This work was supported by the Austrian Research Fund (P31559-B20 to E.E.P.). The authors thank Sarah Bardakji for the excellent technical assistance in the production and reconstitution of mouse UCP1 and UCP3 into proteoliposomes.

1. Production of Recombinant Uncoupling Proteins (UCPs) and the Formation of Planar Bilayer Membranes
<ol>
	<li>Produce recombinant UCP1 and UCP3 as described by Rupprecht et al.9 and Hilse et al.10</li>
	<li>Form planar bilayer membranes on the tips of conventional dispensable plastic pipettes as described by Beck et al.7</li>
</ol>
2. Preparation of the Micro-agar Salt Bridge Electrode
<ol>
	<li>Adjust a microcapillary pipette tip (see Table of Materials) to the appropriate length.
	<ol>
		<li>Mark the position on an empty tip at which the buffer-containing tip will be attached to and use a sliding caliper to measure the length of the micro-agar salt bridge electrode. 
		CAUTION: The microcapillary tip must be long enough to enter the buffer solution for the measurements.</li>
		<li>Cut the microcapillary tip with a sharp knife or blade and clean the cut surface with ethanol and water.</li>
	</ol>
	</li>
	<li>Prepare the AgCl-coated electrode.
	<ol>
		<li>Cut off an Ag wire of approximately 8 cm in length and clean it with ethanol and water.</li>
		<li>Take a piece of sandpaper and smooth down the surface of 1 cm length at the end of the wire, which should be in contact with the salt solution.</li>
		<li>Dip the smoothed end into a 3 M KCl solution and coat it electrochemically with chloride by using a DC supply at 1.5 V for 10 s.</li>
		<li>Clean the electrode with water, dry it, and adjust the length of the AgCl electrode from the uncoated side so that it penetrates the microcapillary tip as deep as possible. 
		Note: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Prepare a 3 M KCl salt solution with 1% (v/v) agarose.
	<ol>
		<li>Weigh out 4.47 g of KCl and dissolve it in 20 mL of water by stirring it in a flask.</li>
		<li>Remove the stirrer, weigh out 0.2 g of agarose, and add it to the flask.</li>
		<li>Heat up the solution to 100 &#176;C to melt the agarose and prevent it from clotting. 
		CAUTION: The flask will be very hot. Do not touch it with bare hands. Use gloves for handling.</li>
	</ol>
	</li>
	<li>Fill the microcapillary tip with the agarose salt solution. 
	&#8203;CAUTION: The solution is hot. Protect hands and work carefully to avoid splashes.
	<ol>
		<li>If the agarose starts clotting, heat up the salt solution to fully melt the agarose again.</li>
		<li>Soak 10 &#181;L of the agar salt solution into the microcapillary tip. Soak it slowly and carefully to avoid air bubbles in the tip.</li>
		<li>Remove the tip from the pipette and push in the AgCl electrode from the broader side of the tip. Ensure that the electrode penetrates the salt solution.</li>
		<li>Cool down the electrode to room temperature and plug it into the amplifier.</li>
	</ol>
	</li>
	<li>Prepare the buffer for the measurements.
	<ol>
		<li>Weigh out 0.710 g of Na2SO4, 0.195 g of MES, 0.121 g of TRIS, and 0.023 g of EGTA, then add 100 mL of distilled water to a beaker and stir the solution.</li>
		<li>Check for the pH value of the buffer by using a pH electrode and adjust the pH value to 7.32.</li>
		<li>Check that the reference electrode and the agar salt bridge electrode are in electrical contact.
		<ol>
			<li>Add 1 mL of buffer to a plastic container.</li>
			<li>Dip in the reference electrode and the agar salt bridge electrode and check for a signal response. If the signal response is correct, proceed to step 2.7.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>If there is a false signal response or no electrical contact at all, perform the following troubleshooting.
	<ol>
		<li>Check if the electrode is in contact with the salt solution and push the electrode into the solution. 
		Note: If the solution is already too sticky, remove the micro-agar salt bridge and prepare a new microcapillary tip.</li>
		<li>Check if there are air bubbles within the salt solution. If yes, prepare a new microcapillary tip.</li>
		<li>Check if the salt solution is in contact with the buffer solution. If not, then cut off another 1 mm of the tube end of the tip. 
		CAUTION: Be sure that the tip is still long enough to penetrate the buffer-containing tip. If there is still no contact, prepare a new microcapillary tip.</li>
		<li>If none of these steps help, prepare a new microcapillary tip.</li>
	</ol>
	</li>
	<li>For storage, dip the agar salt bridge electrode into a 3 M KCl solution. 
