Contribution of Proton Release to the B2 Photocurrent of Bacteriorhodopsin

Misra, Saurav

doi:10.1016/s0006-3495(98)77522-8

Cited by 14 publications

(7 citation statements)

References 33 publications

(62 reference statements)

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“…This study establishes the feasibility of an ERC structure/function investigation applied to the ligand binding pocket, as probed by artificial chromophores or site-specific mutations, or remote regions of the pigment, as probed by engineered mutations or chemically reactive ligands. The ERP has been similarly used in expressed mutant bacteriorhodopsin pigments to understand proton exchange mechanisms (e.g., Moltke et al, 1992;Misra, 1998). The similarity of the visual rhodopsin ERC/ERP and the bacteriorhodopsin ERP is striking and suggests that similar underlying mechanisms of conformational transitions may be conserved over evolutionary time.…”

Section: Discussionmentioning

confidence: 99%

Normal and Mutant Rhodopsin Activation Measured with the Early Receptor Current in a Unicellular Expression System

Shukla

Sullivan

1999

The Journal of General Physiology

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The early receptor current (ERC) represents molecular charge movement during rhodopsin conformational dynamics. To determine whether this time-resolved assay can probe various aspects of structure–function relationships in rhodopsin, we first measured properties of expressed normal human rhodopsin with ERC recordings. These studies were conducted in single fused giant cells containing on the order of a picogram of regenerated pigment. The action spectrum of the ERC of normal human opsin regenerated with 11-cis-retinal was fit by the human rhodopsin absorbance spectrum. Successive flashes extinguished ERC signals consistent with bleaching of a rhodopsin photopigment with a normal range of photosensitivity. ERC signals followed the univariance principle since millisecond-order relaxation kinetics were independent of the wavelength of the flash stimulus. After signal extinction, dark adaptation without added 11-cis-retinal resulted in spontaneous pigment regeneration from an intracellular store of chromophore remaining from earlier loading. After the ERC was extinguished, 350-nm flashes overlapping metarhodopsin-II absorption promoted immediate recovery of ERC charge motions identified by subsequent 500-nm flashes. Small inverted R2 signals were seen in response to some 350-nm flashes. These results indicate that the ERC can be photoregenerated from the metarhodopsin-II state. Regeneration with 9-cis-retinal permits recording of ERC signals consistent with flash activation of isorhodopsin. We initiated structure–function studies by measuring ERC signals in cells expressing the D83N and E134Q mutant human rhodopsin pigments. D83N ERCs were simplified in comparison with normal rhodopsin, while E134Q ERCs had only the early phase of charge motion. This study demonstrates that properties of normal rhodopsin can be accurately measured with the ERC assay and that a structure–function investigation of rapid activation processes in analogue and mutant visual pigments is feasible in a live unicellular environment.

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Section: Discussionmentioning

confidence: 99%

Normal and Mutant Rhodopsin Activation Measured with the Early Receptor Current in a Unicellular Expression System

Shukla

Sullivan

1999

The Journal of General Physiology

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“…With experimental setups essentially different from the one used here, the flash-induced photoelectric signals from oriented purple membranes, probably due to internal charge movement associated with conformational change of bR and/or external charge movements such as proton pumping, have been detected. [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42] The signal has at least three components with duration times lasting from picoseconds to milliseconds. The first component (B1), with a rise time shorter than 100 ps, 30,31 is thought to originate in the charge movement induced by alltrans to 13-cis isomerization of retinal.…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Photocurrent Generation from Bacteriorhodopsin on Gold Electrodes

Saga¹,

Watanabe²,

Koyama³

et al. 1998

J. Phys. Chem. B

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Excitation of bacteriorhodopsin (bR) at an electrode-electrolyte interface generates transient photocurrents as evidenced by the turning of an incident light on and off. By use of a gold electrode as substrate, on which an oxide layer can be formed in a controlled manner, we have found two types of photocurrents, both originating in the excitation of bR. For the first type, arising most probably from the pH response of the surface oxide layer due to proton release/uptake by bR, the magnitude of photocurrent well paralleled the amount of surface oxide up to about one monolayer of Au 2 O 3 and reached a maximum roughly equal to those observed on SnO 2 electrodes. The second type of photocurrent, being 3-to 4-fold smaller than the first one and essentially potential independent, arises presumably from simple charging, again through proton release/uptake by bR, of the electric double layer at the interface.

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“…Gigaohm-seal whole-cell recording had been used to study ERCs in rod and cone photoreceptors (Hestrin and Korenbrot, 1990;Makino et al, 1991) and the small and rapid ionic channel gating currents in unicellular expression systems (e.g., Sheets et al, 1996). In bacteriorhodopsin the combination of ERP (photovoltage) and site-specific mutagenesis/ expression techniques have been used to evaluate the contribution of select amino acid side chains to the vectorial charge motions that underlie the proton pumping mechanism (Moltke et al, 1992;Misra, 1998). Therefore, it was reasonable to explore the ERC as a time-resolved assay of electrically active conformational and chemical states during rhodopsin activation in an expression system where high plasma membrane rhodopsin levels could be achieved.…”

Section: Introductionmentioning

confidence: 99%

Time-Resolved Rhodopsin Activation Currents in a Unicellular Expression System

Sullivan

Shukla

1999

Biophysical Journal

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The early receptor current (ERC) is the charge redistribution occurring in plasma membrane rhodopsin during light activation of photoreceptors. Both the molecular mechanism of the ERC and its relationship to rhodopsin conformational activation are unknown. To investigate whether the ERC could be a time-resolved assay of rhodopsin structure-function relationships, the distinct sensitivity of modern electrophysiological tools was employed to test for flash-activated ERC signals in cells stably expressing normal human rod opsin after regeneration with 11-cis-retinal. ERCs are similar in waveform and kinetics to those found in photoreceptors. The action spectrum of the major R(2) charge motion is consistent with a rhodopsin photopigment. The R(1) phase is not kinetically resolvable and the R(2) phase, which overlaps metarhodopsin-II formation, has a rapid risetime and complex multiexponential decay. These experiments demonstrate, for the first time, kinetically resolved electrical state transitions during activation of expressed visual pigment in a unicellular environment (single or fused giant cells) containing only 6 x 10(6)-8 x 10(7) molecules of rhodopsin. This method improves measurement sensitivity 7 to 8 orders of magnitude compared to other time-resolved techniques applied to rhodopsin to study the role particular amino acids play in conformational activation and the forces that govern those transitions.

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Contribution of Proton Release to the B2 Photocurrent of Bacteriorhodopsin

Cited by 14 publications

References 33 publications

Normal and Mutant Rhodopsin Activation Measured with the Early Receptor Current in a Unicellular Expression System

Normal and Mutant Rhodopsin Activation Measured with the Early Receptor Current in a Unicellular Expression System

Mechanism of Photocurrent Generation from Bacteriorhodopsin on Gold Electrodes

Time-Resolved Rhodopsin Activation Currents in a Unicellular Expression System

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