Single patch chip for planar lipid bilayer assays: Ion channels - - PowerPoint PPT Presentation

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

Mohamed Kreir

April 2008

RTN Mid-Term Activity Molecular basis of antibiotic translocation

Overview Overview

• Planar

Planar lipid lipid bilayers on a chip bilayers on a chip

• Protocole for reconstitution of

Protocole for reconstitution of membrane membrane proteins proteins and and membrane membrane fraction fraction into into bilayers bilayers

• Screening

Screening of OmpF on a

• f OmpF on a chip

chip

• Validation of

Validation of the the approach approach of

• f single

single patch patch chip 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

5 mm

The Port-a-Patch

• One entity device
• Small liquid

consumption: <10 µl

• Integrated fast fluid

exchange

• Higher throughput (up to

50 data points per day)

• Lipid-containing solution, 5 or 10

mM of DPhPC with 10 % cholesterol, dissolved in chloroform

• Non-ionic intracellular solution
• Alternating voltage of 3 V peak to

peak and frequency of 5 Hz apply

• ver a period of 2 hours at room

temperature

Formation of Giant Unilamellar Vesicles (GUV’s): Using electroformation

ITO Slides Lipid Layer intracellular Solution

• 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. Application of alternating electrical fields to the lipid-covered ITO-slides leads to the formation of vesicles.

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

• btained, 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

Critical voltage for gating OmpF porin: 150-200 mV

OmpF properties onto glass chip

• 200
• 100

100 200

• 1000
• 800
• 600
• 400
• 200

200 400 600 800 1000

pA mV

Measurements of OmpF conductance in 1 M KCl, and 10 mM HEPES, pH=5,4 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

Modulation of OmpF channels by applied spermine The perfusion of spermine change the kinetics of the opening and closing events Polyamines (spermine, cadaverine…) inhibit chemotaxis and flux of β-lactam of the outer membrane

spermine 1 mM spermine 0,1 mM control

Condition: 1 M KCl, pH 5,4, V = 50 mV

Antibiotics translocation

control

3,580 3,585 3,590 3,595 3,600 3,605 300 310 320 330 340 350 360 370 380 390 Amplitude (pA) time (s)

5 mM ampicillin 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

2 4 6 8 10 12 14 16 18 20 40 60 80 100 120 140 160 180 200

amplitude (pA) time (s)

5 10 15 20 0,0 20,0p 40,0p 60,0p 80,0p 100,0p 120,0p 140,0p 160,0p 180,0p 200,0p

amplitude (pA) time (s)

5 10 15 20 50 100 150 200 250

Amplitude (pA) time (s)

CTRL 2 mM Norfloxacine 10 mM Ampicillin

R132A OmpF

3.72 ± 0.7 nS

baseline

300 ms 50 pA

+50 mV +150 mV

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

• f all points amplitude histograms

We found a main conductance of 96 pS but there are also other conductances

• ne very small about 30 pS and one high: 150 to 200 pS

Connexin Connexin Cx 26 Cx 26 Reconstitution Reconstitution of

• f hemichannels

hemichannels

Validation of single channel recordings in Validation of single channel recordings in planar lipid bilayers planar lipid bilayers

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)

• 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)

(Shah et al, 2002)

100 mV

baseline

5 pA 25 ms

100 mV

baseline

5 pA 5 ms

Fast opening events

• 150
• 100
• 50

50 100 150

• 15
• 10
• 5

5 10 15 20

96 ± 2 pS current (pA) Voltage (mV)

IV curve for Cx 26: main conductance 96 pS

Time (ms) 5 10 15 20 25 30 35 40 45 Count (N) 5 10 15 20

tau=0.55±0.02 ms

Open time histogram at +100 mV

(tau=0.44 ms, Buehler et al, 1995)

Effect of Quinidine (n=5) IC50 = 0.1 ± 0,05 mM

0,0 0,1 0,2 0,3 0,4 0,5 20 40 60 80 100

% inhibition concentration Quinidine (mM)

Cx 26 is also inhibited by protonized HEPES: effect also done but not successful

KcsA KcsA Potassium Potassium channel channel

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

baseline 2 pA 200 ms

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 100 ms 5 pa

+150 mV

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

• vernight 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

• 100 mV

20 mV

100 mV 150 mV

500 ms 5 pA

0 mV 50 mV

• 100 mV

0 mV

• 100
• 50

50 100 150

• 10
• 5

5 10 15

amplitude pA voltage mV

98.75 ± 1.5 pS IV curve for IP3R main conductance : 99pS (40 to 120 pS in the litterature)

Membrane Membrane fraction 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

• f interest
• Incubation of the membrane fraction with GUVs

– The seal is more difficult to obtain – Recordings of activity start right away

NR1 NR2A NR1 NR2A

• Membrane fraction

from CHO cells containing NMDA receptor activated by glycine-aspartate

Solutions:

• Extracellular: 125 mM

NaCl, 5 mM KCl, 5 mM Tris

• Intracellular: 110 mM KCl,

4 mM NaCl, 1 MgCl2

close

100 ms 2 pA

• 60 mV

clo se

1 0 0 m s 2 p A

• 60 mV

Calcium Calcium channel channel (Cav1.2b, Cav1.2c) (Cav1.2b, Cav1.2c)

• 60 mV
• 60 mV

+10 mV 2000 ms

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 Membrane fraction from CHO cells containing calcium channel

Inward current (+10 mV) corresponding to Ca²+ flux

Cx 43 Cx 43 from from membrane membrane fraction fraction

• Membrane fraction were incubate with GUVs

+100 m V

baseline

20 pA 1 s

No results when we add membrane after bilayer formation

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

Recordings Recordings with with synaptosomes 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

200 ms 1 pA

close c lo s e

+ 60 mV

Summary 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
• n 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

Summary 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
• ther 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