	Note: The protocol can be stopped here. For a pause overnight, store the electrode in 3 M KCl salt solution at 4 &#176;C.</li>
	<li>Prepare the buffer-containing plastic tip. 
	Note: If the electrode was stored overnight, take it out and let it heat up to room temperature for 30 min.
	<ol>
		<li>Take a microcapillary tip and bend the tube, 2 cm from the edge of the narrow part, approximately 90 degrees using a heating wire.</li>
		<li>Use a very sharp knife or blade and cut off the tube around 5 mm from the bend.</li>
		<li>Clean the surface at the end with ethanol and water and measure the diameter of the hole of the tip. From this, calculate the area of the surface.</li>
		<li>Coat the surface of the tip with 85:15 (v:v) hexane:hexadecane.
		<ol>
			<li>Pipette 3 &#181;L of the solvent and remove it from the tip.</li>
			<li>Wait 1 min so that all of the residual solvent in the tip has evaporated.</li>
		</ol>
		</li>
		<li>Fill the measurement tip with 3 &#181;L of buffer and plug it onto the salt bridge electrode. 
		NOTE: Check again if the salt bridge electrode and the reference electrode are in electrical contact by performing step 2.5.3.</li>
		<li>If there is no electrical contact, check for the following:
		<ol>
			<li>Check whether the salt bridge electrode is in contact with the buffer solution. If not, then either increase the volume of the buffer in the tip or prepare a longer microcapillary tip.</li>
			<li>If there is an air bubble, remove the tip from the micro-salt bridge electrode, refill the buffer in the tip, and plug it into the electrode again.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Measurement of the Electrical Parameters of the Membrane Reconstituted with Recombinant Protein
<ol>
	<li>Apply a triangular alternating voltage signal with maximal voltage Umax = 50 mV and &#916;Tramp = 50 ms, which creates a rectangular alternating current response. From the mean values of the positive and negative currents (I+ and I-), calculate the capacity of the membrane according to the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/58552/58552eq1v2.jpg" /></li>
	<li>Apply a voltage ramp ranging from -50 mV to +50 mV and record the current. Fit a linear function to the data - the slope is the conductance - and calculate the x-axis intersection point of the fit.</li>
	<li>Discharge the solution in the plastic tip and fill a new one with a buffer containing an increased substrate concentration.
	<ol>
		<li>If no membrane is formed within the first 20 - 30 s, discharge the volume and refill it. This guarantees that the concentration gradient across the membrane does not significantly change during the membrane formation.</li>
		<li>After the formation of a membrane, perform steps 3.1 and 3.2 again to verify proper membrane formation and to get the x-axis intersection point.</li>
	</ol>
	</li>
</ol>
4. Calculation of the Substrate Turnover Rate
NOTE: See previous work for details3,7.
<ol>
	<li>Estimate the amount of protein in the membrane from the molecular mass of lipid and protein (MLipid and MProtein), the area of the membrane and of one lipid head group (AMembrane and ALipid), and the mass ratio of the protein per lipid (r). 
	<img alt="Equation 2" src="/files/ftp_upload/58552/58552eq2v2.jpg" /></li>
	<li>Calculate the Nernst potential for the transported substrate. R is the gas constant, T the temperature, z the charge of the transported substrate, F the Faraday&#39;s constant, and c1 and c2 the concentrations of the substrate of both sides of the membrane. 
	<img alt="Equation 3" src="/files/ftp_upload/58552/58552eq3v3.jpg" /></li>
	<li>From the current-voltage recordings, take the reverse potential calculated with the difference of the x-axis intersection points from the linear fits in the presence and absence of the substrate gradient.</li>
	<li>Calculate the proportion of substrate conductance, GSubstrate, to the total membrane conductance, Gtotal, by the ratio of reverse potential to Nernst potential11. 
	<img alt="Equation 4" src="/files/ftp_upload/58552/58552eq4v2.jpg" /></li>
	<li>Calculate the substrate turnover number &#916;NSubstrate per time unit &#916;T from the substrate conductance (GSubstrate), the applied voltage U, and the charge of the substrate z: 
	<img alt="Equation 5" src="/files/ftp_upload/58552/58552eq5v2.jpg" /></li>
	<li>From the ratio of transported substrate per time to the number of proteins, calculate the turnover rate &#954;. 
	<img alt="Equation 6" src="/files/ftp_upload/58552/58552eq6v2.jpg" /></li>
</ol>

