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Article Cite This: ACS Omega 2018, 3, 17481−17486
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Quantifying Permeation of Small Charged Molecules across Channels: Electrophysiology in Small Volumes Jiajun Wang, Lorraine Benier, and Mathias Winterhalter* Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen, Germany
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S Supporting Information *
ABSTRACT: A major bottleneck in the development of smallmolecule antibiotics is to achieve good permeability across the outer membrane in Gram-negative bacteria. Optimization with respect to permeability surprisingly lacks appropriate methods. Recently, we proposed to use the diffusion potential for charged molecules created by their difference in electrophoretic mobility while crossing the outer membrane channel under a concentration gradient. The latter provides semiquantitative values, but the current available setups require large volumes and thus exclude several classes of molecules. Here we propose a simple approach of capturing proteoliposomes at the aperture of glass surface (planar aperture or conical glass capillary) by decreasing the necessary volume below 50 μL. We measured the transport of two charged molecules sulbactam and ceftazidime across the two major porins in Escherichia coli. Both molecules were observed to permeate through these porins, with sulbactam having a higher permeability.
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INTRODUCTION Antimicrobial-resistant bacteria are serious threats to public health and entered the agenda of many organizations like WHO and others. To reach the target of antimicrobial agents, bacteria cell walls are the first barrier to be penetrated. In particular, Gram-negative species (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species1) with their double-wall structure often provide an unsurmountable barrier to small molecules. As shown previously, the outer-membrane proteins such as OmpF and OmpC from E. coli facilitate the passive entry of small antibiotic molecules. Both porins are composed of three identical water-filled β-barrel monomers. The structure of OmpF revealed by Rosenbusch and co-worker2,3 revealed that the monomer is composed of 16 antiparallel β-barrels that span in the membrane. There are eight long loops connecting each pair of β-sheet from the extracellular side. Typically, the loop 3 (L3) folds back into the channel lumen and creates a 7−11 Å construction region, which is the narrowest part in the hollow. This loop is mainly composed of charged amino acid groups that make the channel slightly cation selective in potassium chloride solution.4 OmpC, an ortholog of OmpF, shares 60% identical sequence to OmpF.5 The crystal structure indicates a smaller pore size and a larger cation ion selectivity3,4 which corresponds to its high osmotic expression condition.6 Mass spectrometry has recently seen a huge progress in the quantification of small amount of substrates accumulated inside the cells,7 but with limited information on particular porin permeability. To characterize the permeation of small © 2018 American Chemical Society
hydrophilic molecules through a particular channel, the socalled liposome swelling assay5 with a fluorescence readout is often used. This requires large amounts of substances (10 mM) and have requirements on molecule charge states. Following the previous work performed in our laboratory,8−10 here we optimize the method using electrophysiology patch clamp to investigate the permeability flux through the porins that reconstitute liposomes. To quantify the flux of charged compounds through porins, one needs to first generate a concentration gradient across the membrane and measure the transmembrane potential. The membrane potential caused by the unbalanced flux of cations versus anions is due to different ion mobility inside the porins.8,9 A simplified theory treating the flux of ions under external voltage and a concentration gradient gives the relative flux densities and thus allows quantitative conclusions on the permeation. Recording the reversal potential is experimentally easy. Assuming a homogeneous electric field in the channel and no interaction among the ions or with the channel wall allows to use the Goldman−Hodgkin−Katz (GHK) equation11 to describe the ion current under an external electric field and a fixed concentration gradient. Typically, a bi-ionic or a tri-ionic system is applied to simplify the system. In general, a concentration difference of millimolar is the minimum requirement to raise the transmembrane potential to millivolt Received: July 11, 2018 Accepted: October 16, 2018 Published: December 17, 2018 17481
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Figure 1. Schematic diagram of the chip configuration on the Port-a-patch. (A) An agarose salt bridge has been used together with the ground electrode. A salt bridge filled with 1.5% agarose is used to cover the ground electrode to eliminate the polarization. (B) A typical GUV has been captured on a borosilicate chip forming the bilayer.
