Physiological and Pharmacological Manipulations with Light Flashes

Lester, Henry A.; Nerbonne, Jeanne M.

doi:10.1146/annurev.bb.11.060182.001055

Cited by 115 publications

(72 citation statements)

References 55 publications

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“…We used Ϸ1-ms light flashes to jump the concentration (Fig. 3) of the photoisomerizable agonist trans-Bis-Q (19). The flashinduced ⌬I was fitted by a single exponential with ϭ 5.5 and 5.3 ms for ␤19ЈC and WT, respectively.…”

Section: Photoisomerization Of Cis-bis-q Reveals That ⌬F Is Complete mentioning

confidence: 99%

A fluorophore attached to nicotinic acetylcholine receptor βM2 detects productive binding of agonist to the αδ site

Dahan

Dibas

Petersson

et al. 2004

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

110

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To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the ␤19 position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (⌬F͞F) Ϸ10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for ⌬F agreed well with those for epibatidine-induced currents, but were shifted Ϸ100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the ␣␥-binding site than to the ␣␦ site and (ii) ACh binds with reverse-site selectivity, these data suggest that ⌬F monitors an event linked to binding specifically at the ␣␦-subunit interface. In experiments with flash-applied agonists, the earliest detectable ⌬F occurs within milliseconds, i.e., during activation. T he muscle nicotinic acetylcholine receptor (nAChR) is a well studied member of the Cys-loop family of neurotransmittergated ion channels. A 4.6-Å structure (1) shows five Ϸ160-Å-long rod-shaped subunits surrounding a central channel. From the extracellular side the subunits have a counterclockwise order of ␣␥␣␦␤ (2, 3). Each subunit has a large extracellular Nterminal or ligand-binding domain followed by four transmembrane regions, M1-M4 (4). The structure of the extracellular ligand-binding sites, at the ␣␥ and ␣␦ interfaces, resembles that of a homologous molluscan ACh-binding protein (3). Numerous biochemical and electrophysiological experiments indicate that M2 lines the channel (5).The nAChR exists in at least four distinct, interconvertible conformational states: resting, open, fast-onset-desensitized, and slow-onset-desensitized (6). The open and the fast-onsetdesensitized states presumably have moderate affinity for ACh, are metastable (on millisecond time scales), and are present in low concentrations at equilibrium. The supralinear doseresponse relation (Hill coefficient Ͼ1) suggests that the open state of the channel is much more likely to be associated with the presence of two bound agonist molecules than with a single bound agonist (7). In the prevailing kinetic scheme, receptors in the resting state bind two agonist molecules, isomerize to the open state, and in the continued presence of agonist, desensitize.After removal of agonist, the agonist-receptor complex dissociates and the channel closes within milliseconds; but desensitized receptors isomerize more slowly to the resting state (tens of milliseconds to hundreds of seconds). Thermodynamic considerations suggest that the resting state has low affinity for agonist, whereas the slow-onset-desensitized state is the most stable state in the presence of agonist because of its high affinity.Kinetic analyses of single-channel and macroscopic function suggests that in the resting state, the affinity of ACh for the two sites differs by a factor of...

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Section: Photoisomerization Of Cis-bis-q Reveals That ⌬F Is Complete mentioning

confidence: 99%

A fluorophore attached to nicotinic acetylcholine receptor βM2 detects productive binding of agonist to the αδ site

Dahan

Dibas

Petersson

et al. 2004

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

110

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“…We expected that an ultraviolet light flash applied to cell pairs loaded with appropriate membrane permeant esters (7)(8)(9) would create a pH jump (10), uncoupling the cells . In initial experiments on squid blastomeres, we were surprised to find that o-NBA uncoupled cells without a photolyzing light flash ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Because relatively low concentrations are effective, these compounds may be more specific in their action than the weak acids used previously. We noticed this method ofcytoplasmic acidification during attempts to develop compounds that would release protons by flash photolysis, a process that would THE JOURNAL OF CELL BIOLOGY " VOLUME 99 JULY 1984 174-179 ® The Rockefeller University Press -0021-9525/84/07/0174/06 $1 .00 on May 11, 2018 jcb.rupress.org Downloaded from allow study of the kinetics of hydrogen ion action on gap junctions (7,10) . The ester o-nitrobenzyl acetate (o-NBA) and its derivatives are photosensitive, releasing acetic acid upon photolysis (9,10).…”

mentioning

confidence: 99%

Substituted benzyl acetates: a new class of compounds that reduce gap junctional conductance by cytoplasmic acidification.

