β subunits of GABAA receptors form proton-gated chloride channels: Insights into the molecular basis

Gamma-aminobutyric acid type A receptors (GABAARs) are ligand gated channels mediating inhibition in the central nervous system. Here, we identify a so far undescribed function of β-subunit homomers as proton-gated anion channels. Mutation of a single H267A in β3 subunits completely abolishes channel activation by protons. In molecular dynamic simulations of the β3 crystal structure protonation of H267 increased the formation of hydrogen bonds between H267 and E270 of the adjacent subunit leading to a pore stabilising ring formation and accumulation of Cl- within the transmembrane pore. Conversion of these residues in proton insensitive ρ1 subunits transfers proton-dependent gating, thus highlighting the role of this interaction in proton sensitivity. Activation of chloride and bicarbonate currents at physiological pH changes (pH50 is in the range 6- 6.3) and kinetic studies suggest a physiological role in neuronal and non-neuronal tissues that express beta subunits, and thus as potential novel drug target.


1
We greatly appreciate the time you spent carefully reading and giving us proper feedback on our 2 manuscript. We have added a substantial new body of data to address the main concerns and corrected 3 minor typos. We have made changes to several figures and introduced new ones, please find them at the 4 end of this letter.

13
The main concerns are that all experiments were performed in non-neuron cells.
14 There is no validation of the findings in physiological conditions or in-depth of discussion about the 15 significance of this acidic pH-mediated regulation mechanism of β subunits in GABA transmission.

16
Therefore, from this point of view, the study is incomplete, and can be further enhanced by additional

22
There is no validation of the findings in physiological conditions.

24
To provide some evidence for a potential physiological existence of the novel proton gated 25 GABAA receptors built from β subunits, we tested three different cellular systems that are all derived from 26 cells that express β subunits in their native environment. Specifically, a neuroblastoma cell line (SH-27 2 SY5Y), iPSC-derived GABA neurons, and an immortalized T-cell (Jurkat cells) were tested to consider 28 both neuron-derived and immune cell derived systems.

29
In order to be able to compare the current kinetics with the registrations of IH(β) on mammalian cells, the 30 use of planar patch clamps (providing comparable speed of solution exchange as in studies on 31 mammalian cells) was essential. We made therefore use of a Jurkat cell line that is known to express 32 GABAA receptor subunits 1 and can be studies as a suspension with planar patch clamp.

35
In this cell type rapidly activating and desensitizing currents through ASIC overlaid the currents through β 36 homomers. ASIC was blocked by amiloride (200 µM).

37
The proton-activated current remaining in the presence of amiloride displayed kinetics comparable to 38 proton activated currents in CHO cells expressing β subunits (Supplementary Figure 2). There inhibition 39 by picrotoxin provides further evidence that these currents represent IH(β).

46
These data warrant further research as the presence of heteromeric GABAA receptors in these cells 47 (confirmed by IGABA, Supplementary Figure 5b) may reduce formation of homooligomeric receptors 2,3 . In 48 the revised MS we discuss, however, that high expression levels of β subunits is supportive for formation 49 of β-homomers not only in heterologous expression systems. Given the large diversity of neuronal cell 50 types, broad screens would be needed to potentially detect pH sensitive neuron types.

51
In a third series of experiments neuroblastoma cells (SH-SY5Y) were detached and suspensions of these 52 cells studied with planar patch clamp. Experiments were performed on non-differentiated cells as well as 53 on differentiated cells where retinoic acid was used (in this case we carried out the experiments on day 54 10 after the start of differentiation). We observed neither IGABA nor evidence for IH(βx).

56
This is also complicated by the fact that no specific inhibitors for β homomers are currently available.

57
We would, however, like to emphasize that β pentamers have received considerable attention as putative 58 histamine gated anion channels 4,5 .   Figure 5a). These cells express β-subunits at much higher abundance than α GABAA 71 subunits 28 which would be expected to facilitate formation of β homomers. It is tempting to speculate that 72 hyperpolarizing chloride currents upon acidification counterbalances depolarization via ASIC receptors 73 and thus may have a role in immunomodulation 29 . However, this warrants further studies. Different 74 activation states of T-cells might lead to variable expression of proton/ histamine sensitive and GABA 75 sensitive receptors, respectively 29,31,32 .