being detected. Such an approach is label-free, uses only simple theoretical assumption, and is easily accessible. However, standard equipment requires at least a concentration gradient of a few millimolar to accurately measure the membrane potentials within a few millivolt. To date, most of the available instruments require large volume usage and, obviously, this excludes many classes of molecules in terms of the quantity of drug consumption. We hereby seek other approaches using the same concept but miniaturizing the platform to reduce the consumption of the total amount of substrates. In the future, such a design will allow upscaling using a microfluidic array,12,13 leading to higher reproducibility and success rate for our broad-spectrum antibiotic screening purposes.
was then perfused with a buffer solution several times to eliminate the sorbitol introduced from the GUV solution. The volume can be as low as 30−50 μL to achieve the goal of low substrate consumption. Proteoliposomes-containing membrane proteins were introduced to the electrical ground (cis, or upper half cell on the schema) side of the system. In previous studies, we have shown the orientation of the reconstituted protein and channel activity on the Port-a-patch (i.e., the extracellular side of the protein is the same side as the proteoliposome addition).15,16 Such an observation is similar to the protein reconstituted into the traditional planar lipid bilayer.17 A stable baseline of the recording is an indicator of neither the current leakage from the sealing nor the current depletion from the electrodes. The symmetric concentration of 100 mM NaCl pH 6.0 was measured as control experiments (Figure 1b). Whole GUV Patch Clamp. Another approach is to patch GUVs with a micropipette. The micropipette with a 1 μm diameter opening was fabricated by laser pulling of borosilicate glass capillaries (OD = 1.0 mm, ID = 0.58 mm, length = 10 cm, Sutter Instrument Co., Novato, CA) using a P-2000 capillary puller (Sutter Instrument Co., Novato, CA). A two-step process was used for microcapillary fabrication (see the Supporting Information for more detail). The scanning electron microscopy (SEM) characterization was performed by using the Zeiss Ultra Plus scanning electron microscope (Carl Zeiss, Oberkochen, Germany). The pipettes were sputtered with Au for 20 s before SEM characterizations (Figure 2). Previously GUVs with reconstituted OmpF channels were patched using glass capillaries. Single channel activity recording was achieved by optimizing the concentration of the channel protein during GUV incubation.18 Unilamellar vesicles are considered because of their bilayer shell and confined small intracellular volume and a relatively controllable geometry. The GUV patch clamping has been achieved by mounting the pulled borosilicate micropipette to the headstage controlled by a micromanipulator (PCS 6200, Burleigh Instrument, Fishers,
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RESULTS AND DISCUSSION Planar Lipid Bilayer Formation Using Port-a-Patch. The commercially available bilayer setup, the Port-a-patch (Nanion Technologies GmbH, Munich, Germany), requires less than 50 μL solution to make the experiment successful. It is achieved by fusing a giant unilamellar vesicle (GUV) to the aperture of a horizontal planar borosilicate glass chip. To avoid different electrode polarization due to the different ion concentration on each side, we added a salt bridge to eliminate the artifacts from the measuring tools. To achieve this, we coated the electrodes with 1.5% Agarose soaked under measuring condition (Figure 1a). Following previous studies,14,15 we used borosilicate glass chip with a fabricated 1 μm spherical opening and mounted on the Port-a-patch. The bottom side of the chip sees the electrode, which is connected to the headstage (trans), and experiences the atmospheric pressure adjusted from a suction unit. The top surface of the chip is the reference electrode (cis). To initiate the experiment, 5 μL GUV solution were added to the cis side together with another 5 μL NaCl buffer and then a negative pressure (−25 mbar) was applied immediately to pull the vesicles toward the glass chip. The seal resistance above 1 GΩ is obtained when a vesicle is successfully fused on the chip forming a bilayer. The cis side 17482
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without shrinking or rupturing before proceeding with the experiments. After a vesicle is successfully patched by the micropipette in the whole-cell mode, the pressure was decreased to gain the seal resistance above one gigaohm. As illustrated in the Supporting Information, the capacitance of the DPhPC vesicles is stable at 0.42 ± 0.05 μF/cm2. A 25% lower capacitance compared to the DPhPC bilayer capacitance (0.56 μF/cm2)22 is attributed to the system capacitance introduced additionally from capillary, solvent, and electronic components. The vesicle eventually ruptured as soon as the pressure decreased to lower than −30 mbar. Quantifying the Permeation of Small Molecules across Channel. To elucidate the reproducibility of different techniques, we first measured the ion selectivity of both OmpF and OmpC. Under bi-ionic (i.e., Na+, Cl−) condition, using c 150 mM asymmetric concentrations c cis = 100 mM , the reversal poten-
Figure 2. SEM characterization of the microcapillary pulled tip. The pipettes were sputtered with Au for 20 s before SEM characterizations (figure kindly provided by Long’s group, East China University of Science and Technology, Shanghai, China).