<ol>
	<li>Terstappen, G. C., Reggiani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+silico+research+in+drug+discovery.">In silico research in drug discovery.</a> Trends in Pharmacological Sciences. 22 (1), 23-26 (2001).</li><li>Raynauld, J. P., Laviolette, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+silver-silver+chloride+electrode:+A+possible+generator+of+offset+voltages+and+currents.">The silver-silver chloride electrode: A possible generator of offset voltages and currents.</a> Journal of Neuroscience Methods. 19 (3), 249-255 (1987).</li><li>Urbankova, E., Voltchenko, A., Pohl, P., Jezek, P., Pohl, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+kinetics+of+uncoupling+proteins.+Analysis+of+UCP1+reconstituted+in+planar+lipid+bilayers.">Transport kinetics of uncoupling proteins. Analysis of UCP1 reconstituted in planar lipid bilayers.</a> Journal of Biological Chemistry. 278 (35), 32497-32500 (2003).</li><li>Beck, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyunsaturated+fatty+acids+activate+human+uncoupling+proteins+1+and+2+in+planar+lipid+bilayers.">Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid bilayers.</a> The FASEB Journal. 21 (4), 1137-1144 (2007).</li><li>Shao, X. M., Feldman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-agar+salt+bridge+in+patch-clamp+electrode+holder+stabilizes+electrode+potentials.">Micro-agar salt bridge in patch-clamp electrode holder stabilizes electrode potentials.</a> Journal of Neuroscience Methods. 159 (1), 108-115 (2007).</li><li>Kleene, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+intrapipette+salt+bridge.">A simple intrapipette salt bridge.</a> Journal of Neuroscience Methods. 46 (1), 11-16 (1993).</li><li>Beck, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+automated+technique+for+the+reconstitution+of+hydrophobic+proteins+into+planar+bilayer+membranes.+Studies+of+human+recombinant+uncoupling+protein+1.">A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1.</a> Biochimica et Biophysica Acta-Bioenergetics. 1757 (5-6), 474-479 (2006).</li><li>Macher, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+mitochondrial+UCP1+and+UCP3+by+purine+nucleotides+and+phosphate.">Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate.</a> Biochimica et Biophysica Acta. 1860 (3), 664-672 (2018).</li><li>Rupprecht, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+transmembrane+potential+in+the+membrane+proton+leak.">Role of the transmembrane potential in the membrane proton leak.</a> Biophysical Journal. 98 (8), 1503-1511 (2010).</li><li>Hilse, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+UCP3+directly+correlates+to+UCP1+abundance+in+brown+adipose+tissue.">The expression of UCP3 directly correlates to UCP1 abundance in brown adipose tissue.</a> Biochimica et Biophysica Acta. 1857 (1), 72-78 (2016).</li><li>Fuks, B., Homble, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+and+electrical+properties+of+planar+lipid+membranes+from+thylakoid+lipids.">Permeability and electrical properties of planar lipid membranes from thylakoid lipids.</a> Biophysical Journal. 66 (5), 1404-1414 (1994).</li><li>Barry, P. H., Lewis, T. M., Moorhouse, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimised+3+M+KCl+salt-bridge+technique+used+to+measure+and+validate+theoretical+liquid+junction+potential+values+in+patch-clamping+and+electrophysiology.">An optimised 3 M KCl salt-bridge technique used to measure and validate theoretical liquid junction potential values in patch-clamping and electrophysiology.</a> European Biophysics Journal. 42 (8), 631-646 (2013).</li><li>Huang, D., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+feasibility+and+limitation+of+patch-clamp+recordings+from+neonatal+rat+cardiac+ventricular+slices.">The feasibility and limitation of patch-clamp recordings from neonatal rat cardiac ventricular slices.</a> In Vitro Cellular &amp; Developmental Biology. 47 (4), 269-272 (2011).</li></ol>

The authors have nothing to disclose.

