HOW DOES SUB-UNIT COMPOSITION OF GABA-A RECEPTOR AFFECT THE RECEPTOR FUNCTION?
GABA-A receptors [GABAA-R] are the receptors which mediate the fast inhibitory neurotransmission of the gamma-amino butyric acid [GABA], a chief inhibitory neurotransmitter found in vertebrate central nervous system and present in 40% of all neurons. They are entrenched in the cell membrane of the neurons to serve as a channel for the transit of negatively charged ions, usually chloride ions into the cells. GABA or a similar compound activates the receptor by adhering to it which in turn opens the channels and facilitating the passage which diminishes the cell excitation. [John.S.,Adron,1997]. Hence they are functionally, ligand-gated chloride channels and are also called ionotropic receptors. [Richard,Werner,2007]. They are composed of 5 individual protein subunits obtained from six different subunit families with several subtypes namely α1-6, β1-3, ϒ1-3, δ, ε and π which form the ion-channels (as shown in fig 1) .[Haas, Macdonald, 1999]. However the precise composition of GABAA-RS is not completely established.
The subunit composition of GABAA-Rs results in differences in their pharmacological properties. Thus the overall characteristics of the GABAA-Rs are influenced by the subunit composition, or their location in the brain or during the different developmental phases. [ John, Harris,1997]* [MC Kernan, Whiting, P.J., 1996]
GABAA-Rs serve as a main site for the action of various drugs such as benzodiazepines, barbiturates, furosemide, zinc, etc. Although different subtypes manipulate the properties of GABAA-Rs, the subtypes of α-subunit play a significant role in the activation of receptor by GABA and also in the regulation of GABAA-Rs by various allosteric regulators. Among all the subtypes of α-subunit family, α1 & α6 are largely varied contributing the least structural homology (59% of amino acid characteristics) [ Fisher, Zhang, et.al.1997], *[Tyndale,R.F.,1995 ].
Formation of a functional receptor require both α and β subunits [Korpi,et. al.,2002]. And these both subunits are responsible for the effects of barbiturates on GABAA-Rs [ Thompson,et.al.,1996].
Ø The β-subunit has a positive influence on several anaesthetics including barbiturates in which β2 and β3 are more active when compared to β1. Whereas the influence of α-subunit is less established however, it is known that pentobarbital shows greater efficacy as an agonist than GABA in the presence of α6-subunit compared to other α1-α5 subunits or in combination with β and ϒ subunits. [Drafts, C., Fisher, L.,2006]*[Thompson et.al.,1999] . The methods used to determine the structures in α6-subunit responsible for the high barbiturate efficacy are by the
[Drafts, C., Fisher, l., 2006]
By these tests, it is obtained that
ü “pentobarbital is a more effective agonist than GABA only at receptors containing the α6-subunit”.
ü “Higher efficacy of pentobarbital is associated with the N-terminal domain of the α6-subunit”.
ü “The α6 mutation alters direct activation but not allosteric modulation by pentobarbital”. [Jones,A.,Korpi,E.R.,et.al.,1997].
Ø The effect of the β-subunit on the receptor's pharmacology can be found by expressing recombinant human GABAA-Rs containing diverse β-subunits in both transfected cells and Xenopus oocytes. The methods used to determine the β-subunit effect includes the following. [Haldingham, K.L.,et.al.,1993]
Ø From these methods, it is established that β-subunit does influence neither the efficacy nor the affinity of many compounds such as benzodiazepines, barbiturates and various neurosteroids by which it suggest that the pharmacology of these compounds are not affected at the GABAA-R site in the humans.[Haldingham, K.L.,et.al.,1993]
Ø The effect of α6-subunit on δ subunit can be studied by using mutant cerebellar granule cells in the mouse. These cells express 6 GABAA-R subunits (α1, α6, β2, β3, ϒ2 and δ) copiously. In this study, it is been observed that removal of α6 protein results in the disruption of δ subunit. The methods used are: [Jones,A.,Korpi.E.R.,et.al.,1997]
ü The obtained results showed that inactivation of α6-subunit results in the inhibition of δ-subunit expression. [Jones,A.,Korpi,E.R.,et.al.,1997]
According to a hypothesis, it is been manifested that diversity in GABAA-R channel subtypes may vary the assembly of specified set of subunits into different configurations. On the other hand, assembly of subunits into GABARs turn out to an ideal form of receptor channel. For e.g., the assembly of α1β1 and α1β1ϒ2s subunits generated unique ion-channels with different single-channel properties. The methods used for this are: [Anjelotti, P.T.,Macdonald,L.R.,1993]
o Plasmid construction
o Cell culture and DNA transfection
o β-galactosidase staining
o Electrophysiological analysis
o Single channel data collection and analysis
The obtained results showed that α1β1 and α1β1ϒ2s possess different conductance and gating properties. It also emerged that α1β1ϒ2s GABARs are formed superior to α1β1 GABARs which when recorded showed that α1β1ϒ2s GABARs opened 3 times the duration as α1β1 GABARs due to the difference in open and burst properties which is showed by the single channel analysis.