trans
NY). The capillary was filled with an intercellular solution and attached to the Axon headstage connected to an Ag/AgCl electrode.18,19 Electro-osmotic shock can cause GUV rupture,20,21 and the lipid diffuse inside the capillary due to the lack of a membrane skeleton (Figure 3B,C). Bovine serum albumin (BSA) pretreatment is necessary to prevent lipid diffusion in the capillary19 (i.e., GUVs formed in presence of 2 mg/mL BSA and, in addition, coating the interior of the pipette with BSA solution). As an example, we successfully patched a 20 μm GUV using our homemade device (Figure S2). Before patching a GUV, a minor positive pressure (+5 mbar) was applied to keep the interior clean. The headstage was controlled via the micromanipulator, which allowed the manipulation of the vesicle under a microscope. A negative pressure (from −5 to −15 mbar) was then applied to move the vesicle toward the tip of the micropipette. At this moment, the value of resistance measured by the PClamp (Axon Instruments, Forest City, CA) gradually exceeded 2 GΩ. The vesicle should remain stable at the capillary tip for at least 1 min
tial is obtained directly from the I−V plot (Figure S3). The reversal potential of OmpF and OmpC at such an asymmetric condition read 5.9 and 7.0 mV, respectively, using Port-apatch, whereas 6.2 and 7.2 mV, respectively, using the patch clamp technique. Introducing the experimental parameter into eq 2 gives the P relative permeability through OmpF PNa (port‐a‐patch) = 3.7 Cl
and are
PNa (GUV) = 4.0. PCl PNa (port‐a‐patch) PCl
However, for OmpC, the permeability = 5.3 and
PNa (GUV) PCl 8,9,23
= 5.6 and in
agreement with the published results. In a second set of experiment, we recorded ceftazidime, an antibiotic, and sulbactam, a β-lactamase inhibitor (Figure 4). Both molecules are negatively charged in our condition with Na+ as a counterion. After the determination of the relative permeation rate of sodium and chloride through both OmpF and OmpC under bi-ionic conditions, the permeation rate of
Figure 3. (A) Schematic diagram of the whole GUV patch clamp. (B) A vesicle got captured at the microcapillary tip. (C) An unbalanced osmotic pressure causes the vesicle to shrink 240 s after patching, reducing the size of the vesicle to less than 5 μm. The seal resistance exceeded gigaohm gradually. (D) BSA treatment: A vesicle formed in a BSA solution is patched with a BSA coated microcapillary shows no obvious shrinkage (see Whole GUV Patch Clamp for details). 17483
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might vary. This observation suggests that in both cases, the interaction with the channel surface is less pronounced and other effects like electro-osmosis may have a stronger impact.24,25
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CONCLUSIONS To quantify the translocation of charged small molecules across the membrane channel, we measured the zero-current transmembrane potential. As this method is based on concentration gradients, reduction of volume to the value below the required quantity (below mg) is a prerequisite to test certain class of molecules, particularly for screening purposes. Using the simple concept of reversal potential measurement, we have characterized the relative permeability ratio of small antimicrobial molecules, sulbactam, and ceftazidime. Both negatively charged molecules have a surprisingly fast diffusion. Sulbactam has a generally stronger permeability through channels compared to ceftazidime mainly due to its small molecular structure. Both molecules have a preference to OmpC over OmpF, although former is more cation selective than the latter. Further details of the molecule−protein interaction could be investigated via in situ analysis on a microscopic time scale. In all, this method has great potential for fast screening of small charged molecules through membrane channels compared to other in vitro electrochemical characterizations. Last but not least, one of the drawbacks of such a method is the substrate consumption issue. In this manuscript, we also optimized our homemade devices with respect to volume. We show that for the commercially available Port-a-patch, the volume can be decreased to 20−50 and 150 μL using whole GUV patch clamping with a homemade device. The volume consumption in the GUV patch clamping is dominated by the O-ring, which accumulates the buffer. In the future the required volume could be reduced using multiple pipettes sharing one reservoir. The parallelization of the vesicle patch clamp could be achieved by using several pipettes in parallel or implementing into a microfluidic device to reach the low volumetric consumption.26 The pros and cons are summarized in Table 2.
