Single patch chip for planar lipid bilayer assays: Ion channels characterization and screening

Transcription

1 RTN Mid-Term Activity Molecular basis of antibiotic translocation Single patch chip for planar lipid bilayer assays: Ion channels characterization and screening Mohamed Kreir April 2008

2 Overview Planar lipid bilayers on a chip Protocole for reconstitution of membrane proteins and membrane fraction into bilayers Screening of OmpF on a chip Validation of the approach of single patch chip Recordings of others proteins Connexin Cx 26 and Cx 43 KcsA Potassium channel IP3 receptor NMDA receptor CaV1.2b calcium channel Mutant OmpF R132A Screening of single channels

3 The Port-a-Patch 5 mm One entity device Small liquid consumption: <10 µl Integrated fast fluid exchange Higher throughput (up to 50 data points per day)

4 Formation of Giant Unilamellar Vesicles (GUV s): Using electroformation Lipid-containing solution, 5 or 10 mm of DPhPC with 10 % cholesterol, dissolved in chloroform Lipid Layer ITO Slides intracellular Solution ~ Non-ionic intracellular solution Application of alternating electrical fields to the lipid-covered ITO-slides leads to the formation of vesicles. Alternating voltage of 3 V peak to peak and frequency of 5 Hz apply over a period of 2 hours at room temperature Typical diameters of the vesicles is in the tenths of microns (scale bar: 25 µm). GUV preparation by Markus Sondermann, Group of Prof. Behrends, University Freiburg.

5 Reconstitution of OmpF into the vesicles Experimental procedure - Formation of GUV's by electroformation - Incubation with OmpF solubilized in detergent with GUVs solution - Removal of detergent with Biobeads SM-2 (BioRad) - Centrifugation and discarted Biobeads

6 Formation of a planar lipid bilayer containing purified proteins 1-3 microliters of the proteoliposomes solution pipetted onto the patch clamp chip The chip contains an aperture approximately 1 micron in diameter. The GUVs were positioned onto the aperture in the chip by application of a slight negative pressure, 10 mbars, for reliable positioning within a few seconds after GUV addition

7 Planar lipid bilayers formation on glass surface When the GUVs touch the glass surface of the chip, they burst and form planar bilayers with formation of gigaseal. When proteoliposomes are used, a planar lipid bilayer is immediately obtained, with the reconstituted protein present (d) so that the patch clamp recording can start right away.

8 OmpF properties onto glass chip Representative current traces of the OmpF channel in 1 M KCl at a transmembrane potential of +50 mv

9 OmpF properties onto glass chip pa mv Measurements of OmpF conductance in 1 M KCl, and 10 mm HEPES, ph=5,4 Critical voltage for gating OmpF porin: mv We determined the trimeric conductance at 4,06 ns (I-V curve) and 1,35 ns for the monomeric conductance

10 Interaction of compound with OmpF control spermine 0,1 mm Polyamines (spermine, cadaverine ) inhibit chemotaxis and flux of β-lactam of the outer membrane spermine 1 mm The perfusion of spermine change the kinetics of the opening and closing events Modulation of OmpF channels by applied spermine Condition: 1 M KCl, ph 5,4, V = 50 mv

11 Antibiotics translocation control 5 mm ampicillin Amplitude (pa) ,580 3,585 3,590 3,595 3,600 3,605 time (s) Penetrating ampicillin molecules modulate the ionic current through OmpF channel reconstituted in the planar lipid bilayer (1 M KCl, ph 5,4)

12 Kinetics of interaction of ampicillin with OmpF at 50mV.Dwell time histograms of 2.5mM ampicillin OmpF were fitted by an exponential with characteristic time of ± 0.05 ms for ampicillin. Power spectral densities of current fluctuations at four different ampicillin concentrations at +50mV applied voltage, 1MKCl, ph=5.4. Each spectrum was analyzed by Lorentzian fitting with characteristic time of s = ± ms

