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B Birnir

Publications and source records attributed to B Birnir.

At least 19 recordsLinked to original sources
PubMed2003

Effects of pentobarbital on GABA-activated currents in acutely-isolated rat dentate gyrus granule neurons.

Granule neurons from the rat dentate gyrus were acutely isolated and whole-cell currents recorded. Maximal enhancement of 7 microM gamma-aminobutyric acid (GABA; EC30) evoked currents was obtained with 100 microM pentobarbital where the peak-current was 2.1+/-0.2 of control. One hundred microM pentobarbital alone evoked no current response whereas 1 mM pentobarbital elicited a current response that was 0.4+/-0.2 of the 100 microM GABA-activated peak current. In 100 microM pentobarbital, the GABA EC50 value shifted from 14 to 3 microM but the peak-saturating-current value was not altered. An off-current was recorded on removal of 100 microM and higher pentobarbital concentrations. Ten mM pentobarbital abolished the peak-current response to 7 and 100 microM GABA. The results show that in the granule neurons the drug potency differs for the different effects of pentobarbital at GABAA receptors with the modulatory and inhibitory effects requiring lower concentrations than the direct activation of the receptors.

Animals↗
PubMed2001

GABA concentration sets the conductance of delayed GABAA channels in outside-out patches from rat hippocampal neurons.

GABAA channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches). The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased with GABA concentration from less than 10 pS (0.5 microm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC50 value of 33 microm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of 1 microm diazepam, the GABA EC50 decreased to 0.2 microm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 microm GABA the bicuculline IC50 was 19 microm. The effect of GABA on channel conductance shows that the role of the ligand in GABAA receptor channel function is more complex than previously thought.

Animals↗
PubMed2001

Heterogeneity of functional GABA(A) receptors in rat dentate gyrus neurons revealed by a change in response to drugs during the whole-cell current time-course.

We examined if the drug sensitivity of GABA(A) receptors in dentate gyrus granule neurons changed during the whole-cell current time-course. Effects of drugs on currents evoked immediately (the peak current) upon drug application and currents remaining about two seconds later (semi-plateau current) were compared. The apparent affinity for GABA (EC(50)) of the peak and the semi-plateau current were 14 and 4 microM, respectively. Bicuculline inhibited 50% of the peak and the semi-plateau current (IC(50)) at 7 and 36 microM, respectively, while 100 microM was required for full inhibition of the 100 microM GABA-evoked current. Zinc inhibited about 50% of the peak current with an IC(50) value of 94 microM whereas biphasic, but complete inhibition of the semi-plateau current was recorded with IC(50) values of 3 and 558 microM. The decay phase of the 100 microM GABA-evoked current was fitted by a fast (tau(1), 100-300 ms) and a slow (tau(2), 1-2 s) time-constants in all cells. The relative current amplitude associated with the fast (A1) and the slow (A2) component varied. The A1 current amplitude appeared more sensitive to bicuculline than the A2 current while the opposite was true for zinc. The results are consistent with heterogenous population of functional GABA(A) receptors in the dentate gyrus granule neurons.

Action Potentials↗
PubMed2000

Spontaneously opening GABA(A) channels in CA1 pyramidal neurones of rat hippocampus.

Spontaneous, single channel, chloride currents were recorded in 48% of cell-attached patches on neurones in the CA1 region of rat hippocampal slices. In some patches, there was more than 1 channel active. They showed outward rectification: both channel conductance and open probability were greater at depolarized than at hyperpolarized potentials. Channels activated by gamma-aminobutyric acid (GABA) in silent patches on the same neurones had similar conductance and outward rectification. The spontaneous currents were inhibited by bicuculline and potentiated by diazepam. It was concluded that the spontaneously opening channels were constitutively active, nonsynaptic GABA(A) channels. Such spontaneously opening GABA(A) channels may provide a tonic inhibitory mechanism in these cells and perhaps in other cells that have GABA(A) receptors although not having a GABA(A) synaptic input. They may also be a target for clinically useful drugs such as the benzodiazepines.

Animals↗
PubMed2000

Bicuculline, pentobarbital and diazepam modulate spontaneous GABA(A) channels in rat hippocampal neurons.