The role of the individual subunits of GABARs can be studied by using their behavioural actions on Benzodiazepines (BZs) and anaesthetics. Using the studies regarding amino acid residues leading to ligand-binding areas and channel function at receptor stage and merging that with crystallographic information and molecular modelling, the structure of a protein can be speculated. Mutations can be made by joining these findings with recombinant genetics in mice. [Burt, D.R., Kamachi,1991] *[Chen C.,Okaayama, 1987]
* The presence of ϒ-subunit is vital in which ϒ2 subunit provide a high level of sensitivity than the other two (ϒ1 and ϒ3) subunits which alter in the presence of BZ ligands.
* It is considered that the BZ-binding site is formed in between the ϒ2 and α-subunits of a GABAR.[Colquhoun,D.,Sigworth,1983]as they are present in the homologous position implying that binding sites structural analogous allocated between two subunits[Gu,Y.,Forsayeth, J.R.,1983]
* When studied invitro, intravenous anaesthetic etomidate illustrated selectivity for β2 and β3 restraining receptors.[Puia, G.,Santi,M.R.,et.al.,1990]
* It is disclosed that β3 possessing receptors contribute to most of the anaesthetic affect of etomidate when followed by the administration of anaesthesia by measuring the sleeping time. And it was confirmed by mutating the β3 subunit which resulted in the eradication of anaesthetic activity.
A major influence of the subtypes of GABARs can be observed even in the temporal lobe epilepsy which is related to impaired inhibitory neurotransmission. It was noticed in CA1 and CA3 that the expression structure of α-subunits are strikingly modified in the surviving neurons after subjecting to subunit staining. Whereas in CA2, the arrangement of all three subtypes of α-subunit took place. The expression pattern of other subunits β2,3 and ϒ2 complied with that of α-subunit mutants implying the exclusive expression specific to the GABAARs in human hippocampus. These results are obtained by approaching the following process: [Loup, F., Weiser, H.G.,2000]
I. Patient selection
II. Tissue preparation
IV. Data analysis
VI. Neuron counts
VII. Densitometric measurements
VIII. Statistical analysis
HOW DOES THE SUBUNIT MUTATIONS AFFECT THE ETHANOL ACTIONS ON GABAA RECEPTORS IN XENOPUS OOCYTES? [Ueno, S., Wick, J.M.,2000]
The GABAA-R function can be illustrated by using alcohol as an example which is the most usually consumed drug with behavioural effects. And the action of ethanol in brain requires several neurotransmitter receptors and ion channels. Mutagenesis and heterologous expression system methods facilitated to study the cellular and molecular mechanisms of ethanol's action on GABAA-Rs.
Mutation of specific amino acids in the transmembranes [ TMs] of α and β subunits can abolish the action of ethanol without abolishing the action of GABARs. To establish the importance of ϒ-subunit, α1 and β2 subunits are subjected to mutation and expressed along with ϒ2L subunit in xenopus oocytes by ranging the concentration of ethanol.
Ueno and Wick done the experiments on female frogs of Xenopus laevis . GABA, DImethyl-sulphoxide, collagenase type 1A, picrotoxin and flunitrazepam, ethanol were obtained from the respective chemicals and other chemicals were of reagent grade.
They conducted mutations on cDNAs of mammalian GABAAR α2 and β1 subunits. Mutations were initiated on ϒ2L subunits by using mutagenesis kit. And then Xenopus oocytes are separated and cDNAs are introduced and bi-electrode voltage clamp recordings were done, after desensitization, capacity of ethanol that increases the effect of GABA concentration that produce 5% of maximal effect was determined individually on each oocyte. The oocytes are immersed in the ethanol before subjecting to GABA and observed for the effect of GABA and then statistical measurements are done.
The effects of ethanol are determined in which the co-expression of α and ϒ subunit along with the mutant β-subunit result in the formation of a wild type of gated-subunit which in turn results in the depletion of ethanol effect on GABARs.
Finally, it can be summarized as the subunit composition of GABA-A receptors affect the function of GABAR in a variety of ways. But still, the exact mechanism and composition of the GABA is not known completely.
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