeta 3gamma 2s (black-square GABAA receptors. Data represents the mean ± S.E.M. of at least four individual cells.

Interestingly, the threonine residue is conserved in all other alpha -subunits, including the alpha 4 subunit, that has intermediate furosemide sensitivity (Wafford et al., 1996), and so cannot account for the higher affinity of alpha 4-containing receptors for furosemide. Mutation of this conserved threonine to isoleucine in alpha 4 produced a 10-fold increase in furosemide sensitivity with an IC50 of 22.3 (18.5, 26.9) µM, similar to alpha 6beta 3gamma 2s receptors. (Fig. 7).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 7.   Concentration-inhibition curves for furosemide versus a GABA EC50 response on Xenopus oocytes expressing human GABAA receptors composed of alpha 4beta 3gamma 2s (open circle ), alpha 4T234Ibeta 3gamma 2s (), alpha 6beta 3gamma 2s (), and alpha 6I228Tbeta 3gamma 2s (black-square). Data represents the mean ± S.E.M. of four individual cells.

The location of this residue in TM1 suggests that TM1 may form part of the ion channel with the residue providing a binding site for furosemide. The residue may also have a role in ion-channel gating, possibly making the receptor more amenable to block by furosemide. Thus mutation of a single amino acid within the alpha 1 subunit from threonine to the alpha 6 equivalent isoleucine at position 230 produced a 20-fold increase in furosemide sensitivity. This single amino acid change, however, did not shift the IC50 completely to that observed on alpha 6beta 3gamma 2 receptors, suggesting that other residues are also involved.

Additional Determinants Affecting Furosemide Sensitivity. Our results from the chimera studies identified a possible second domain (131-160) within the alpha 6 subunit, which may contribute to the high furosemide sensitivity. Within the transmembrane domain of alpha 1, a single amino acid changed to the alpha 6 equivalent, alpha 1T230I, increased the furosemide sensitivity of alpha 1 by 20-fold. A further 5-fold increase in sensitivity is required to bring the furosemide IC50 to that seen on alpha 6beta 3gamma 2s or chimera 2. Single amino acid mutations or insertion of the region 131 to 160 into alpha 1 however, did not affect the furosemide IC50 (see Table 1a, chimera 6), so it is currently unclear how this small additional component is conferred. The action of furosemide has also been shown to depend on the beta -subunit variant, being weaker on beta 1-containing receptors than on beta 2- and beta 3-containing receptors (Korpi et al., 1995). Potentiation of GABAA receptors by the anticonvulsant loreclezole has been shown to be dependent on the beta -subunit (Wafford et al., 1994) and is dependent on the presence of asparagine 265 in the beta 2 or beta 3 subunit (Wingrove et al., 1994). We have compared the effects of furosemide on alpha 6beta 1gamma 2 and alpha 6beta 3gamma 2 receptors, confirming selectivity for alpha 6beta 3gamma 2. We have also used beta  point mutants, beta 1S265N and beta 3N265S, coexpressed with alpha 6 and gamma 2s to demonstrate that the beta -subunit selectivity is determined by the same asparagine residue as loreclezole (Fig. 8). Mutation of the serine within beta 1 to asparagine (the beta 3 counterpart) increased furosemide sensitivity [from an IC50 of 66.5 (63.3, 70.0) µM to 12.3 (11.8, 12.9) µM]. Conversely, mutation of the asparagine within beta 3 to serine decreased furosemide sensitivity [from an IC50 of 12.4 (11.4, 12.9) µM to 224 (190, 263) µM]. It is interesting to note that mutation within the beta 1 subunit revealed an identical IC50 as alpha 6beta 3gamma 2s whereas mutation within the beta 3 subunit produced a significantly higher IC50 than alpha 6beta 1gamma 2s. Like the threonine/isoleucine we have identified in TM1, the asparagine/serine is located on the extracellular end of TM2 and it is possible that these two amino acids are located close to each other at the extracellular face of the channel.


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 8.   Concentration-inhibition curves for furosemide versus a GABA EC50 response on Xenopus oocytes expressing human GABAA receptors composed of alpha 6beta 1gamma 2s (open circle ), alpha 6beta 1S265Ngamma 2s (), alpha 6beta 3gamma 2s (), and alpha 6beta 3N265Sgamma 2s (black-square). Data represents the mean ± S.E.M. of at least four individual cells.