Spontaneously opening, chloride-selective channels that showed outward rectification were recorded in ripped-off patches from rat cultured hippocampal neurons and in cell-attached patches from rat hippocampal CA1 pyramidal neurons in slices. In both preparations, channels had multiple conductance states and the most common single-channel conductance varied. In the outside-out patches it ranged from 12 to 70 pS (Vp=40 mV) whereas in the cell-attached patches it ranged from 56 to 85 pS (-Vp=80 mV). Application of GABA to a patch showing spontaneous channel activity evoked a rapid, synchronous activation of channels. During prolonged exposure to either 5 or 100 microM GABA, the open probability of channels decreased. Application of GABA appeared to have no immediate effect on single-channel conductance. Exposure of the patches to 100 microM bicuculline caused a gradual decrease on the single-channel conductance of the spontaneous channels. The time for complete inhibition to take place was slower in the outside-out than in the cell-attached patches. Application of 100 microM pentobarbital or 1 microM diazepam caused 2 - 4 fold increase in the maximum channel conductance of low conductance (<40 pS) spontaneously active channels. The observation of spontaneously opening GABA(A) channels in cell-attached patches on neurons in slices suggests that they may have a role in neurons in vivo and could be an important site of action for some drugs such as benzodiazepines, barbiturates and general anaesthetics.

Animals↗
PubMed2000

Mutating the highly conserved second membrane-spanning region 9' leucine residue in the alpha(1) or beta(1) subunit produces subunit-specific changes in the function of human alpha(1)beta(1) gamma-aminobutyric Acid(A) receptors.

The properties of the human alpha(1)beta(1) gamma-aminobutyric acid (GABA)(A) receptors were investigated after mutation of a highly conserved leucine residue at the 9' position in the second membrane-spanning region (TM2). The role of this residue in alpha(1) and beta(1) subunits was examined by mutating the 9' leucine to phenylalanine, tyrosine, or alanine. The mutations were in either the alpha(1) subunit (alpha*beta), the beta(1) subunit (alphabeta*), or in both subunits (alpha*beta*), and the receptors were expressed in Sf9 cells. Our results show that the rate of desensitization is increased as the size and hydrophobicity of the 9' residue in the alpha(1) subunit is increased: Y, F > L > A, T. Mutation of L9' in only the beta(1) subunit (alphabeta*) to either phenylalanine or tyrosine increased the EC(50) value for GABA at least 100 times, but the EC(50) was unchanged in alphabeta* alanine mutants. In the 9' alpha(1) mutants (alpha*beta, alpha*beta*) the GABA EC(50) was minimally affected. In alpha*beta and alpha*beta*, but not alphabeta*, the peak currents evoked by millimolar concentrations of GABA were greatly reduced. The reduction in currents could only be partially accounted for by decreased expression of the receptors These findings suggest different roles for the two types of subunits in GABA activation and later desensitization of alpha(1)beta(1) receptors. In addition, an increase in the resting membrane conductance was recorded in alanine but not in phenylalanine and tyrosine mutants, indicating that the side chain size at the 9' position is a major determinant of current flow in the closed conformation.

Animals↗
PubMed2000

Pentobarbital modulates gamma-aminobutyric acid-activated single-channel conductance in rat cultured hippocampal neurons.

We examined the effect of a range of pentobarbital concentrations on 0.5 microM gamma-aminobutyric acid (GABA)-activated channels (10 +/- 1 pS) in inside-out or outside-out patches from rat cultured hippocampal neurons. The conductance increased from 12 +/- 4 to 62 +/- 9 pS as the pentobarbital concentration was raised from 10 to 500 microM and the data could be fitted by a Hill-type equation. At 100 microM pentobarbital plus 0.5 microM GABA, the conductance seemed to reach a plateau. The pentobarbital EC(50)(0.5 microM GABA) value was 22 +/- 4 microM and n was 1.9 +/- 0.5. In 1 mM pentobarbital plus 0.5 microM GABA, the single-channel conductance decreased to 34 +/- 8 pS. This apparent inhibition of channel conductance was relieved by 1 microM diazepam. The channel conductance was 64 +/- 6 pS in the presence of all three drugs. The channels were open more in the presence of both GABA and pentobarbital than in the presence of either drug alone. Pentobarbital alone (100 microM) activated channels with conductance (30 +/- 2 pS) and kinetic properties distinct from those activated by either GABA alone or GABA plus pentobarbital. Whether pentobarbital induces new conformations or promotes conformations observed in the presence of GABA alone cannot be determined from our study, but the results clearly show that it is the combination of drugs present that determines the single-channel conductance and the kinetic properties of the receptors.