13 R132A OmpF +50 mv 2 mm Norfloxacine CTRL ,0p 180,0p ,0p ,0p amplitude (pa) ,0p 100,0p 80,0p 60,0p 40 40,0p 20 20,0p 0 0, time (s) time (s) 10 mm Ampicillin Amplitude (pa) amplitude (pa) 3.72 ± 0.7 ns baseline 50 pa ms mv 5 10 time (s) 15 20

14 Validation of single channel recordings in planar lipid bilayers Connexin Cx 26 Reconstitution of hemichannels Planar lipid bilayers are formed from GUVs: 5mg/ml DPhPC (10% cholesterol) Connexin are added to the solution containing GUVs (in 1 M sorbitol) and the mix are incubate overnight (Different time of incubation, 2 tests: with and without BioBeads, no real difference) The biobeads can be added just once during 1 hour. The recordings solution is 200 mm KCl, 10 mm HEPES, 0.02 mm EDTA, ph 7.4 and are done at 20 khz sampling, 3 khz bessel filter. The recordings were done at different voltages (-150 to +150 mv) Mean conductance values for single channels were obtained from Gaussian fits of all points amplitude histograms We found a main conductance of 96 ps but there are also other conductances one very small about 30 ps and one high: 150 to 200 ps

15 Cx 26 Connexins (gap junctions proteins) are a family of structurally-related transmembrane proteins Connexins are formed by 2 hemichannels 6 α-helical domains Organized in hexameric structure Monomere: 26 kda Each connexin has four predominantly hydrophobic, membrane spanning regions (M1 M4) The hydrophilic domains between M1 and M2 and between M3 and M4 form two extracellular loops (Shah et al, 2002) The conductance of hemichannel: 2 main conductances (subconductance): ps, ps in 200 mm KCl. (Buehler et al, 1995) The conductance of gap junction: 0,3 to 2 ns (Shah et al, 2002)

16 100 mv 100 mv baseline baseline 5 pa 25 ms 5 pa 5 ms Fast opening events

17

18 current (pa) ± 2 ps Voltage (mv) IV curve for Cx 26: main conductance 96 ps

19 20 15 tau=0.55±0.02 ms Count (N) Time (ms) Open time histogram at +100 mv (tau=0.44 ms, Buehler et al, 1995)

20 % inhibition ,0 0,1 0,2 0,3 0,4 0,5 concentration Quinidine (mm) Effect of Quinidine (n=5) IC50 = 0.1 ± 0,05 mm Cx 26 is also inhibited by protonized HEPES: effect also done but not successful

21 KcsA Potassium channel K+ channel from Streptomyces lividans (KcsA channel) 4 identical subunits: each subunit containing two alphahelices connected by an approximately 30 amino acids long loop proofreading into the pore region Theoretical and Computational Biophysics, NIH, MD simulation movement of K+ ions across the potassium channel

22 KcsA Planar lipid bilayers are formed from GUVs: 5mg/ml DPhPC (10% cholesterol) KcsA (solubilized in 400 nm Imidazol, 200 mm NaPO4, 150 mm KCl, ph 7.8 at concentration of mg/ml) are added to the solution containing GUVs (in 100 mm sorbitol) and the mix are incubate 1 hours. BioBeads is added and incubate overnight to remove detergent. KcsA could used directly on the top of the chip containing bilayers. Internal solution: 100 mm KCl, 10 mm HEPES ph 7 External solution: 100 mm KCl, 10 mm MES ph mv baseline 2 pa 200 ms

23 +150 mv baseline 5 pa 100 ms Different conductances were observed: KcsA present different patterns of channel activity. (Molina et al, 2006, Clustering and coupled gating modulate the activity in KcsA, a potassium channel model.)

