The γ-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the brain, and its homeostasis is mainly regulated by the GABA transporter hGAT1 in the central nervous system (CNS). GAT1 is a secondary-active transport protein that exploits the Na+ gradient to energize the uphill re-uptake of GABA from the synaptic cleft into the presynaptic neuron. Since dysregulations in GABA transport are linked to many neurological disorders, hGAT1 is an important target for their medical treatment.
The solid supported membrane-based electrophysiology (SSME) is a technique that allows the measurement of electrogenic events in transporters, pumps, and channels. The sample (i.e., native membrane vesicles or proteoliposomes) is adsorbed on an artificial bilayer on top of a gold-coated sensor and a capacitive-coupled system is generated. The transport is triggered by substrates concentration gradients as driving force, while membrane voltage is zero.
Through SSME we detected GABA-induced currents on CHO membrane vesicles overexpressing hGAT1, with a maximum amplitude of 3-5 nA, that showed a triphasic behavior leading us to identify three different electrogenic events. The transport component has been identified, showing a KM of 15-20 μM. We could also assess the stoichiometry of GABA versus Na+, which was found to be in agreement with the currently most accepted one of GABA:Na+=1:2.
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So um the next speaker is PhD candidate and my soon to be colleague uh Roco serot uh Rocco uh from Parma Italy uh has a master de degree in biomolecular chemist chemistry from the University of Parma and is currently industrial Marie K PhD student at nanan with the academic
Affiliation uh at the University of Regensburg in Germany so Roco over to you okay thank you Cy for the nice introduction so today I would like to talk to you about the functional characterization of the human gut one that I did mainly with the surfer and one uh technology so the solid support
And membran based electrophysiology uh as a quick agenda of the talk uh first I would like to briefly introduce to you the technique in case some of you that know it and also the target so the human gut one then I would like to briefly go to the sample
Validation then uh I would like to have a look a deep look into the Gaba induced current with the sh solid supported mem electrophysiology and in the end just the functional characterization so the km and the Gaba sodium stochiometry Okay so about uh some Basics on the technique uh we have as
You can see here on the left we have our our gold uh sensor and we functionalize the surface with um an artificial bil layer that is composed by a th layer and a lipid layer on top then we let the sample that can be composed by uh Native membrane fragments or prot liposomes
That are um expressing the protein of interest of course and we let it absorb on top of this artificial by layer here and this generates a capacitive couple system so basically what happens is that all the currents that we see are transient because once we charge the capacitor uh completely the current
Stops flowing and so the case uh another important thing is that we don’t have any kind of direct voltage control so the uh measurement starts at 0 molt uh so here just the general um workflow uh for the for the surfer so we start start with a non-activating we
Start perusing a non-activating solution that doesn’t contain the main substrate and this is important because uh basically we get rid of the mechanical artifact that is um generated by just the fluidics going into action and we also get the Baseline this can also be used though to generate an ion gradient
Or a CO substrate gradient uh if this is needed then we perfuse the activating solution that con contains our main substrate and hopefully we activate and and see a current uh going on generated then in the end we restore the initial condition by perfusing again the non-activating solution so we have
Basically the reverse transport reaction okay just to stress on some basic key concept uh again the current recording happens current recordings happen at 0 molt and the driving force is just a substrate gadient no so it’s not voltage driven uh all the currents are transient
And as you can see here uh just some example on the left are the currents that you can see on the ssme and on the right are the same currents reconstructed are as they should look like in a normal conventional PCH clamp environment so if we have as low transient this is usually
Associated to the stationary transport current and you should see something like this in a p clamp environment if we have a fast transient that usually is associated with a pready state current pready state current is uh actually an out of equilibrium um confirmation arrangement of the transporter um so we have basically
Charges that are crossing the electri field of the membrane um but not in in a dynamic equilibrium as the transport is or we can have more licated situation like the one that you can see below so basically we can have a combination of par State currents and transport
Current again some terminology I guess everybody is uh I mean all of you know them very much but ec50 is just the sub subside concentration that is needed to reach half the maximum uh the maximal uh amplitude of a certain parameter so it can be charge or it can be the current
Um current to current at a certain time point or whatsoever and km is just a particular case of vc50 that is the meis m constant okay just uh for um for the Target itself so the human gut one uh human gut one is main its main function