Spray¹,

Nerbonne²,

Carvalho³

et al. 1984

The Journal of Cell Biology

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Conductance of gap junctions in many preparations has been shown to be sensitive to cytoplasmic pH, decreasing as pH decreases below 7.5 in fish and amphibian embryos and below 7.1 in crayfish septate axon . We have found a new class of compounds, benzyl acetate derivatives, that reversibly decrease junctional conductance, gJ, when applied in low concentration (-1 mM) . Simultaneous intracellular pH (pH ;) measurements show that the ester effects are attributable to reduction in pHi. The sensitivity of gJ to these compounds and the relative lack of side effects make these agents attractive for studies of the role played by gap junctions in normal tissue function . In addition, the finding of cytoplasmic acidification in response to cell exposure to esters suggests caution in interpretation of results obtained using esterified compounds for intracellular loading .Gap junctions are common intercellular structures and the electrical coupling and molecular transfer that they mediate suggest that they play an important role in cell and tissue function (2,17) . To analyze their role, one would like to have a compound that specifically blocked gap junction channels. Such a compound might act directly or operate through an intermediate that affects gating processes of the channel (19).Gap junction conductance (gj)' is markedly decreased by treatments expected to lower intracellular pH (pHi) in many tissues (e.g., liver, D. Meyer and J. P. Revel, personal communication; heart [12]; embryonic cells [ 15,23]; crayfish axon [3,6]; cortex [4]). (Junctions may be insensitive to lowering pHi in adult lens [13; but see reference 11 ]; and retina [Griff and Pinto, personal communication] .) For frog and fish embryos and crayfish septate axon, where the short term relation between intracellular pH (pHi) and gj has been measured, a simple titration curve has been found with an apparent pK 'Abbreviations used in this paper:g;,junctional conductance; o-NBA, o-nitrobenzyl acetate ; pH;, intracellular pH.close to the normal cytoplasmic pH and a sufficiently steep slope that acidification by a few tenths of a pH unit significantly decreases gj (3, 15) . For cardiac Purkinje fibers (12) and ventricular myocytes (unpublished observations), normal pHi is in a region of the curve in which small pHi changes in either direction alter gj. One mode ofcytoplasmic acidification is to bathe the tissue in C02-equilibrated saline or with salts of weak acids at or near neutrality. C02 or the undissociated acid permeates the cell membrane, dissociates, and thereby acidifies the cytoplasm . Although cytoplasmic acidification may have multiple effects, a drug that liberates protons intracellularly might be generally useful in the study of the significance of gap junctional communication .We report here that a group of membrane permeant esters can be used to acidify the cytoplasm in several cell types. Because relatively low concentrations are effective, these compounds may be more specific in their action than the weak acids used previously....

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“…; protection/deprotection in organic synthesis [1][2][3][4] and uncaging of chemically masked or caged molecules). [5][6][7][8][9][10][11][12][13] To date, the photolysis of sulfonic esters, [14][15][16][17][18][19][20][21][22][23][24] sulfonamides, [25][26][27][28][29][30][31][32] and sulfones 33,34) has been applied to photoresist materials, 15,26) protecting groups, 8,19,20,[27][28][29][30][31][32] and other chemical reactions. 14,[35][36][37] Based on the above findings, the development of new photocleavage reactions of sulfonic acid derivatives and related mechanistic ...…”

mentioning

confidence: 99%

“…39,40) During a study of the hydrolytic uncaging reaction of 4, we found that 3 undergoes photolysis to yield 1, whose emission increased upon the addition of Zn . In this study, we report on the photolysis of 3, 4, 8-quinolinyl sulfonate derivatives that contain no metal chelating group (5)(6)(7)(8), and related compounds in aqueous solution (Chart 2). Some mechanistic aspects of this photolysis reaction are also discussed.…”

mentioning

confidence: 99%

Photochemical Cleavage Reactions of 8-Quinolinyl Sulfonates in Aqueous Solution

Kageyama

Ohshima

Sakurama³

et al. 2009

CHEMICAL & PHARMACEUTICAL BULLETIN

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Photochemical cleavage reactions of 8-quinolinyl benzenesulfonate derivatives and related sulfonates in aqueous solutions are reported. The 8-quinolinyl benzenesulfonates undergo photolysis upon photoirradiation at 300-330 nm to give the corresponding 8-quinolinols and benzenesulfonic acids with the production of only negligible amounts of byproducts. The effects of substituent groups of the 8-quinolinyl moiety and the benzene ring on the photolysis reactions were examined. Based on steady-state mechanistic studies using a triplet sensitizer, a triplet quencher, and electron donors, it was suggested that the photolysis proceeds mainly via the homolytic cleavage of S-O bonds in the excited triplet state.

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Physiological and Pharmacological Manipulations with Light Flashes

Cited by 115 publications

References 55 publications

A fluorophore attached to nicotinic acetylcholine receptor βM2 detects productive binding of agonist to the αδ site

A fluorophore attached to nicotinic acetylcholine receptor βM2 detects productive binding of agonist to the αδ site

Substituted benzyl acetates: a new class of compounds that reduce gap junctional conductance by cytoplasmic acidification.

Photochemical Cleavage Reactions of 8-Quinolinyl Sulfonates in Aqueous Solution

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