24 Ramp: -200 mv to 200 mv

25 Ramp: -200 mv to 200 mv Rectification of the current: Potassium channel behavior

26 IP3R Membrane glycoprotein complex acting as Calcium channel activated by IP3 (inosotol triphosphate) 4 α-helical subunits 100 kda Planar lipid bilayers are formed from GUVs: 5mg/ml DPhPC (10% cholesterol) IP3R are added to the solution containing GUVs (in 100 mm sorbitol) and the mix are incubate 1 hours. BioBeads is added and incubate overnight to remove detergent. Recordings were obtained in the presence of 0.2 mm Calcium and 1 mm Na2ATP, 140 mm KCl. Addition of 2 mm InsP3 on the cis (cytosolic) side evoked openings of InsP3R

27 0-100 mv 0 mv 20 mv 0 50 mv 100 mv mv 0 5 pa 500 ms

28 98.75 ± 1.5 ps amplitude pa voltage mv -10 IV curve for IP3R main conductance : 99pS (40 to 120 ps in the litterature)

29 Membrane fraction There were used for the recording of Calcium channel, NMDA receptor and for connexin 43 A small amount of membrane fraction (0.2 µl) were directly deposit on the top of the chip after formation of bilayer Inconvenient: the fusion can take a while; after fusion the bilayer could become instable, presence of others channels that the channel of interest Incubation of the membrane fraction with GUVs The seal is more difficult to obtain Recordings of activity start right away

30 Membrane fraction from CHO cells containing NMDA receptor activated by glycine-aspartate NR1 NR2A Solutions: Extracellular: 125 mm NaCl, 5 mm KCl, 5 mm Tris Intracellular: 110 mm KCl, 4 mm NaCl, 1 MgCl2

31 close -60 mv 2 pa 100 m s c lo s e -60 mv 2 p A m s

32 Calcium channel (Cav1.2b, Cav1.2c) Membrane fraction from CHO cells containing calcium channel Solutions: Cis side: 100 mm BaCl2, 50 mm NaCl, 10 mm HEPES, 5 µm ATP Trans side: 145 KCl, 2 mm MgCl2, 2 mm EGTA, 20 mm saccharose Protocole +10 mv 2000 ms -60 mv -60 mv

33 Inward current (+10 mv) corresponding to Ca²+ flux

34 Cx 43 from membrane fraction Membrane fraction were incubate with GUVs +100 m V 20 pa baseline 1 s No results when we add membrane after bilayer formation

35 Vesicles/Bilayers fusion SUV-Bilayers fusion: no real success with the reconstitution of proteins via SUV fusion Native vesicles fusion using synaptosomes: The fusion is done adding a small amount of synaptosomes on the chip after formation of bilayers Patch synaptosomes: No success (gigaseal were obtained but no recordings) Synaptosomes similarity with proteoplast

36 Recordings with synaptosomes Synaptosomes are formed from the phospholipid layer of the cell membrane and synaptic proteins such as receptors. Synaptosomes from mossy fibers prepared from hyppocampi and cerebella of C57BL/6J mice. Synaptosomes formed vesicles with a size in order to 1 µm containing NMDA receptor activated by glycine-aspartate concentration: 1,57 mg/ml The synaptosomes were added to the bath solution after formation of planar lipid bilayer onto the chip to obtain membrane fusion. Patching direcly the synaptosomes on the chip were done: gigaseal but no recordings Solutions: Extracellular: 125 mm NaCl, 5 mm KCl, 5 mm Tris Intracellular: 110 mm KCl, 4 mm NaCl, 1 MgCl2

37 close 1 pa 200 ms + 60 mv c lo s e

38 Summary Reconstitution of different proteins (more complexe, α- helical) were done (validate the approach of single patch chip for planar lipid bilayer, possible to screen for single channel on chip) Purified proteins for the reconstitution and the recordings on the glass chip is more successful than membrane fraction or liposomes fusion Patching synaptosomes was possible (possibility for proteoplasts also?) but no recordings were done

39 Summary The microstructured glass chip offer a high resolution Single molecule translocation can be well define Screening of different concentration of antibiotic using the same bilayer can be done The competitive action of 2 compounds on OmpF were also tested (polyamine and ampicillin) The success with the other proteins open the research in other field: Connexins are involved in different syndromes and abnormalities (Keratitis-Icthyosis-Deafness, cancers ) IP3 Receptor is very important in the cell signaling and is also involved in the proliferation of tumors