The implementation of the micro-agar salt bridge with the electrode minimizes its diffusion potential and allows more precise measurements of membrane potential generated by a pH gradient. In the presence of various transmembrane pH gradients, the potential shift of both electrodes was acceptable at &#916;pH = 0.35 when comparing to the theoretical value of the Nernst equilibrium potential (&#934;Nernst =23.8 mV for &#916;pH = 0.4). However, at more physiological pH gradients, as for instance in mitochondria between the matrix and intermembrane space, the standard AgCl electrode failed to precisely measure the potential shift at &#916;pH = 1 (Figure 3A). The electrode bridged with micro-agar salt delivered the values which were much more comparable to the theory.
Diffusion potential may also occur at the AgCl reference electrode if the buffer solution is changed during the experiment. Chloride-free buffer solution was used in the experiments since uncoupling proteins were suggested to transport chloride ions, and the pH was adjusted using Tris or MES. The electrode potential, in the absence of a substantial concentration of chloride, primarily depends on chloride impurities in the buffer solution. As its composition is unchanged during the experiments, it will simply result in a constant offset potential. However, for the measurement of an absolute potential difference between the two electrodes, a simple agar salt-bridge system (Ag/AgCl 3 M KCl) could also be used for the reference electrode.
A micro-agar salt bridge balances the diffusion potential by an equilibration of the electrode potential. In order to stabilize the salt solution, 1% (w/v) agarose was added to prevent the mixing of the salt solution with the buffer solution. The salt ions K+ and Cl- have similar mobilities in liquid and balance the electrode potential. To properly install the salt bridge, the agar salt solution has to be sufficiently heated up to fill the microcapillary tip without any air bubbles and to cover the AgCl electrode. Before further usage, electrical contact between the salt bridge electrode and the reference electrode has to be checked. Depending on the time the salt bridge is used, the salt solution has to be sufficiently gelled to prevent any mixing of the salt solution with the buffer. This is especially critical if K+ or Cl- transporters are investigated. The salt bridge was used for a very short time and the elution of agarose is negligible in this time range. A higher concentration of agarose of up to 5%, or of agar (3% - 5%), allows using the salt bridge for a longer period of time6,12.
This method allows determining the transport kinetics of a membrane transporter (i) with low turnover rates and (ii) of mitochondrial proteins of the inner membrane, which can hardly be investigated in standard patch clamp set-ups13. Its precision is mainly dependent on the reverse potential measurement, which accuracy is decreased at a low total membrane conductance and small concentration gradients which induce a membrane potential below the noise of recording.
Using this set-up, the turnover rates of UCP1 and UCP3 as produced under the same conditions were measured. Due to the higher pH gradient, the obtained rates seem to be more precise and unperturbed by artefacts resulting from the minor electrode potential shift. It can be used to further analyze and compare mitochondrial membrane transporters produced under similar conditions.