77
In studies on GABA-sensitive iPS cell-derived GABANeurons expressing heteropentamers we did not find

84
preparations was obtained from studies with alternative agonists such as histamine 5,18,44 .

86
Our results imply that GABAA β receptors are activated at physiological and pathological pH changes. It 87 is therefore tempting to speculate that such pH sensitive channels formed by ubiquitously expressed 88 subunits in the brain may exist and serve crucial functions under acidotic conditions such as stroke and 89 ischemia where they can enhance neuroprotective hyperpolarization. Intriguingly, in an experimental 90 model of stroke, upregulation of GABRB3 was connected with a putative neuroprotective role 45 . Although 91 macroscopic changes in extracellular pH in the brain are tightly controlled, pH fluctuations in specific

100
The pH sensitivity of the receptors can serve as a novel tool to analyze the tissue specific expression of This manuscript presents a new claim that GABAA receptors form proton gated chloride channels.

114
The authors also perform an MD simulation capturing a crucial H267-E270 interaction, many details of the 115 said interaction are revealed by the simulation were anticipated by extensive prior mutagenesis studies 116 coupled with functional and biochemical analysis, the direct visualization of these mechanisms is a major 117 advance, and refreshingly the authors do a reasonable job of discussing the MD simulation in context of 118 prior work, although this could be improved.

119
Also, appropriately acknowledged is the fact that it is unclear whether there could be GABA present in 120 trace quantities during the experiment that could impact the major proton gated claim of this manuscript.

121
Also the authors should address the physiological relevance of the pH range tested, as far as my 122 understanding goes the pH even in synapses seldom goes below 6.5.

123
Lastly I am not sure why the authors chose to ignore an array of publications related to

131
Results, Section : Functional properties of proton activated chloride channels, para 2, "Even a small 132 decrease from pH 7.2 to 6.5"-According to me between these two pH values the concentration of H3O+ 133 ion changes exponentially as pH is in log scale.

154
There may be a similar picture to that of the GLIC, which has been described to have two electrostatic   This question was addressed in Fig. 2 where we illustrate IH(β) evoked by pH changes from 7.2 to 6.5.

178
Furthermore, physiological and pathophysiological pH changes are now discussed in more detail on p.

189
In studies on GABA-sensitive iPS cell-derived GABANeurons expressing heteropentamers we did not find

196
In fact, evidence for the existence of GABA insensitive receptors formed by β-subunits in neuronal 197 preparations was obtained from studies with alternative agonists such as histamine 5,18,44 .
is therefore tempting to speculate that such pH sensitive channels formed by ubiquitously expressed  227 specifically to align beta subunits, the structure of β1 subunit (6DW0) from the PMID:30044221, of β2 229 subunits (6X3T) from PMID: 32879488 was used alongside with two β3 subunits (4COF and 7A5V).

230
Their discussion (and additional discussion on GLIC touching on the PMID: 30541892 paper) are now 231 included into the new paragraphs on p. 10, l. 243-264.

239
We propose that interaction between H267 and E270 might play a crucial role in the direct activation of 240 homomeric GABAA β3 receptors by protons. Mutational studies on the GLIC channel suggests that proton 241 activation occurs also in GLIC allosterically to the orthosteric site, but at the level of multiple loci 41 and we 242 thus cannot exclude that other residues than H267 contribute to proton-induced activation of β3 243 receptors. Furthermore, additional evidence suggests that other amino acid residues may be involved in 244 proton-induced gating of GLIC where more detailed mechanisms of receptor activation are known 42,43 .

245
There may be a similar picture to that of the GLIC, which has been described to have two electrostatic 246 triads of amino acid residues in the extracellular domain (structurally highly conserved between pLGIC),   Indeed, the word "small" was misleading as change of pH by 0.7 (from 7.2 to 6.5) corresponds to a 5-fold 272 increase of the H+ concentration {10 0.7 ≈ 5}. Correspondingly we removed the word small and write on p.