Isoleucine 228 in alpha 6 Also Confers Higher GABA Affinity. Interestingly, concentration-response curves for GABA expressing the wild-type alpha 1beta 3gamma 2, alpha 6beta 3gamma 2, and the corresponding Thr/Ile mutants revealed significant differences in GABA affinity. The GABA EC50 for alpha 1T230Ibeta 3gamma 2s receptors [0.84 (0.77, 0.91) µM] in HEK cells was significantly lower than alpha 1beta 3gamma 2s receptors [3.29 (2.50,5.37) µM] but not different from alpha 6beta 3gamma 2s receptors [0.89 (0.74, 1.08) µM (Fig. 9; Table 1b]. However, the equivalent mutation in alpha 6 (I228T) did not affect GABA EC50 [0.71 (0.59, 0.86) µM]. Hence, this mutation could also account for the higher GABA affinity of alpha 6-containing receptors. GABA concentration-response curves in Xenopus oocytes were also carried out on the mutant alpha 1 and alpha 6 receptors, as well as all the alpha 1/alpha 6 chimeras, however, the greater intrinsic variability in the oocyte expression system precluded the significant detection of such a 5-fold difference. The location in TM1 makes it unlikely that this residue forms part of the GABA binding site, which has been shown to be formed by residues in the alpha  and beta -subunit N-terminal regions (Sigel et al., 1992; Amin and Weiss, 1993). The EC50 value is a function of both the GABA binding affinity and the isomerization rate constants for transitions between the various closed, open, and desensitized states. Channel gating involves conformational changes in the membrane-spanning domains and we hypothesize that mutation from threonine 230 to isoleucine within TM1 alters the transduction process, resulting in a lower EC50 value. The high GABA affinity of alpha 6-containing receptors has recently been shown to be critical to their function in granule cells, as mediating a tonic inhibition via spillover of GABA from Golgi to granule cell synapses (Brickley et al., 1996; Rossi and Hamann, 1998).


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 9.   GABA concentration-response curves on transiently transfected HEK cells expressing alpha 1beta 3gamma 2s (open circle ), alpha 1T230Ibeta 3gamma 2s (), alpha 6beta 3gamma 2s (), and alpha 6I228Tbeta 3gamma 2s (black-square) GABAA receptors. Data represents the mean ± S.E.M. of at least eight individual cells.

Similar to GABA and furosemide, direct activation by pentobarbital displays alpha 6 selectivity (Thompson et al., 1996) and was therefore examined on Xenopus oocytes expressing alpha 1beta 3gamma 2s and alpha 1T230Ibeta 3gamma 2s receptors. No differences were observed in either the EC50 or maximum response as a percentage of the maximum GABA response (189 µM and 75% for alpha 1beta 3gamma 2 compared with 191 µM and 66% for alpha 1T230Ibeta 3gamma 2s). Additionally, potentiation of a GABA EC20 by the benzodiazepine flunitrazepam (1 µM) was unaffected by the threonine to isoleucine mutation (104 ± 13% for alpha 1beta 3gamma 2s and 90 ± 8% for alpha 1T230Ibeta 3gamma 2s). Although mutation of Thr230 to Ile within the alpha 1 subunit significantly increased furosemide and GABA affinity, it did not alter the direct activation of pentobarbital or the potentiation elicited by flunitrazepam.

The role of the putative membrane spanning TM1 has also been investigated in the muscle nicotinic receptor (Akabas and Karlin, 1995) using cysteine substitution experiments. They suggest that the top third (N terminal) of TM1 contributes to the lining of the ion channel and hypothesize that in the closed state, TM1 segments intercalate between TM2 at the extracellular end. On receptor activation, movements of TM1 and TM2 could flip a gate, possibly formed by the cytoplasmic loop between them. If the same is true in the homologous GABAA receptor, by interacting directly with TM1, furosemide could be stabilizing this closed state of the ion channel gate. The position of the asparagine in beta 2 and beta 3, however, is hypothesized to be facing away from the lumen of the channel (Xu and Akabas, 1996); if this is the case, it may interact with the residues identified within TM1 in this study. Further study of the effects of this mutation at the single channel level will enhance our understanding of how this residue affects channel function and the mechanism of furosemide antagonism.

    Footnotes

Received September 28, 1998; Accepted March 24, 1999

Send reprint requests to: Dr. K.A. Wafford, Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, U.K. CM20 2QR. E-mail: keith-wafford{at}merck.com

    Abbreviations

TM, transmembrane-domain; HEK, human embryonic kidney.