Membrane proteins are targeted by up to 60% of all known pharmaceutical drugs1. Electrophysiological measurements of membrane proteins are a powerful but delicate tool to analyze the electrogenic transport of substrates mediated by membrane carrier proteins. The modulation of the transmembrane current by the application of constant voltages or voltage ramps allows assessing the pharmacological and physical properties of the carriers, for instance, the activation and inhibition by substrates or the transport kinetics. Of special interest is the substrate turnover number, which displays the amount of substrate that is translocated by a membrane protein per time unit. It is a major parameter when comparing the kinetics of various membrane proteins. Establishing a concentration gradient of the charged substrate across the membrane generates an electromotive force from which the turnover number of the substrate is deduced.
Using an AgCl electrode, the presence of a chloride-free buffer creates a diffusion potential that alters electrode potential and leads to a shift in current-voltage measurements2. Although always present, it is negligible for standard conductance and capacity measurements since these parameters are either dependent on the slope of the current-voltage recording (conductance) or are the difference of a single recording (capacity), which cancels the potential. However, the recording of the reverse potential, which is created by the transport of substrate, can be significantly disturbed by the diffusion potential. Thus, for exact measurements of the reverse potential, the electrode potentials have to be kept constant.
The diffusion potential can be minimized by two methods: (i) in the presence of a bilayer membrane, a substrate concentration has to be increased on one side of the membrane3,4, or (ii) a salt bridge balances the electrode potential5. The first method is highly dependent on the stability of the measurements. The membrane has to survive for several minutes, from the addition of substrate under stirring until the substrate is almost equally distributed in the solution. If the membrane ruptures in between, the substrate gradient is altered by the free exchange of charged molecules, and measurements turn inaccurate. The latter method balances the diffusion potential but is limited by the size of the set-up. Implementing a small but functioning salt bridge in a micro range to an electrophysiological set-up is challenging6. For the latter method, the salt solution is filled into a microcapillary tip and stabilized by the addition of agarose to prevent diffusion of the salt solution to the buffer solution.
In this protocol, a straightforward production of a micro-agar salt bridge and implementation into an electrophysiological set-up based on the pipette set-up7 is described. A microcapillary tip is adjusted both to contain a 3 M KCl solution with 1 mol% (w/v) agarose and to bridge an AgCl electrode and buffer solution. The advantage of the micro-salt bridge is displayed by time recordings of the electrode potential shift and the more precise measurements of membrane potential at various pH gradients. In the model system of recombinant proteins reconstituted into liposomes, the turnover rates of mitochondrial carriers UCP1 and UCP3 produced under similar conditions are reinvestigated and compared to previous results3,8.

<table><tbody><tr><td>Microloader tips</td><td>Eppendorf</td><td>5242956.003</td><td>Microcapillary pipette tip</td></tr><tr><td>Ethanol 99%</td><td>AustrAlco &amp;Ouml;sterr. Agrar-Alkohol Handelsges.m.b.H</td><td>AAAH-5020-07025-230317</td><td></td></tr><tr><td>Kaliumchlorid</td><td>Carl Roth GmbH + Co. Kg</td><td>6781.3</td><td></td></tr><tr><td>DC supply</td><td>Voltcraft</td><td>V10/CPG 1940 -01</td><td></td></tr><tr><td>Agarose Standard</td><td>Carl Roth GmbH + Co. Kg</td><td>3810.2</td><td></td></tr><tr><td>Patch Clamp Amplifier</td><td>Heka</td><td></td><td></td></tr><tr><td>Sample tube</td><td>Carl Roth GmbH + Co. Kg</td><td>5863.1</td><td></td></tr><tr><td>Na2SO4</td><td>Carl Roth GmbH + Co. Kg</td><td>8560.3</td><td></td></tr><tr><td>MES</td><td>Carl Roth GmbH + Co. Kg</td><td>4256.2</td><td></td></tr><tr><td>TRIS</td><td>Carl Roth GmbH + Co. Kg</td><td>AE15.2</td><td></td></tr><tr><td>EGTA</td><td>Carl Roth GmbH + Co. Kg</td><td>3054.1</td><td></td></tr><tr><td>Hexane</td><td>Sigma-Aldrich</td><td>296090-100ML</td><td></td></tr><tr><td>Hexadecane</td><td>Sigma-Aldrich</td><td>296317-100ML</td><td></td></tr><tr><td>Heating wire</td><td>Voltcraft</td><td>USPS-2250</td><td></td></tr></tbody></table>

a micro-agar salt bridge electrode for analyzing the proton turnover rate of recombinant membrane proteins