334
In studies on GABA-sensitive iPS cell-derived GABANeurons expressing heteropentamers we did not find

341
In fact, evidence for the existence of GABA insensitive receptors formed by β-subunits in neuronal 342 preparations was obtained from studies with alternative agonists such as histamine 5,18,44 .

343
Our results imply that GABAA β receptors are activated at physiological and pathological pH changes. It 344 is therefore tempting to speculate that such pH sensitive channels formed by ubiquitously expressed

372
All recordings of IGABA were made at physiological pH (pH= 7.2). This is now clarified in the subscript of

410
and IGABA during long lasting exposure to pH 5 or to GABA 100 μM. Desensitization half time for IH(β3) is induced by switching the pH from 7.2 to 6.5. d The pH-dependence of steady-state desensitization and of 413 activation of receptors composed of the indicated β-subunits. Oocytes were conditioned for 10 minutes at 414 the indicated pH, and receptors were subsequently activated by shifting the pH rapidly to 4.5 in order to 415 estimate the the fraction of available (not yet desensitized) channels. The normalized current response is 416 plotted as a function of the conditioning pH (superimposed data for current activation (dash lines) are 417 taken from Fig. 1). Half-maximal desensitisation occurs at pH 6.4 ± 0.02 for β1(S265N), at pH 6.97 ± 0.06 418 for β2 and at pH 6.6 ± 0.15 (n= 4) for β3 homomers. pH was reduced from 9 to the indicated values. The       I agree with the authors addition of new body of data, and it addresses any concerns that I had. The manuscript is in a much better shape and provides a more comprehensive view of the question at hand. Please accept my congratulations on this wonderful piece of work.
Reviewer #4 (Remarks to the Author): The authors have addressed all of my comments. This manuscript is now suitable for publication.
We greatly appreciate the time you spent carefully reading and giving us proper feedback on our manuscript. We have added a substantial new body of data to address the main concerns and corrected minor typos. We have made changes to several figures and introduced new ones, please find them at the end of this letter.

Reviewer #2 (Remarks to the Author):
This is a new study characterizing acidic pH-mediated stimulation of β3 subunit in GABARs chloride current in Oocytes expressing the recombinant DNAs. The main findings included reversal of the low pHmediated channel activation in cells expressing β3 H267A mutation, gain of the function in cells expressing ρ1 (G331H-A334E), and simulation analysis of a hydrogen bond formation between H267 and E270, accumulation of Cl-near the pore structure with protonated H267. Overall, the data are clearly presented, and convincing.

The main concerns are that all experiments were performed in non-neuron cells.
There is no validation of the findings in physiological conditions or in-depth of discussion about the significance of this acidic pH-mediated regulation mechanism of β subunits in GABA transmission. Therefore, from this point of view, the study is incomplete, and can be further enhanced by additional work.

Reviewers comment (1,2) The main concerns are that all experiments were performed in non-neuron cells.
There is no validation of the findings in physiological conditions.