Is the reuptake of Gaba from the synaptic left uh inside the pr synaptic neuron and to do that it has to exploit the energy that comes from the inward directed sodium gradient that is normally existing in uh nature and so for the stomry we have that for each
Gaba molecule that is re uptaken two sodium ions and one chloride ion is are transported so this is the most currently accepted uh actually there are some debating about the number of sodium ions that can be uh reup taken and also if the chloride is actually always coupled in this
Way uh the transport anyway is electrogenic so that’s very good and we can use electrophysiology to uh characterize this protein and also this is an important T Target in Pharmacology because um a lot of diseases are associated with disregulation in Gaba homeostasis okay so uh moving on to the sample
Validation here what I just did uh is I had my um vesicles CH vesicles over expressing youat one uh filled with pottassium chloride then I perfuse with sodium chloride so I generate the sodium gent without activating transport and then in the end I perfuse with a solution containing the same amount of
Sodium chloride plus over saturating Gaba so now I am activating the transport so here on the right you can see the results of um this experiment uh cond conducted on uh on CHR uh vesicles over expressing one and you can see I have this nice Peak here then for
Control I also used CH vesicles empty so not expressing the the protein of interest and of course I mean luckily I didn’t see any kind of current uh rising and I also wanted to assess if uh actually the Gaba so if uh this transport was um related to sodium and
Chloride as it be so I just perused with Gaba but in absence of sodium and chloride and again I didn’t see any current Rising still I wasn’t happy so I wanted to also check the functionality of the the living cells so I used the patch liner that we have in house here here
And since I felt very lucky I just went straight for um a do response curve I mean a do response experiment uh with different Gaba concentrations so I used the the pass clamp technique I performed it in the whole Cell mode and I kept the voltage hold at minus 70 molt as usually
Is the neuron resting potential and luckily again I was able to see uh these nice currents here um I did uh the heill plot of the of the currents and I found out this ec50 or km of around 12 microl that this is very good agreement with the literat literator
Results okay so now the tricky part so I wanted to really dissect the currents that I could see uh in SS SM so here again this is the same current we saw before so basically high sodium gradient and then um over saturating Gaba perfused so I wanted to have a close
Look at the current and I what I could observe is that what I could see uh is two Rising pH two two different Rising phase that here I don’t know if you can notice them well but there is one that is very fast and one is lower and then
Again for the Decay I have two different Decay times so one slower very low here and one that was faster so actually here I asked myself some question because uh if the peak would represent the transport as usually can happen with the surface supported M electrophysiology then I should not see
All this kind of different Rising time and DEC time so what I did uh was like let’s try and kill the sodium gradient and see what happens so here I just um filled up my bices with very high sodium chloride and then I generate a very small sodium gradient because I had 135
MIM sodium chloride inside and then I perus outside with 150 M so the gradient is like just 50 m sodium choride then again I activate with uh oversaturating Gaba and what you can see here is that now we lose the low Rising phase and we have a very fast rising phase uh
Followed by again a very lowc phase to all the activating uh time again I was not happy because at this point I asked myself where did the slow Rising phase and the otherc phase end up so I say probably that uh can be something that is related to the sodium
Gradient so let’s just go back keep a high sodium gradient and just lower Gaba let’s see what happens this so basically I could see uh three different uh phases in the current so one is the first current that we could see in absence of sodium gradiate in nominal absence of sodium gradient
The second one basically has a this Decay uh time that is consistent with the with the first decaying time I I could observe and again the third Rising phase so at this point I wanted to check what all these paks were about so I just did a deconvolution of and I tried to
Dissect really dissect all these these processes I was able to do that and here you can see just the convolution of the paks and I also try to say what these paks are individually so the first Peak actually is very similar to uh a process that has been observed in P clamp in
Literature um they actually uh um managed to achieve a fast uh solution exchange by um using photo cage Gaba and just using a pulse of light and fre this Gaba very quickly in the solution and the both Rising time rise and Decay time of this PE are very consistent with uh
The one that this guy F find found and so basically this should be either G one occlusion or the full carrier flipping from the outward facing uh confirmation to the inward facing confirmation then the second Peck the second Peck is quite tricky still I don’t have an
Answer for that so my uh ideas is that this could be either sodium release inside or a sodium leak so there are also in literature many examples of um Channel like behavior of Transporters so this could be it since it’s anyway something that is um affected by sodium