To date, more than 50% of all pharmacological drugs target the transport kinetics of membrane proteins. The electrophysiological characterization of membrane carrier proteins reconstituted in lipid bilayer membranes is a powerful but delicate method for the assessment of their physicochemical and pharmacological properties. The substrate turnover number is a unique parameter that allows the comparison of the activity of different membrane proteins. In an electrogenic transport, the gradient of the translocated substrate creates a membrane potential that directly correlates to the substrate turnover rate of the protein. By using silver chloride electrodes, a diffusion potential, also called liquid junction potential, is induced, which alters electrode potential and significantly disturbs precise membrane potential measurements. Diffusion potential can be minimized by a salt bridge, which balances electrode potential. In this article, a micro-agar salt bridge is designed to improve the electrophysiological set-up, which uses micropipettes for the membrane formation. The salt solution is filled into a microcapillary pipette tip, stabilized by the addition of agarose, and can be easily mounted to a standard electrode. The electrode potential of a micro-salt bridge electrode is more stable compared to a standard electrode. The implementation of this system stabilizes electrode potential and allows more precise measurements of membrane potential generated by a pH gradient. Using this system, the proton turnover rates of the mitochondrial carriers UCP1 and UCP3 are reinvestigated and compared to earlier measurements.

To verify the minimization of the diffusion potential, the stability of the current-voltage measurements of an intact membrane was measured. In Figure 2A, representative-current voltage recordings are depicted in the presence (white dots) and absence (black dots) of a pH gradient. According to the Nernst equation, the pH gradient induces a shift in voltage. From the x-axis intersection point of a linear fit to the data, the potential shift is calculated. In order to test both electrodes, the shift in x-axis intersection point was analyzed for a standard AgCl electrode (Figure 2B; white dots) and a micro-agar salt bridge (Figure 2B; black dots). A voltage ramp was recorded ten times in a row and the mean shift in the x-axis is depicted against time. Whereas the agar salt bridge electrode had a maximum shift of less than 5 mV even after 300 seconds of measurement, the standard electrode varied up to 30 mV in unpredictable, random, behavior.
Next, both electrodes were tested at different pH gradients (Figure 3A). For the standard electrode, a pH gradient of 0.35 and 1.0 (white dots) was generated; for the agar salt bridge electrode, pH gradients of 0.35, 0.7, and 1.0 (black dots). The shift in potential was analyzed in three independent measurements. In contrast to a gradient of 0.35, where the measured shift only varies slightly, the voltage shift significantly alters at the pH gradient of 1.0 in the absence of the micro-agar salt bridge. From a linear fit to the data, the slope of the function is 26.4 &#177; 2.3 mV/&#916;pH for the standard electrode and 50.1 &#177; 4.6 mV/&#916;pH for the micro-agar salt bridge electrode. According to the Nernst equation, the calculated potential shift is 60.7 mV/&#916;pH at T = 32 &#176;C.
Using the micro-agar salt bridge, the proton turnover number, &#954;, of mitochondrial UCP1 and UCP3 was measured and compared to previous measurements (Figure 3B). Similar to Figure 3A, &#916;pH = 1.0 was generated and the reverse potential was measured. The amount of protein in the membrane was estimated according to the formula provided in section 4 of the protocol, with a protein to lipid ratio of 4 &#181;g/(mg of lipid), a molecular mass of 33,000, and 750 Da for the protein and lipid, a membrane surface area of 3.53 x 10-4 cm2, and an area per lipid of 7.8 x 10-15 cm2. The obtained &#954;was 5.56 &#177; 0.38 s-1 and 4.10 &#177; 0.71 s-1 for UCP1 and UCP3, respectively (Figure 3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58552/58552fig1.jpg" /> 
Figure 1: The electrophysiological set-up with the micro-agar salt bridge. (A) This panel shows a sketch of the set-up. The microcapillary tip containing the agar salt solution (depicted in orange) is placed between the electrode (black) and the buffer-containing tip (blue). The membrane is formed at the surface at the end of the buffer-containing tip (indicated by the arrow). (B) This panel shows an image of the electrophysiological set-up with the implementation of the micro-agar salt bridge. The arrows point to the electrode, the micro-agar salt bridge, the reference electrode, and the container with buffer solution. <a href="https://www.jove.com/files/ftp_upload/58552/58552fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58552/58552fig2.jpg" /> 
Figure 2: The comparison of a micro-agar salt bridge and a standard AgCl electrode. (A) This panel shows a representative current-voltage measurement in the presence (grey dots) and absence (white dots) of a pH gradient of 1. The lines represent a linear fit to the data, from which conductance and x-axis intersection values are obtained. The voltage shift is evaluated by the difference of the intersection values of both recordings.&#160;(B) This panel shows the shift of membrane potential of a standard AgCl electrode (white dots) to a micro-agar salt bridge electrode (black dots) in time. Ten current-voltage measurements were recorded in a row and the mean voltage shift by diffusion potential is plotted against time. In all experiments, the membrane was made of 45:45:10 mol% DOPC:DOPE:CL reconstituted with 15 mol% arachidonic acid at a concentration of 1.5 mg/mL. The buffer contained 50 mM Na2SO4, 10 mM MES, 10 mM TRIS, and 0.6 mM EGTA at pH = 7.34 and T = 32 &#176;C. <a href="https://www.jove.com/files/ftp_upload/58552/58552fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58552/58552fig3.jpg" /> 
Figure 3: Proton turnover number of UCP1 and UCP3 calculated from the reverse potential in the presence of a pH gradient. (A) This panel shows the shift in potential of a UCP1-containing membrane of various pH gradients of a standard AgCl electrode (white dots) and a micro-agar salt bridge electrode (black dots). The lines represent a linear fit to the data. (B) This panel shows the proton turnover number of UCP1 (first data set) and UCP3 (second data set) as calculated from the ratio of voltage shift to Nernst potential according to the formulas in section 4 of the protocol. The first bar of each set represents turnover rates measured with the micro-agar salt bridge. The second bar of each data set represents previous measurements using a standard AgCl electrode. Values for UCP1 and UCP3 were taken from Urbankovaet al.3 and Macher et al.8. In all measurements, the membrane was made of 45:45:10 mol% DOPC:DOPE:CL reconstituted with 15 mol% AA and UCP1/UCP3. The concentration of lipid and protein was 1.5 mg/mL and 4 &#181;g/mg of lipid, respectively. The buffer contained 50 mM Na2SO4, 10 mM MES, 10 mM TRIS, and 0.6 mM EGTA at pH = 7.34 and T = 32 &#176;C. The pH of the buffer for the gradient measurements was increased to 7.66, 8.00, or 8.33 by adding TRIS and was changed in the solution-containing pipette. The values are the mean &#177; standard deviation of three independent measurements. <a href="https://www.jove.com/files/ftp_upload/58552/58552fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A Micro-agar Salt Bridge Electrode for Analyzing the Proton Turnover Rate of Recombinant Membrane Proteins