Response
To provide some evidence for a potential physiological existence of the novel proton gated GABAA receptors built from β subunits, we tested three different cellular systems that are all derived from cells that express β subunits in their native environment. Specifically, a neuroblastoma cell line (SH-both neuron-derived and immune cell derived systems. In order to be able to compare the current kinetics with the registrations of IH(β) on mammalian cells, the use of planar patch clamps (providing comparable speed of solution exchange as in studies on mammalian cells) was essential. We made therefore use of a Jurkat cell line that is known to express GABAA receptor subunits 1 and can be studies as a suspension with planar patch clamp.
These new data are summarized in an additional Supplementary Figure 5a in Supplementary Information on p. 6, l. 51 (also please find the figure at the end of this document) In this cell type rapidly activating and desensitizing currents through ASIC overlaid the currents through β homomers. ASIC was blocked by amiloride (200 µM).
The proton-activated current remaining in the presence of amiloride displayed kinetics comparable to proton activated currents in CHO cells expressing β subunits (Supplementary Figure 2). There inhibition by picrotoxin provides further evidence that these currents represent IH(β).
A physiological role of the hyperpolarizing IH could therefore induce (counteract depolarization) inhibition of the proton-induced depolarization by ASIC and slower desensitization suggest a more long-lasting effect compared to the transient ASIC currents.
Suspensions of GABA-sensitive iPS cell-derived GABANeurons were also suitable for planar patch clamp. These cells expressed heteropentamers (evident from IGABA in Supplementary Figure 5a). We did, however, not find evidence for IH(βx) after 4-6 days in differentiating media (Supplementary Figure 5b).
These data warrant further research as the presence of heteromeric GABAA receptors in these cells (confirmed by IGABA, Supplementary Figure 5b) may reduce formation of homooligomeric receptors 2,3 . In the revised MS we discuss, however, that high expression levels of β subunits is supportive for formation of β-homomers not only in heterologous expression systems. Given the large diversity of neuronal cell types, broad screens would be needed to potentially detect pH sensitive neuron types.
In a third series of experiments neuroblastoma cells (SH-SY5Y) were detached and suspensions of these cells studied with planar patch clamp. Experiments were performed on non-differentiated cells as well as on differentiated cells where retinoic acid was used (in this case we carried out the experiments on day This is also complicated by the fact that no specific inhibitors for β homomers are currently available.
We would, however, like to emphasize that β pentamers have received considerable attention as putative histamine gated anion channels 4,5 .

Response
According to the reviewer's suggestion we discuss the possible role of pH mediated regulation mechanism of β-subunits in more detail.  Figure 5a). These cells express β-subunits at much higher abundance than α GABAA subunits 28 which would be expected to facilitate formation of β homomers. It is tempting to speculate that hyperpolarizing chloride currents upon acidification counterbalances depolarization via ASIC receptors and thus may have a role in immunomodulation 29 . However, this warrants further studies. Different activation states of T-cells might lead to variable expression of proton/ histamine sensitive and GABA sensitive receptors, respectively 29,31,32 .
In studies on GABA-sensitive iPS cell-derived GABANeurons expressing heteropentamers we did not find evidence for IH(βx) after 4-6 days in differentiating media (Supplementary Figure 5b)  In fact, evidence for the existence of GABA insensitive receptors formed by β-subunits in neuronal preparations was obtained from studies with alternative agonists such as histamine 5,18,44 .
Our results imply that GABAA β receptors are activated at physiological and pathological pH changes. It is therefore tempting to speculate that such pH sensitive channels formed by ubiquitously expressed subunits in the brain may exist and serve crucial functions under acidotic conditions such as stroke and ischemia where they can enhance neuroprotective hyperpolarization. Intriguingly, in an experimental model of stroke, upregulation of GABRB3 was connected with a putative neuroprotective role 45  This manuscript presents a new claim that GABAA receptors form proton gated chloride channels.
The authors also perform an MD simulation capturing a crucial H267-E270 interaction, many details of the said interaction are revealed by the simulation were anticipated by extensive prior mutagenesis studies coupled with functional and biochemical analysis, the direct visualization of these mechanisms is a major advance, and refreshingly the authors do a reasonable job of discussing the MD simulation in context of prior work, although this could be improved.
Also, appropriately acknowledged is the fact that it is unclear whether there could be GABA present in trace quantities during the experiment that could impact the major proton gated claim of this manuscript.
Also the authors should address the physiological relevance of the pH range tested, as far as my understanding goes the pH even in synapses seldom goes below 6.5. Results, Section : Functional properties of proton activated chloride channels, para 2, "Even a small decrease from pH 7.2 to 6.5"-According to me between these two pH values the concentration of H3O+ ion changes exponentially as pH is in log scale. Figure 3c: readers might also benefit from having at least one Gamma subunit in the alignment, I further recommend testing the effects on a tri-heteromeric receptor and comparing the Gamma versus the Rho subunit.

