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Introduction to Solid Supported Membrane Based Electrophysiology
Published on: May 11, 2013
Delineating electrogenic reactions during lactose/H+ symport.
Juan J Garcia-Celma1, Julian Ploch, Irina Smirnova
1Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, D-60438 Frankfurt/M, Germany.
Biochemistry
|June 24, 2010
Summary
The lactose permease (LacY) in E. coli facilitates lactose and proton transport. Electrophysiology reveals two key charge movements, including a rate-limiting proton release step crucial for symport.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- The lactose permease (LacY) from Escherichia coli is a model symporter.
- Understanding its transport mechanism involves characterizing electrogenic steps.
Purpose of the Study:
- To investigate the electrogenic reactions of LacY during lactose/H(+) symport.
- To delineate the kinetic mechanism of charge translocation with high time resolution.
Main Methods:
- Solid-supported membrane-based electrophysiology was employed.
- Purified LacY reconstituted into proteoliposomes was analyzed across a pH range (5.2–8.5).
Main Results:
- Two distinct electrogenic steps were characterized: a sugar-binding-triggered step and a major, pH-dependent step.
- The major step, accounting for 94% of charge translocation, is rate-limited by proton release (rate ~30 s⁻¹, pK 7.5).
- An intermediate "occluded" state was proposed, linked to the weaker electrogenic step.
Conclusions:
- The study provides a detailed kinetic mechanism for LacY-mediated symport.
- Electrophysiology successfully delineated the electrogenic steps involved in lactose and proton translocation.
- Findings clarify the role of proton release in the LacY transport cycle.
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