Figure 4. Molecular structure of (A) sulbactam (MW = 233) and (B) ceftazidime (MW = 546).
the substrates becomes accessible to be calculated under triionic condition. To settle the tri-ionic condition, 50 mM substrates were dissolved in 100 mM NaCl to yield a total of 150 mM sodium (+1e) counter with 50 mM substrate (−1e) and 100 mM chloride (−1e) at the cis side. Along with the 100 mM NaCl at the trans side of the bilayer, the asymmetric condition generates a transmembrane potential corresponding to permeability. Moving ahead to the two compounds, the transmembrane voltage generated by 50 mM sulbactam (sodium salt) through OmpF using Port-a-patch and GUV patch clamp is 4.5 and 5.0 mV, respectively, whereas that generated through OmpC is 1.0 and 2.0 mV, respectively. The 50 mM ceftazidime (sodium salt) yielded transmembrane voltage of 5.2 and 5.0 mV using Port-a-patch and GUV patch clamp approach, respectively. We again obtained lower transmembrane voltage of 3.5 and 3.3 mV through the OmpC channel. The same chloride concentration on both sides of bilayer makes the net flux negligible. The permeability of sodium and the tested substrates was normalized to chloride, ex ante. According to the determination of sodium permeation rate in previous section, the relative permeability of substrates were then calculated via eq 3 and summarized in Table 1. The permeability of cation (i.e., sulbactam and ceftazidime) obtained from both Port-a-patch and GUV patch clamp methods is similar to that of counterion (i.e., Cl−), in agreement with previous studies. A slight variation does not change the conclusion that the permeability of sulbactam is generally lower than that of sodium ions. Moreover, sulbactam permeates faster through both porins compared to ceftazidime. Interestingly, although OmpC is somewhat smaller and more cation selective, both negatively charged ions show higher rates compared to OmpF, although the net substrate conductivity
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METHODS Goldman−Hodgkin−Katz Equation Application. To estimate the permeability of charged molecules, we analyze the permeability ratio of the ions. In the simple case of a 1:1
Table 1. Zero-Current Transmembrane Voltage Measurements of Both Porins from E. coli Using Different Techniquesa techniques d
black lipid membrane (BLM) porins
conductance (nS)b
OmpF
0.4
OmpC
0.3
asymmetric condition ([cis]−[trans])c 50 50 50 50 50 50
mM mM mM mM mM mM
NaCl sulbactam-Na ceftazidime-Na NaCl sulbactam-Na ceftazidime-Na
transmembrane potential (mV)
p+/p−
5.5 ± 1.2
3.3:1
7.0 ± 1.5
5.2:1
Port-a-patch
GUV patch
transmembrane potential (mV)
p+/p−/psub
± ± ± ± ± ±
3.7:1 3.7:1:1.5 3.7:1:1.2 5.3:1 5.3:1:4.6 5.3:1:2.9
5.9 4.5 5.2 7.0 1.0 3.5
2.0 1.2 3.0 2.5 2.0 1.2
transmembrane potential (mV)
p+/p−/psub
± ± ± ± ± ±
4:1 4:1:1.5 4:1:1.5 5.6:1 5.6:1:4.1 5.6:1:3.2
6.2 5.0 5.0 7.2 2.0 3.3
1.7 1.2 2.7 2.0 1.0 0.9
a
The NaCl concentrations were buffered with 20 mM MES and pH of 6.0 was measured at room temperature. BLM technique is measured as a control. All measurements were repeated at least thrice. bElectrical conductance measured for single channel under 100 mM NaCl, pH 6.0. c Asymmetric condition generated from 100 mM NaCl, pH 6.0. Additional 50 mM analysts are added from the cis (ground) side. dBLM was not used to measure valuable substrates due to the large volume assumption. 17484
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applied through the ITO slides and, after 2 h of formation, both the voltage and frequency are gradually decreased to zero within 3 min. Finally, the ITO slide was uncovered and the GUV solution was collected in Eppendorf tubes. Purified membrane protein either OmpF or OmpC (1.5 mg/ mL), as previously described,28 see also supplement for details, was added directly to the GUV solution. The protein was diluted in 1% n-octyl-oligo-oxyethylene (Octyl-POE) and then added to the GUV solution for incubation. A final concentration of 10−13 mg/mL as described was reached to obtain few channel activities per vesicle fusion as described previously.15,18,29 The protein−GUV mixture was gently vortexed and placed at room temperature for 2−4 h and then 500 mg/mL biobeads (Bio-Rad Laboratories, Inc., Hercules, CA) was added to the solution and further incubated at 4 °C overnight for the removal of excess detergent. Finally, after brief centrifugation, the proteoliposome-rich supernatant was collected.