Response
According to the reviewers comment we have extended the discussion on MD simulations in the context of what is already known from prior work on the GLIC receptor and added a new paragraph on p.10, l.

249-264
"We propose that interaction between H267 and E270 might play a crucial role in the direct activation of homomeric GABAA β3 receptors by protons. Mutational studies on the GLIC channel suggests that proton activation occurs also in GLIC allosterically to the orthosteric site, but at the level of multiple loci 41 and we thus cannot exclude that other residues than H267 contribute to proton-induced activation of β3 receptors. Furthermore, additional evidence suggests that other amino acid residues may be involved in proton-induced gating of GLIC where more detailed mechanisms of receptor activation are known 42,43 .
There may be a similar picture to that of the GLIC, which has been described to have two electrostatic triads of amino acid residues in the extracellular domain (structurally highly conserved between pLGIC), which are part of a continuous network governing the gating transitions of the receptor. However, due to the absence of open/closed β3 homomeric receptor structures, it is difficult to speculate about the exact determinants and this question remains open for further investigation. The HxxE motif we describe here is unique to the β subunits (Fig. 3c) and absent in GLIC, and is necessary and sufficient for proton sensitivity as demonstrated by the conversion mutants, irrespective of the details of transduction. While MD simulations of GLIC have been conducted 42 , a side-by-side comparison with the same method would be needed to get more insight into similarities and differences in the proton interaction sites and transduction mechanisms."

Reviewers comment
(2) "Also, appropriately acknowledged is the fact that it is unclear whether there could be GABA present in trace quantities during the experiment that could impact the major proton gated claim of this manuscript" No traces of GABA were present in the experiments shown in Fig, 1, Fig. 2, Fig.3

Reviewers comment
(3) "Also the authors should address the physiological relevance of the pH range tested, as far as my understanding goes the pH even in synapses seldom goes below 6.5"