Gradient and in the end the last phase um I thought it was transport so now switching to the functional characterization I started this very slow Decay phase uh so here you just see two different way of um of measuring this so in the case of a of a very low
Sodium gradient I was able to reconstruct the current as basically uh they could see they could um appear as a stationary transport current and then just of course read uh the current amplitude and in this case I just read the the current at a time point in which
Both processes uh so the both pre states that we saw before were at zero in both cases I was um able to uh obtain these uh km values that are in very good agreement uh with literature results uh coming from um two electrod voltage clamp uh measured at um a voltage close
To zero so conditions that are let’s say close to the one that we have on the ccer okay lastly um I wanted to shed a bit of light uh on on the sodium gasometry so here I will spare you the physics but uh basically what happens is
That um the charge can be seen as a function of the ratio of the chemical potentials of sodium and Gaba here and most importantly when the charge is zero the Delta G is zero and so basically this ratio equals the ratio of the stochiometric U the stochiometric ratio
Of the two substrates so here I just played around with the with the concentration in order to obtain uh specific um chemical potential ratios and as you can see here at zero charge we have a two ratio stochiometry between sodium and Gaba so at least for what I
Am able to see sodium mava has a stary of 2 one uh as is the most accepted um in literator so uh again for quick summary um ssme is a suitable technique uh for measuring um currents on human gut one the currents the Gaba induced
Current on uh human gut one show a very complex Behavior because they are basically triphasic triphasic and again lastly sodium gasometry appears to be confirmed as 2:1 so that is all and thank you for the attention thank you very much Roo uh I think we have time for one
Question have a question like more more more on the technical side so basically just to get a good idea of your Technique the dimensions spatial and temporal so the cells there is no cell per se but how close like you can put like the typical size of your vesicles
How intact are those vesicles how close the electrodes so first let’s go to do the spatial component okay so I don’t know about the the actual size of the vesicles um we know that we have uh on on top of the gold surface we can have access to mil of
Transporters and the electrode uh is actually um some millimeters some millimeters away because normally you would want them to because conceptually this is like end plate potential the same principle it’s just kind of on steroid with recent technical the advances normally you want to be as close as possible so I would expect
Something like 100 microns or less preferentially much smaller I I never measured act but maybe and and then related question would be like temporal resolution the best timing you show was like couple of milliseconds yes but I wonder because I’m not talking about resolution of electronics obviously it
Can do micros seconds and less but because of the positioning of all the geometry would have a lot of straight capacitance and some DeLay So how much that influences can you even go like below 1 millisecond with this setup yeah so so uh for it depends on the
Sensor type so if you go to with 3 mm sensors then uh the time resolution the maximum time resolution is about 30 milliseconds but if you use 1 mm sensor so you have a surface area that is actually smaller or the same size of the ion jet then uh the maximum time
Resolution that you can achieve is 1 millisecond sorry 3 milliseconds ah okay yeah so you basically on a millisecond time scale so the your best the fastest that was my impression that maybe you are limited because there is a sodium iron Channel voltage GED so involved there you would expect sub millisecond
Rise time there yes and exactly like I could see the the the triphasic thing only with 1 mm that TR specifically was that you are limited by your equipment actually there yes of course it’s the time resolution of the of the technique so you know about the elect
Yeah for the size of the vesicles I did do DLS measurements and they’re usually 100 to 200 nanometers yeah small yeah we usually prepare them ourselves and we’re doing tip sonication so um by that no we uh purify the membrane from cells and then we reconstitute them into those
Uh vesicles by tips but then was like remark is that you you said that it is at zero molts but since you have working Transporters there that’s a 0 MTS P preparation at like 0 Z in time but once you have Transporters going they will create they
Will build voltage yeah so that will develop certain potential on the membrane you can May well be looking at 50 to 100 MTS of potential across the membrane actually you can you have no way to measure it but with like Transporters at high concentration in this membrane they will develop gradient
Salt gradient across you would have difference and sodium definitely in your case yes that will by itself like 60 MTS easily or more of membrane potential in those small vesicles so they’re not strictly zero molts no no no so the potential will build up Vier in the
Measurement but like some 100 Mill after the anyway so basically at the beginning you don’t have a significant voltage build up yes I think we have to continue these discussions uh afterwards thank you very much for your contribution