SSM-based electrophysiology

Schulz, Patrick; García-Celma, Juan J.; Fendler, Klaus

doi:10.1016/j.ymeth.2008.07.002

Cited by 98 publications

(149 citation statements)

References 28 publications

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“…Proteoliposomes containing reconstituted LacY were immobilized on an SSM-coated gold electrode (the sensor), and charge displacement induced by downhill sugar/H ϩ symport into the proteoliposomes was detected by capacitive coupling (13). Transport was initiated at 1.5 s by a sugar concentration jump using rapid solution exchange (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Electrogenic transport by a reconstituted protein leads to transient currents in the capacitively coupled system (13,17). With wild-type LacY, downhill sugar/H ϩ symport into the proteoliposomes generates an insidepositive potential, which acts to decelerate the downhill symport reaction catalyzed by LacY, leading to transient currents.…”

Section: Resultsmentioning

confidence: 99%

“…This operation requires a transfer function for the specific measurements determined as described (30). The transfer function is the derivative of the substrate concentration rise at the surface of the SSM, and corresponds to the time resolution of the measurement (15 ms) (13,30). However, significantly faster processes (k Ͻ 200 s Ϫ1 ; see Fig.…”

Section: Resultsmentioning

confidence: 99%

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Electrophysiological characterization of LacY

García-Celma

Смирнова

Kaback

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Electrogenic events due to the activity of wild-type lactose permease from Escherichia coli (LacY) were investigated with proteoliposomes containing purified LacY adsorbed on a solid-supported membrane electrode. Downhill sugar/H ؉ symport into the proteoliposomes generates transient currents. Studies at different lipidto-protein ratios and at different pH values, as well as inactivation by N-ethylmaleimide, show that the currents are due specifically to the activity of LacY. From analysis of the currents under different conditions and comparison with biochemical data, it is suggested that the predominant electrogenic event in downhill sugar/H ؉ symport is H ؉ release. In contrast, LacY mutants Glu-3253 Ala and Cys-1543 Gly, which bind ligand normally, but are severely defective with respect to lactose/H ؉ symport, exhibit only a small electrogenic event on addition of LacY-specific substrates, representing 6% of the total charge displacement of the wild-type. This activity is due either to substrate binding per se or to a conformational transition after substrate binding, and is not due to sugar/H ؉ symport. We propose that turnover of LacY involves at least 2 electrogenic reactions: (i) a minor electrogenic step that occurs on sugar binding and is due to a conformational transition in LacY; and (ii) a major electrogenic step probably due to cytoplasmic release of H ؉ during downhill sugar/H ؉ symport, which is the limiting step for this mode of transport.bioenergetics ͉ membrane proteins ͉ permease ͉ solid-supported membrane ͉ transport

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Electrophysiological characterization of LacY

García-Celma

Смирнова

Kaback

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Proteoliposomes were adsorbed to a sensor equipped with a SSM, and the BetP transporter was activated via a rapid substrate concentration jump (65). Transient currents corresponding to the electrogenic activity of BetP were recorded via capacitive coupling.…”

Section: Methodsmentioning

confidence: 99%

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Khafizov¹,

Pérez²,

Koshy³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodiumbinding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuTlike (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K m and K d for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22 Na + binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.secondary transport | symmetry | membrane protein | alkali metal ion | osmoregulation

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“…ATP, in the presence of buffer solutions of various compositions. If at least one electrogenic step is involved in the relaxation process after activation, a current transient can be recorded along the external circuit (26,27). Numerical integration of each transient is related to a net charge movement that depends upon the particular electrogenic event.…”

Section: Methodsmentioning

confidence: 99%

Distinctive Features of Catalytic and Transport Mechanisms in Mammalian Sarco-endoplasmic Reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases

Lewis

Pilankatta

Inesi

et al. 2012

Journal of Biological Chemistry

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Background: SERCA and ATP7A/B are P-type ATPases entailing phosphoenzyme intermediate (E-P) formation. Results: Biochemical characterization, however, reveals distinctive features. Conclusion: A Hϩ -gated Ca 2ϩ pathway coupled to E1-P to E2-P transition is SERCA-specific. Headpiece interactions with an N-metal binding extension (NMBD) and related conformational constraints are ATP7A/B-specific. Significance: SERCA and ATP7A/B mechanisms present significant differences even though they are both P-type.

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SSM-based electrophysiology

Cited by 98 publications

References 28 publications

Electrophysiological characterization of LacY

Electrophysiological characterization of LacY

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Distinctive Features of Catalytic and Transport Mechanisms in Mammalian Sarco-endoplasmic Reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases

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