Response
This question was addressed in Fig. 2 where we illustrate IH(β) evoked by pH changes from 7.2 to 6.5.
Furthermore, physiological and pathophysiological pH changes are now discussed in more detail on p.
"β-homomers form readily in recombinant expression systems, their existence in mammalian cells is currently unknown. First evidence for picrotoxin-sensitive IH(βx) was obtained in Jurkat T cells (Supplementary Figure 5a). These cells express β-subunits at much higher abundance than α GABAA subunits 28 which would be expected to facilitate formation of β homomers. It is tempting to speculate that hyperpolarizing chloride currents upon acidification counterbalances depolarization via ASIC receptors and thus may have a role in immunomodulation 29 . However, this warrants further studies. Different activation states of T-cells might lead to variable expression of proton/ histamine sensitive and GABA sensitive receptors, respectively 29,31,32 .
In studies on GABA-sensitive iPS cell-derived GABANeurons expressing heteropentamers we did not find evidence for IH(βx) after 4-6 days in differentiating media (Supplementary Figure 5b). These data warrant further research as the presence of heteromeric GABAA receptors in these cells (confirmed by IGABA, Supplementary Figure 5b) may reduce formation of homooligomeric receptors 31,32 . It seems plausible that high expression levels of β-subunits are supportive for formation of β-homomers not only in heterologous expression systems. Given the large diversity of neuronal cell types, broad screens would be needed to potentially detect pH sensitive neuron types.
In fact, evidence for the existence of GABA insensitive receptors formed by β-subunits in neuronal preparations was obtained from studies with alternative agonists such as histamine 5,18,44 .
is therefore tempting to speculate that such pH sensitive channels formed by ubiquitously expressed subunits in the brain may exist and serve crucial functions under acidotic conditions such as stroke and ischemia where they can enhance neuroprotective hyperpolarization. Intriguingly, in an experimental model of stroke, upregulation of GABRB3 was connected with a putative neuroprotective role 45  specifically to align beta subunits, the structure of β1 subunit (6DW0) from the PMID:30044221, of β2 subunits (6X3T) from PMID: 32879488 was used alongside with two β3 subunits (4COF and 7A5V).
Their discussion (and additional discussion on GLIC touching on the PMID: 30541892 paper) are now included into the new paragraphs on p. 10, l. 243-264.
"In order to compare the similarity of the three β subunits in the H267 -E270 region, we also examined structures with β1 and β2 subunits. Of these, only heteromeric structures are available 39,40 . Thus, the individual β subunits were superposed for the best fit of the M2 segment. The amino acids of interest are structurally equivalent, and only slightly differ in sidechain rotamers. Thus, we expect the residues H267 and E270 play a structurally equivalent role in β1 and β2 homopentamers (Supplementary Figure 6).
We propose that interaction between H267 and E270 might play a crucial role in the direct activation of homomeric GABAA β3 receptors by protons. Mutational studies on the GLIC channel suggests that proton activation occurs also in GLIC allosterically to the orthosteric site, but at the level of multiple loci 41 and we thus cannot exclude that other residues than H267 contribute to proton-induced activation of β3 receptors. Furthermore, additional evidence suggests that other amino acid residues may be involved in proton-induced gating of GLIC where more detailed mechanisms of receptor activation are known 42,43 .
There may be a similar picture to that of the GLIC, which has been described to have two electrostatic triads of amino acid residues in the extracellular domain (structurally highly conserved between pLGIC), which are part of a continuous network governing the gating transitions of the receptor. However, due to the absence of open/closed β3 homomeric receptor structures, it is difficult to speculate about the exact determinants and this question remains open for further investigation. The HxxE motif we describe here is unique to the β subunits (Fig. 3c) and absent in GLIC, and is necessary and sufficient for proton sensitivity as demonstrated by the conversion mutants, irrespective of the details of transduction. While MD simulations of GLIC have been conducted 42 , a side-by-side comparison with the same method would be needed to get more insight into similarities and differences in the proton interaction sites and transduction mechanisms." This is an interesting report showing a novel function of GABA receptor beta subunit, although their existence in vivo has not been shown. The research is well-designed and the results are clear. I think it will be of interest to the people in the field. I want to point out several minor points to consider. 1. A potential significance of the co-expression of beta homomers and heteropentamers in the physiological and pathological conditions should be discussed, although the existence of beta homomers in mammalian cells has not yet been investigated.
2. Figure 2ab: The pH on the figure legend is 5.5. Please make an adequate correction in the manuscript.
In addition, the pH used to examine IGABA should be clarified in the manuscript since pH modulates GABAactivated responses on the GABAA receptors.

Reviewers comment
(1) A potential significance of the co-expression of beta homomers and heteropentamers in the physiological and pathological conditions should be discussed, although the existence of beta homomers in mammalian cells has not yet been investigated.

Response
In order to address the reviewer's suggestion, we have performed additional experiments on Jurkat cells expressing predominantly GABAA receptor β-subunits and iPS cell-derived GABANeurons expressing heteropentamers. Jurkat cells grow as a suspension and GABANeurons can be transferred to a suspension after detaching from the substrate. Both cell types were therefore suitable for studies using the planar patch clamp technique.  Figure 5a). These cells express β-subunits at much higher abundance than α GABAA subunits 28 which would be expected to facilitate formation of β homomers. It is tempting to speculate that hyperpolarizing chloride currents upon acidification counterbalances depolarization via ASIC receptors and thus may have a role in immunomodulation 29 . However, this warrants further studies. Different activation states of T-cells might lead to variable expression of proton/ histamine sensitive and GABA sensitive receptors, respectively 29,31,32 .
In studies on GABA-sensitive iPS cell-derived GABANeurons expressing heteropentamers we did not find evidence for IH(βx) after 4-6 days in differentiating media (Supplementary Figure 5b). These data warrant further research as the presence of heteromeric GABAA receptors in these cells (confirmed by IGABA, Supplementary Figure 5b) may reduce formation of homooligomeric receptors 31,32 . It seems plausible that high expression levels of β-subunits are supportive for formation of β-homomers not only in heterologous expression systems. Given the large diversity of neuronal cell types, broad screens would be needed to potentially detect pH sensitive neuron types.
In fact, evidence for the existence of GABA insensitive receptors formed by β-subunits in neuronal preparations was obtained from studies with alternative agonists such as histamine 5,18,44 .
Our results imply that GABAA β receptors are activated at physiological and pathological pH changes. It is therefore tempting to speculate that such pH sensitive channels formed by ubiquitously expressed subunits in the brain may exist and serve crucial functions under acidotic conditions such as stroke and ischemia where they can enhance neuroprotective hyperpolarization. Intriguingly, in an experimental model of stroke, upregulation of GABRB3 was connected with a putative neuroprotective role 45 . Although The pH sensitivity of the receptors can serve as a novel tool to analyze the tissue specific expression of homomeric β-receptors in excitable and not excitable cells. To clarify the existence of proton-gated β homomers and their physiological and pathophysiological role in neuronal and/or non-neuronal cells is thus an important goal. In tissues expressing ASIC receptors they might counterbalance a pH dependent depolarisation under acidic conditions (Supplementary Figure 5). Such receptors (which are already known to be sensitive e.g. to histamine and barbiturates) may form a class of novel potential drug targets."