Animals↗
PubMed1999

Mutant human alpha(1)beta(1)(T262Q) GABA(A) receptors are directly activated but not modulated by pentobarbital.

Pentobarbital activates GABA(A) receptors and enhances GABA-activated currents. A threonine residue (262) in the second membrane spanning region at the 12' position in the beta(1) subunit, alpha(1)beta(1)(T12'Q), is necessary for the potentiating action of pentobarbital. We examined whether T12'Q-mutated receptors expressed in Spodoptera frugipedra (Sf 9) cells responded to direct activation by pentobarbital. In both mutant and wild type receptors, pentobarbital (100 microM to 1 mM) evoked a current response. The pentobarbital EC(50) values were similar; 119 and 158 microM for alpha(1)beta(1) and alpha(1)beta(1)(T12'Q) receptors, respectively. The results show it is possible to discriminate between agonistic and potentiating effects of pentobarbital, suggesting these actions involve separate mechanisms.

Amino Acid Substitution↗
PubMed1999

A threonine residue in the M2 region of the beta1 subunit is needed for expression of functional alpha1beta1 GABA(A) receptors.

Although there is a high degree of homology in the M2 transmembrane segments of alpha1 and beta1 subunits, subunit-specific effects were observed in alpha1beta1 GABA(A) receptors expressed in Spodoptera frugipedra (Sf9) cells when the conserved 13' threonine residue in the M2 transmembrane region was mutated to alanine. When threonine 263 (13') was mutated to alanine in the beta1 subunit, high-affinity muscimol binding and the response to GABA were abolished. This did not occur when the threonine 263 (13') was mutated to alanine in the alpha1 subunit, but the rate of desensitisation increased and the effect of bicuculline, a competitive inhibitor, was reduced. The results show differential effects of subunits on receptor function and support a role for M2 in desensitisation.

Action Potentials↗
PubMed1998

Two threonine residues in the M2 segment of the alpha 1 beta 1 GABAA receptor are critical for ion channel function.

The role of three threonine residues in the M2 hydrophobic region of the GABAA receptor has been investigated by replacing these polar residues with alanine at the 6', 10' and 13' positions of M2 in the GABAA alpha 1, and beta 1 subunits and co-expressing the mutated subunits in the baculovirus Sf9 insect cell system. GABA did not elicit a current in cells expressing either the 6' or 13' threonine to the alanine mutants. The mutant subunits formed intact heteromeric GABAA receptors as judged by the binding of [3H] muscimol or the relative level of alpha 1 protein present in the plasma membrane. In contrast, a chloride current was generated by GABA in cells expressing the 10' mutant receptor. However, the current decayed more rapidly to baseline in the continued presence of GABA in the 10' mutant receptor than in the wild type receptor. The results are discussed in terms of the possible roles of the threonine residues in the ion conduction pathway.

Alanine↗
PubMed1997

Hippocampal GABA(A) channel conductance increased by diazepam.

Benzodiazepines, which are widely used clinically for relief of anxiety and for sedation, are thought to enhance synaptic inhibition in the central nervous system by increasing the open probability of chloride channels activated by the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Here we show that the benzodiazepine diazepam can also increase the conductance of GABAA channels activated by low concentrations of GABA (0.5 or 5 microM) in rat cultured hippocampal neurons. Before exposure to diazepam, chloride channels activated by GABA had conductances of 8 to 53pS. Diazepam caused a concentration-dependent and reversible increase in the conductance of these channels towards a maximum conductance of 70-80 pS and the effect was as great as 7-fold in channels of lowest initial conductance. Increasing the conductance of GABAA channels tonically activated by low ambient concentrations of GABA in the extracellular environment may be an important way in which these drugs depress excitation in the central nervous system. That any drug has such a large effect on single channel conductance has not been reported previously and has implications for models of channel structure and conductance.

Animals↗
PubMed1997

A structural determinant of desensitization and allosteric regulation by pentobarbitone of the GABAA receptor.

Functional properties of the alpha1beta1 GABAA receptor changes in a subunit-specific manner when a threonine residue in the M2 region at the 12' position was mutated to glutamine. The rate and extent of desensitization increased in all mutants but the rate of activation was faster in the beta1 mutants. A negligible plateau current and abolition of potentiation by pentobarbitone of the GABA-activated current depended on the Thr 12' Gln mutation being present in the beta1 subunit. The Hill coefficient of the peak current response to GABA was reduced to less than one also in a beta1 subunit-specific manner. It was concluded that the beta1 subunit dominated conformational changes activated by GABA.

