Application Note

Channels:endogenous
Recorded from: Spheroplasts
Tools:
Port-a-Patch®

Preparation and recordings on Nanion´sPort-a-Patch® of native E. coli Spheroplasts
The electrophysiology team at Nanion Technologies GmbH, Munich.

Summary
Bacterial membranes are not easy to patch clamp.

E. coli spheroplasts showed an endogenous current withSince bacterial ion channels are of increasing interest,

slow kinetics and rectifying properties (fig. 2). In Nanion’s

we started to optimize the protocols for patch clamping

standard solutions with internal potassium, the averaged

bacterial spheroplasts with the Port-a-Patch.

maximum current response to a ramp protocol from -100

Bacterial spheroaplasts can be prepared up to a size ofmV to 100 mV was 880 ± 560 pA at +100 mV, with a slightly

5 µm. They consist of the inner bacterial membrane. This

positive reversal potential.

technique was first used for patch clamp experiments
mechanosensitive channels in E. coli. Here we describe1

the preparation of Spheroplasts out of E. coli and show
ion channel currents recorded with the Port-a-Patch.

2nA

2nA

by Boris Matrinac in 1987 and let to the discovery of010-1-1-2Results
The E. coli strain C41 was used for the preparation of bacterial spheroplasts. To be able to patch clamp the small
membranes, NPC-1 chips with a high resistance (10-15MΩ) were used. When patch clamping, success rates of
70 % were reached (90% catching, 70 % reaching a GigaOhm seal).

-2
0.00

0.10

s

0.20

0.30

0.0

0.2

0.4

0.6
s

0.8

1.0

1.2

Figure 2:
Endogenous spheroplast currents. Left: Ramp from -100 mV to +100 mV in
200 ms. Right: Voltage steps from 0 mV to 100 mV. Holding potential in bothcases was -80 mV. Nanions standard solutions, with additional 400 mM sucrose in the external solution, were used.

Also single channels with a conductance of about 1 nS
could be observed, possibly native MscL (fig. 3). Spheroplasts reacted also sensitive to external pH (data not50 pAshown).

500 msFigure 1: Spheroplast preparation out of C41 E. coli strain.

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Figure 3: Singe channel activity in spheroplasts with a conductance ofabout 1 nS.

Application Note
Recordings with solutions containing a reduced chloride
concentraton indicated that the observed current

500in Nanion’s standard solutions was +15.5 mV (fig. 4),

pA

is mainly caused by chloride. The reversal potential

0meaning that chloride can’t be the only permeable-500

ion. Calculated reversal potentials in these solutions are:Potassium -83 mV, sodium +66 mV, chloride -23 mV andcalcium >> +100 mV. Based on this insight, solutions were

0.00

designed which repress the endogenous current. Theseconditions could then be used to study ion channels
which were overexpressed in E. coli (fig. 5).

0.10

Internal Solution:
120 mM KF
20 mM KCl
10 mM HEPES (pH 7)

1.0nA

0.50.050 mV-50-0.5

0.200.30 sExternal Solution 1:
3 mM KCl
80 mM NaCl
10 mM MgCl2
35 mM CaCl2
10 mM HEPES+ agar bridge
External Solution 2:
50 mM K-MSA
35 mM Ca-MSA
80 mM Na-MSA
10 mM HEPES+ agarms
bridgeVrev= 15.5 mV-1.0
0.00.2s0.40.6Figure 4: Calculation of the reversal potential by tail current analysis in
Nanion’s standard solutions.

Figure 5: Solutions designed to repress the endogenous current. They canbe used to study other channels expressed in E. coli. The current shown inresponse to a voltage ramp (200 ms ramp from -100 mV to +100 mV) isbased on potassium.

References

30 µl 1 M Tris-HCl, pH 8, 24 µl of 5 mg/ml lysozyme, 6 µl

1. Boris Martinac, Matthew Buenchner, Anne Delcour, Ju-

8 and mix in between by inverting. Incubate 15 min atlius Adler and Ching Kung, 1987. Pressure-sensitive ionroom temperature. Add 100 µl stop solution (10 mM Tris-channels in Escherichia coli. Proc. Natl. Acad. Sci. USA,HCl, pH 8, 0.7 sucrose, 20 mM MgCl2). Spheroplasts canVol. 84, pp. 2297-2301.

be stored at -80 °C.

of 5 mg/ml DNase and 6 µl of 125 mM EDTA-NaOH, pH

Electrophysiology: Experiments were performed withMethods

the Port-a-Patch, using NPC-1 borosilicate glass chips

Preparation of Spheroplasts: Inoculate a single colony

with a resistance of 10-15 MΩ. If nothing else is indicatedfrom a prepared agar plate in 5 ml modified LB medium

Nanion’s standard solutions with additional 400 mM

and incubate aerobically at 250 rpm and 37 °C until A600

sucrose in the external solution were used: Internal: 50

reached ~0.5. Add 60 µg/ml cephalexine for about 1 hour,mM KCl, 10 mM NaCl, 60 mM KF, 20 mM EGTA, 10 mM

control length of cells by microscope. Harvest 2 ml of theHepes, KOH pH 7.2 External: 140 mM NaCl, 4 mM KCl, 1

culture at 3*10 xg for 1 min in 2 ml tube. Resuspend pellet

mM MgCl2, 2 mM CaCl2, 5 mM Glucose, 10 mM Hepes,in 500 µl of 1 M glucose by inverting. Add successively

400 mM sucrose, NaOH pH 7.4.

3

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