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H⁺-Pumping Rhodopsin from the Marine Alga Acetabularia

Satoshi P. Tsunoda ^*, David Ewers , Sabrina Gazzarrini , Anna Moroni , Dietrich Gradmann  and Peter Hegemann ^*

^* Experimentelle Biophysik, Fachbereich für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany; A.-v.-Haller-Institut der Universität, 37073 Göttingen, Germany; and Dipartimento di Biologia and Consiglio Nazionale delle Ricerche Istituto di Biofisica, Università degli Studi di Milano, 20133 Milan, Italy

Correspondence: Address reprint requests to Dietrich Gradmann, Tel.: 49-7071-550005; Fax: 49-7071-869001; E-mail: dgradma{at}uni-goettingen.de.

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 transmembrane 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 transmembrane 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 eukaryotic, H⁺-pumping rhodopsin and of microbial-type rhodopsins in general.

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