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Light-driven Na(+) pump from Gillisia limnaea: a high-affinity Na(+) binding site is formed transiently in the photocycle.

Balashov SP, Imasheva ES, Dioumaev AK, Wang JM, Jung KH, Lanyi JK - Biochemistry (2014)

Bottom Line: However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+).A greater concentration of Na(+) is needed for switching the reaction path at lower pH.Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of California , Irvine, California 92697, United States.

ABSTRACT
A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

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Light-induced pH changes in a suspension of E.coli cells with G. limnaea rhodopsin expressedand reconstitutedwith all-trans-retinal: trace 1, absence of pH changein sodium free medium [in 100 mM KCl (pH 7.5)]; trace 2, proton uptake(alkalinization) in 100 mM NaCl; trace 3, same as trace 2 but afteraddition of a protonophore (50 μM CCCP), which increases therate and extent of passive proton influx.
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fig1: Light-induced pH changes in a suspension of E.coli cells with G. limnaea rhodopsin expressedand reconstitutedwith all-trans-retinal: trace 1, absence of pH changein sodium free medium [in 100 mM KCl (pH 7.5)]; trace 2, proton uptake(alkalinization) in 100 mM NaCl; trace 3, same as trace 2 but afteraddition of a protonophore (50 μM CCCP), which increases therate and extent of passive proton influx.

Mentions: Figure 1 shows light-induced pH changesproduced by GLR expressed in E. coli. Illuminationof the cells in the absence of Na+ (in 100 mM KCl) doesnot produce significant pH changes, which suggests that unlike KR2,17 GLR does not transport protons, or if thereis such transport it is extremely weak. In 100 mM NaCl, the light-inducedincrease in pH is observed, and it is not abolished by CCCP as inthe H+ pumps, BR,42 XR,9 or ESR,38 but is enhancedby this proton conductor. This indicates that the light-induced alkalinizationis from passive H+ uptake in response to the active electrogenictransport of Na+, similar to what was observed for KR2from Krokinobacter,17 andthe main difference from KR2 is that the latter transports protonsin the absence of Na+ whereas in GLR no such activity isseen. The light-induced pH changes were suppressed by 10 mM TPP (notshown), a membrane penetrant cation, confirming the electrogenic natureof the Na+ transport, as in KR2.17 These are the same kind of observations that had identified halorhodopsinas a light-driven pump not for protons but for chloride ions.10


Light-driven Na(+) pump from Gillisia limnaea: a high-affinity Na(+) binding site is formed transiently in the photocycle.

Balashov SP, Imasheva ES, Dioumaev AK, Wang JM, Jung KH, Lanyi JK - Biochemistry (2014)

Light-induced pH changes in a suspension of E.coli cells with G. limnaea rhodopsin expressedand reconstitutedwith all-trans-retinal: trace 1, absence of pH changein sodium free medium [in 100 mM KCl (pH 7.5)]; trace 2, proton uptake(alkalinization) in 100 mM NaCl; trace 3, same as trace 2 but afteraddition of a protonophore (50 μM CCCP), which increases therate and extent of passive proton influx.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263435&req=5

fig1: Light-induced pH changes in a suspension of E.coli cells with G. limnaea rhodopsin expressedand reconstitutedwith all-trans-retinal: trace 1, absence of pH changein sodium free medium [in 100 mM KCl (pH 7.5)]; trace 2, proton uptake(alkalinization) in 100 mM NaCl; trace 3, same as trace 2 but afteraddition of a protonophore (50 μM CCCP), which increases therate and extent of passive proton influx.
Mentions: Figure 1 shows light-induced pH changesproduced by GLR expressed in E. coli. Illuminationof the cells in the absence of Na+ (in 100 mM KCl) doesnot produce significant pH changes, which suggests that unlike KR2,17 GLR does not transport protons, or if thereis such transport it is extremely weak. In 100 mM NaCl, the light-inducedincrease in pH is observed, and it is not abolished by CCCP as inthe H+ pumps, BR,42 XR,9 or ESR,38 but is enhancedby this proton conductor. This indicates that the light-induced alkalinizationis from passive H+ uptake in response to the active electrogenictransport of Na+, similar to what was observed for KR2from Krokinobacter,17 andthe main difference from KR2 is that the latter transports protonsin the absence of Na+ whereas in GLR no such activity isseen. The light-induced pH changes were suppressed by 10 mM TPP (notshown), a membrane penetrant cation, confirming the electrogenic natureof the Na+ transport, as in KR2.17 These are the same kind of observations that had identified halorhodopsinas a light-driven pump not for protons but for chloride ions.10

Bottom Line: However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+).A greater concentration of Na(+) is needed for switching the reaction path at lower pH.Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of California , Irvine, California 92697, United States.

ABSTRACT
A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

Show MeSH
Related in: MedlinePlus