    References
Top
Summary
Introduction
Materials and Methods
Results and Discussion
References

0026-895X/99/060993-07$3.00/0
MOLECULAR PHARMACOLOGY, 55:993-999 (1999).
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics


This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
C. J. Herden, N. E. Pardo, R. K. Hajela, Y. Yuan, and W. D. Atchison
Differential Effects of Methylmercury on {gamma}-Aminobutyric Acid Type A Receptor Currents in Rat Cerebellar Granule and Cerebral Cortical Neurons in Culture
J. Pharmacol. Exp. Ther., February 1, 2008; 324(2): 517 - 528.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
B. C. Drafts and J. L. Fisher
Identification of Structures within GABAA Receptor {alpha} Subunits That Regulate the Agonist Action of Pentobarbital
J. Pharmacol. Exp. Ther., September 1, 2006; 318(3): 1094 - 1101.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Serantes, F. Arnalich, M. Figueroa, M. Salinas, E. Andres-Mateos, R. Codoceo, J. Renart, C. Matute, C. Cavada, A. Cuadrado, et al.
Interleukin-1beta Enhances GABAA Receptor Cell-surface Expression by a Phosphatidylinositol 3-Kinase/Akt Pathway: RELEVANCE TO SEPSIS-ASSOCIATED ENCEPHALOPATHY
J. Biol. Chem., May 26, 2006; 281(21): 14632 - 14643.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
M. Ernst, S. Bruckner, S. Boresch, and W. Sieghart
Comparative Models of GABAA Receptor Extracellular and Transmembrane Domains: Important Insights in Pharmacology and Function
Mol. Pharmacol., November 1, 2005; 68(5): 1291 - 1300.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
D. M. Jones-Davis, L. Song, M. J. Gallagher, and R. L. Macdonald
Structural Determinants of Benzodiazepine Allosteric Regulation of GABAA Receptor Currents
J. Neurosci., August 31, 2005; 25(35): 8056 - 8065.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
X. Jin, J. R. Huguenard, and D. A. Prince
Impaired Cl- Extrusion in Layer V Pyramidal Neurons of Chronically Injured Epileptogenic Neocortex
J Neurophysiol, April 1, 2005; 93(4): 2117 - 2126.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
H.-J. Feng and R. L. Macdonald
Proton Modulation of {alpha}1{beta}3{delta} GABAA Receptor Channel Gating and Desensitization
J Neurophysiol, September 1, 2004; 92(3): 1577 - 1585.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
B. C. Drafts and J. L. Fisher
Structural Determinants of the Pharmacological Properties of the GABAA Receptor {alpha}6 Subunit
J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1108 - 1115.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
F. Minier and E. Sigel
Positioning of the {alpha}-subunit isoforms confers a functional signature to {gamma}-aminobutyric acid type A receptors
PNAS, May 18, 2004; 101(20): 7769 - 7774.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
B. Buffin-Meyer, M. Younes-Ibrahim, G. El Mernissi, L. Cheval, S. Marsy, M. Grima, J.-P. Girolami, and A. Doucet
Differential Regulation of Collecting Duct Na+,K+-ATPase and K+ Excretion by Furosemide and Piretanide: Role of Bradykinin
J. Am. Soc. Nephrol., April 1, 2004; 15(4): 876 - 884.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
M. T. Bianchi and R. L. Macdonald
Neurosteroids Shift Partial Agonist Activation of GABAA Receptor Channels from Low- to High-Efficacy Gating Patterns
J. Neurosci., November 26, 2003; 23(34): 10934 - 10943.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. T. Sinkkonen, S. Mansikkamaki, T. Moykkynen, H. Luddens, M. Uusi-Oukari, and E. R. Korpi
Receptor Subtype-Dependent Positive and Negative Modulation of GABAA Receptor Function by Niflumic Acid, a Nonsteroidal Anti-Inflammatory Drug
Mol. Pharmacol., September 1, 2003; 64(3): 753 - 763.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
B. K. Hoffpauir and E. L. Gleason
Activation of mGluR5 Modulates GABAA Receptor Function in Retinal Amacrine Cells
J Neurophysiol, October 1, 2002; 88(4): 1766 - 1776.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
J. L. Fisher
Amiloride Inhibition of gamma -Aminobutyric AcidA Receptors Depends upon the alpha Subunit Subtype
Mol. Pharmacol., June 1, 2002; 61(6): 1322 - 1328.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S.-A. Thompson, P. B. Wingrove, L. Connelly, P. J. Whiting, and K. A. Wafford
Tracazolate Reveals a Novel Type of Allosteric Interaction with Recombinant gamma -Aminobutyric AcidA Receptors
Mol. Pharmacol., April 1, 2002; 61(4): 861 - 869.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
B. X. Carlson, A. C. Engblom, U. Kristiansen, A. Schousboe, and R. W. Olsen
A Single Glycine Residue at the Entrance to the First Membrane-Spanning Domain of the gamma -Aminobutyric Acid Type A Receptor beta 2 Subunit Affects Allosteric Sensitivity to GABA and Anesthetics
Mol. Pharmacol., March 1, 2000; 57(3): 474 - 484.
[Abstract] [Full Text]

Home page