In electrophysiological measurements, the presence of a diffusion potential disturbs the precise measurement of the reverse potential by altering the electrode potential. Using a micro-agar salt bridge, the impact of the diffusion potential is minimized, which allows a more precise measurement of substrate turnover numbers of reconstituted recombinant membrane proteins.

To date, more than 50% of all pharmacological drugs target the transport kinetics of membrane proteins. The electrophysiological characterization of ...

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8.8K Views.  University of Veterinary Medicine. Deze methode kan helpen bij het onderzoek van membraaneiwitten zoals transporters en kanalen. De informatie over substraatomzetverlies dat eiwitactiviteit en specificiteit vergelijkt met de verschillende fysiologische en pathologische omstandigheden. Het belangrijkste voordeel van deze techniek is dat het minimaliseert potentiële verschuiving variatie en chloride vrije buffer voorwaarden.En verhoogt de nauwkeurigheid van de bepaling van de omloopsnelheid aanzienlijk als gevolg van nau...

Video: A Micro-agar Salt Bridge Electrode for Analyzing the Proton Turnover Rate of Recombinant Membrane Proteins

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single &#945;-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.&#160;&#160;

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

Chemistry

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Biochemistry

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Biology

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Differential Radial Capillary Action of Ligand Assay: A High-Throughput Technique to Identify Bacterial Nucleotide Second Messenger-Binding Intracellular Proteins

Source: Schicketanz, M. L., et al. <a href="https://app.jove.com/v/62331">Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA).</a> J. Vis. Exp. (2021)
This video demonstrates the differential radial capillary action of ligand assay &#8212; a technique for systematic high-throughput screening of intracellular nucleotide second messenger-binding proteins. This technique allows for direct binding of the messengers to the overexpressed target protein in the cell lysate &#8212; bypassing the need for protein purification.

A Micro-agar Salt Bridge Electrode for Analyzing the Proton Turnover Rate of Recombinant Membrane Proteins

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