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J Gen Physiol
2011 Dec 01;1386:641-9. doi: 10.1085/jgp.201110723.
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Expression and characterization of the bacterial mechanosensitive channel MscS in Xenopus laevis oocytes.
Maksaev G, Haswell ES.
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We have successfully expressed and characterized mechanosensitive channel of small conductance (MscS) from Escherichia coli in oocytes of the African clawed frog, Xenopus laevis. MscS expressed in oocytes has the same single-channel conductance and voltage dependence as the channel in its native environment. Two hallmarks of MscS activity, the presence of conducting substates at high potentials and reversible adaptation to a sustained stimulus, are also exhibited by oocyte-expressed MscS. In addition to its ease of use, the oocyte system allows the user to work with relatively large patches, which could be an advantage for the visualization of membrane deformation. Furthermore, MscS can now be compared directly to its eukaryotic homologues or to other mechanosensitive channels that are not easily studied in E. coli.
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Figure 1. Suitability of Xenopus oocytes for the expression of bacterial mechanosensitive channel MscS. (A) Inactivation of endogenous mechanosensitive channels upon patch excision. Traces from the same water-injected oocyte are shown in cell-attached configuration (top) and excised-patch (bottom). Pipettes with BN 7 at a potential of +40 mV were used. (B) Confocal scan showing a portion of an oocyte expressing MscS-GFP 4 d after injection. Bright field and GFP signal (in green) are superimposed. (C) MscS activation by negative and positive pressure in the pipette, as recorded from the same excised patch in symmetric ND96 buffer. Pipette potential was +30 mV with BN 6.
Figure 2. MscS conductance in oocytes is comparable to that in E. coli. (A) MscS and MscL channel activities in spheroplasts, recorded in 200 mM KCl plus 90 mM MgCl2. (B) MscS channel activity in oocytes, recorded in 96 mM NaCl plus 2 mM KCl (ND96). (C) Opening and closing of single MscS channels in response to stretch in an excised Xenopus oocyte patch. Pipette, BN 5. (D) Single-channel conductance measured in 98 mM TEA-Cl. Pipette, BN 7. Pipette potential was +40 mV in A and B, +30 mV in C and D.
Figure 3. MscS exhibits low voltage dependence when expressed in oocytes. (A) The current–voltage relationship for MscS in symmetric ND96 (squares; n = 7 patches) and in TEA-Cl (diamonds; n = 4 patches). Error bars indicate standard deviation. (B) Example of conductive substates as recorded at +40-mV pipette potential. (C) Nearly symmetric activation curves recorded at opposite potentials in the same excised patch.
Figure 4. Activation and inactivation behavior of MscS expressed in oocytes. (A) Representative trace from a short triangle stimulation ramp; pipette, BN 7. (B) Hysteresis of MscS is more prominent in small patches (pipette, BN 5). In A and B, slope fits for channel opening are shown in red and for channel closing in blue; dashed and dotted lines indicate threshold and midpoint tensions, respectively. Pipette potential was +40 mV. (C) Slow inactivation at subsaturating pressure with subsequent recovery during saturating tension pulses.
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