Table 2. Comparison of Different Techniques technique features minimum volume (cis side) accessibility (trans side) parallelization
consumables
BLM
Port-a-patch
GUV patch clamp
2500 μL
40 μL
150 μL
yes
yes (addition device) yes14,27 (microfluidic approach) disposable microfluidic chamber
hardly
no
reusable Teflon thin film
complex26 (multiple patch tips) disposable borosilicate glass capillary
electrolyte (e.g., NaCl), this can be obtained by applying a concentration gradient and recording the voltage needed to zero net ion current. The Goldman−Hodgkin−Katz (GHK) provides a simple relation that allows us to obtain the permeability ratio11
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+ − RT ijj PNa[Na ]cis + PCl[Cl ]trans yzz lnjj z + zF jk PNa[Na ]trans + PCl[Cl−]cis zz{ (1) By measuring the experimental zero-current transmembrane voltage at the desired asymmetric condition, the relative permeability (Px (x = Na+, Cl−)) ratio between cation (Na+) and anion (Cl−) gives
S Supporting Information *
Vm =
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01611. Patched vesicles electrical behavior and microscopic image; pipette pulling parameters as well as the permeability calculation approaches; purification of membrane proteins (PDF)
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( zVF )
exp RT [Cl−]cis − [Cl−]trans PNa = zVF PCl [Na +]cis − exp RT [Na +]trans
( )
(2)
*E-mail:
[email protected]. ORCID
Jiajun Wang: 0000-0003-3420-6376 Notes
The authors declare no competing financial interest.
I x(V , Px , z , ccis , ctrans) = Pxz
2
RT
(c ·
x,cis
( −RTzVF )) −zVF 1 − exp( RT )
AUTHOR INFORMATION
Corresponding Author
In case multivalent ions are involved, we rather use the GHK equation for the ion current I and solve the permeability ratio numerically. The ion current I is then given by the sum of all the individual ion currents Ix
2 VF
ASSOCIATED CONTENT
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− c x,trans·exp
ACKNOWLEDGMENTS J.W. is supported from the Innovative Medicines Initiative Joint Undertaking under Grant Agreement No. 115525, resources which are composed of financial contribution from European Union’s seventh framework programme (FP7/20072013), and EFPIA companies. We thank Prof. Yi-Tao Long’s group from East China University of Science and Technology for providing home-designed nanopore fabrication and detection instrumentation. We further acknowledge support from the project “Boomer” - Bacterial periplasmic organelles and outer membrane vesicles (ZF4176703AJ6) by the Federal Ministry for Economic Affairs and Energy (BMWi) under the framework of the Central Innovation Programme for SMEs (ZIM).
(3)
where V is the transmembrane voltage, Px is the permeability for the ion x, z is the valence, F (Faraday constant) = 9.6 × 104 mol−1, and R (gas constant) = 8.3 J/(mol K). The experimental input cx,cis and cx,trans are the ion concentrations on the two sides of the membrane. Proteoliposome Preparation. The whole process of electroformation was controlled by the Vesicle Prep Pro (Nanion Technology GmbH, Munich, Germany) with programmable parameters (i.e., amplitude, frequency, main time, rise time, and fall time). We used indium tin oxide (ITO) slides as electrodes for the GUV electroformation. Ten microliters of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (5 mg/mL) dissolved in chloroform was deposited and spread carefully on the conductive part of the ITO slide. After complete evaporation of chloroform, an O-ring (1 mm thickness) was placed around the dried lipid residue. One molar sorbitol was added to fill the O-ring space. Here, we added 275 μL in accordance with the O-ring size. Another pair of the ITO slide was then covered above the O-ring, with the conductive sides facing each other. During the electroformation period, an AC current (Vpp = 3 V, f = 5 Hz) is
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