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
Overview Overview Planar lipid lipid bilayers on a chip bilayers on a chip Planar • • Protocole for reconstitution of membrane membrane proteins proteins and and Protocole for reconstitution of • • membrane fraction fraction into into bilayers bilayers membrane Screening of OmpF on a of OmpF on a chip chip Screening • • Validation of the the approach approach of of single single patch patch chip chip Validation of • • – 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
The Port-a-Patch •One entity device • Small liquid 5 mm consumption: <10 µl • Integrated fast fluid exchange • Higher throughput (up to 50 data points per day) �
Formation of Giant Unilamellar Vesicles (GUV’s): Using electroformation • Lipid-containing solution, 5 or 10 mM of DPhPC with 10 % ITO Slides � intracellular Solution Lipid cholesterol, dissolved in chloroform Layer Application of alternating electrical fields to the lipid-covered ITO-slides • Non-ionic intracellular solution 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.
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
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
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.
OmpF properties onto glass chip Representative current traces of the OmpF channel in 1 M KCl at a transmembrane potential of +50 mV
OmpF properties onto glass chip 1000 pA 800 600 400 200 -200 -100 100 200 -200 mV -400 -600 -800 -1000 Measurements of OmpF conductance in Critical voltage for gating OmpF porin: 1 M KCl, and 10 mM HEPES, pH=5,4 150-200 mV We determined the trimeric conductance at 4,06 nS (I-V curve) and 1,35 nS for the monomeric conductance
Interaction of compound with OmpF control Polyamines (spermine, cadaverine…) inhibit chemotaxis and flux of β -lactam of the outer spermine 0,1 mM 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
Antibiotics translocation 5 mM ampicillin control 390 380 370 360 Amplitude (pA) 350 340 330 320 310 300 3,580 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)
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 0.262 ± 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 = 0.224 ± 0.012 ms
R132A OmpF +50 mV 3.72 ± 0.7 nS 2 mM Norfloxacine CTRL 200 200,0p 180 180,0p 160 160,0p 140 140,0p amplitude (pA) amplitude (pA) 120 120,0p 100 100,0p 80 80,0p 60 60,0p 40 40,0p 20 20,0p 0 0,0 0 2 4 6 8 10 12 14 16 18 0 5 10 15 20 time (s) time (s) 10 mM Ampicillin 250 200 Amplitude (pA) 150 100 50 baseline 50 pA 0 300 ms 0 5 10 15 20 +150 mV time (s)
Validation of single channel recordings in Validation of single channel recordings in planar lipid bilayers planar lipid bilayers Connexin Cx 26 Cx 26 Connexin Reconstitution of of hemichannels hemichannels Reconstitution 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
Cx 26 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) ( Shah et al, 2002 ) • The hydrophilic domains between M1 and M2 and between M3 and M4 form two extracellular loops The conductance of hemichannel: 2 main conductances (subconductance): 35-45 pS, 90-110 pS in 200 mM KCl. ( Buehler et al, 1995 ) The conductance of gap junction: 0,3 to 2 nS ( Shah et al, 2002 )
100 mV 100 mV baseline baseline 5 pA 5 pA 5 ms 25 ms Fast opening events
96 ± 2 pS 20 current (pA) 15 10 5 -150 -100 -50 50 100 150 -5 Voltage (mV) -10 -15 IV curve for Cx 26: main conductance 96 pS
20 15 tau=0.55±0.02 ms Count (N) 10 5 0 0 5 10 15 20 25 30 35 40 45 Time (ms) Open time histogram at +100 mV (tau=0.44 ms, Buehler et al, 1995 )
100 80 60 % inhibition 40 20 0 0,0 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
KcsA Potassium Potassium channel channel KcsA K+ channel from Streptomyces lividans (KcsA channel) 4 identical subunits: each subunit containing two alpha- helices 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
KcsA 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 1-1.5 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 4 +100 mV baseline 2 pA 200 ms
+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.)
Ramp: -200 mV to 200 mV
Ramp: -200 mV to 200 mV Rectification of the current: Potassium channel behavior
IP3R 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
0 0 -100 mV -100 mV 0 mV 0 mV 20 mV 0 50 mV 100 mV 0 150 mV 0 5 pA 500 ms
98.75 ± 1.5 pS 15 10 amplitude pA 5 -100 -50 50 100 150 voltage mV -5 -10 IV curve for IP3R main conductance : 99pS (40 to 120 pS in the litterature)
Recommend
More recommend