Reviewers comment
(2) Figure 2ab: The pH on the figure legend is 5.5. Please make an adequate correction in the manuscript. In addition, the pH used to examine IGABA should be clarified in the manuscript since pH modulates GABA-activated responses on the GABAA receptors.

Response
We have made an appropriate correction of the figure legend, included the pH in the subscript, see Fig. 2 on p. 25, l. 617, l.619 and l. 622.
All recordings of IGABA were made at physiological pH (pH= 7.2). This is now clarified in the subscript of   and IGABA during long lasting exposure to pH 5 or to GABA 100 μM. Desensitization half time for IH(β3) is a Location of H267 in the pore region of the crystal structure of β3 homomers (the position of H267 is indicated with violet spheres) 9 . b Mutation H267A in the human subunits β3 subunit prevented activation of IH(β3(H267A)) but, instead, induced pH-dependent inhibition of large baseline currents (upper traces).
μM pentobarbital, 1mM histamine and 100 μM bicuculline). c Sequence alignment of the transmembrane M2 α-helix of α1, β1-3 (human), γ2 (human) and ρ1 (rat) subunits (segment does not differ between rat and human). The location of H267 and E270 in β subunits and the corresponding homologous positions in the ρ1 subunit (double mutant ρ1(G331H-A334E)) are illustrated. d Histogram of the number of hydrogen bonds between protonated and deprotonated H267 and E270. e Snapshots of the ring structure observed in the MD runs. composed of concatenated subunits α1-β2/β2-α1-β2 (rat) (a), α1-β3/β3-α1-β3 (rat) (b) and α1-β3-α1/γ2-β3 (rat) (c) are not activated by changing the pH from 9 to indicated values (a, b and c, respectively) but activated by GABA (pH 7.2). a Representative currents recorded from Jurkat cells upon shifting the pH from 7.2 to 5 alone (left) or in the presence of 200 μM amiloride (middle overlaid currents, blue). Application of pH 5 produced ASIC-like fast activating currents, whereas co-application of pH 5 with amiloride (an ASIC blocker 2 ) resulted in slower activating and desensitising currents that were completely blocked by picrotoxin (100 µM).
Application of GABA did not activated IGABA in these cells. Average mean current amplitudes are 440 ± 118 pA for pH 5-induced currents and 34 ± 4 pA for pH 5 in presence of 200 μM amiloride (n = 11, the data are presented as mean values ±SEM).
b Representative currents recorded from iPS cell-derived iCell GABAergic neurons cells. Application of pH 5 resulted in a current (left) which was completely abolished by co-application with amiloride (middle trace), consistent with an ASIC current. IGABA (induced by 100 μM GABA) is shown on the right (indicating the presence of heteromeric GABAA receptors in these neurons). Average current amplitudes are 225 ± 44 pA for pH 5-induced ASIC currents and 308 ± 35 pA for GABA-induced currents (n = 5, the data are presented as mean values ±SEM).