Allosteric Regulation↗
PubMed1997

Nature of the 5' residue in the M2 domain affects function of the human alpha 1 beta 1 GABAA receptor.

The effects on the functional properties of the alpha 1 beta 1 GABAA receptor when the 5' (alpha 1 Val260; beta 1 Ile255) hydrophobic amino acids in the second transmembrane (M2) region were changed to threonine were examined. In response to a saturating concentration of GABA, the current evoked in mutant receptors showed a decreased rate of desensitization and at equilibrium was a greater fraction of the peak current than in wild-type receptors. The half-saturation concentration of the peak current response to GABA in mutant receptors was comparable to that in wild-type receptors, but the Hill coefficient was reduced to less than one. It was concluded that the 5' amino acids in the M2 region have a role in the conformational changes that occur within the alpha 1 beta 1 GABAA receptor in response to GABA.

Animals↗
PubMed1996

Effects of mutating leucine to threonine in the M2 segment of alpha1 and beta1 subunits of GABAA alpha1beta1 receptors.

The conserved leucine residues at the 9' positions in the M2 segments of alpha1 (L264) and beta1 (L259) subunits of the human GABAA receptor were replaced with threonine. Normal or mutant alpha1 subunits were co-expressed with normal or mutant beta1 subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher "resting" chloride conductance than cells expressing wild-type alpha1beta1 receptors. This chloride conductance was blocked by 10 mM penicillin, a recognized blocker of GABAA channels, but not by bicuculline (100 microm) or picrotoxin (100 microm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 microM). In cells expressing wild-type beta1 with mutated alpha1 subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type alpha1beta1 receptors. In cells expressing mutated beta1 subunits with wild-type or mutated alpha1 subunits (alphabeta(L9'T) and alpha(L9'T)beta(L9'T)), no response to GABA could be elicited: this was not due to an absence of GABAA receptors in the plasmalemma because the cells bound [3H]-muscimol. It was concluded that in GABAA channels containing the L9'T mutation in the beta1 subunit, GABA-binding does not cause opening of channels, and that the L9'T mutation in either or both subunits gives an open-channel state of the GABAA receptor in the absence of ligand.

Baculoviridae↗
PubMed1995

Rapid desensitization of alpha 1 beta 1 GABA A receptors expressed in Sf9 cells under optimized conditions.

alpha 1 and beta 1 subunits of human GABA A receptors were expressed in Sf9 cells using the Sf9-baculovirus system. Better expression was obtained by manipulating the system. Cell growth phase at the time of infection determined the practical range of virus titre, the period postinfection during which cells were useful for signal detection and the maximal current obtained. Cells in the early exponential phase were relatively insensitive to multiplicity of infection (MOI) whereas cells in the mid- to late-exponential phase were highly dependent on MOI and they responded with the largest Cl- current generated by GABA. Channels activated by GABA were chloride-selective. Half the maximum peak whole-cell current was obtained with 11 microM GABA. The time course of Cl- currents activated by saturating GABA concentrations in cells infected with alpha 1 beta 1-recombinant viruses was examined employing a rapid perfusion system which allowed whole-cell solution exchange in less than 1 msec. The current decay could be fitted by 3 to 4 exponentials for the first 8 sec. The initial fast current decrease had a time constant of about 23 msec. No voltage dependence of time constants was detected but the whole-cell IV relation showed outward rectification. Currents were depressed by bicuculline, penicillin and picrotoxin and potentiated by pentobarbitone.

Animals↗
PubMed1994

Characteristics of GABAA channels in rat dentate gyrus.

Single channel currents were activated by GABA (0.5 to 5 microM) in cell-attached and inside-out patches from cells in the dentate gyrus of rat hippocampal slices. The currents reversed at the chloride equilibrium potential and were blocked by bicuculline (100 microM). Several different kinds of channel were seen: high conductance and low conductance, rectifying and "nonrectifying." Channels had multiple conductance states. The open probability (Po) of channels was greater at depolarized than at hyperpolarized potentials and the relationship between Po and potential could be fitted with a Boltzmann equation with equivalent valency (z) of 1. The combination of outward rectification and potential-dependent open probability gave very little chloride current at hyperpolarized potentials but steeply increasing current with depolarization, useful properties for a tonic inhibitory mechanism.

Action Potentials↗