H+ pumping rhodopsin from the marine alga Acetabularia
Satoshi P. Tsunoda 1, David Ewers 2, Sabrina Gazzarrini 3, Anna Moroni 3, Dietrich Gradmann 4* and Peter Hegemann 1
1 Experimentelle Biophysik, HU Berlin
2 Medizinische Hochschule Hannover
3 Istituto di Biofisica, Università degli Studi di Milano
4 A.-v.-Haller-Institut, Universität Goettingen
* To whom correspondence should be addressed. E-mail: dgradma{at}uni-goettingen.de.
Submitted on April 3, 2006
Revised on April 20, 2006
Accepted on 18 May 2006
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Abstract |
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An opsin encoding cDNA was cloned from the marine alga Acetabularia acetabulum. The cDNA was expressed in Xenopus oocytes into functional Acetabularia rhodopsin (AR) mediating H+ carried outward photocurrents of up to 1.2 µA with an action spectrum maximum at 518 nm (AR518). AR is the first ion pumping rhodopsin found in a plant organism. Steady-state photocurrents of AR are always positive and rise sigmoidally from negative to positive trans-membrane voltages. Numerous kinetic details (amplitudes and time constants), including voltage dependent recovery of the dark state after light-off, are documented with respect to their sensitivities to light, internal and external pH, and the trans-membrane voltage. The results are analyzed by enzyme kinetic formalisms using a simplified version of the known photocycle of bacteriorhodopsin (BR). Blue-light causes a shunt of the photocycle under H+ reuptake from the extracellular side. Similarities and differences of AR with BR are pointed out. This detailed electrophysiological characterization highlights voltage dependencies in catalytic membrane processes of this eucaryotic, H+ pumping rhodopsin and of microbial-type rhodopsins in general.
Key Words:
Xenopus oocytes, bacteriorhodopsin, electrophysiology, enzyme kinetics, rhodopsin photocurrents
