Development of a Novel Thiol Reagent for Probing Ion Channel Structure:  Studies in a Model System

Abstract

We have synthesized a novel thiol reagent, 2-[(methylsulfonyl)thio]ethyl [N-(N,N-dimethylamino)ethyl]carbamate (MTSAC), that contains a carbamate functional group as well as a (positively charged) terminal amino group. The carbamate C-N bond isomerizes on a millisecond time scale and significantly alters the three-dimensional shape of the reagent. The behavior of this reagent was contrasted with that of the commonly used thiol reagent, [(methylsulfonyl)thio]ethylamine MTSEA [Akabas, M. H., & Karlin, A. (1995) Biochemistry 34, 12496-12500], with respect to its effect on single-channel currents passing through modified gramicidin channels. While both reagents decreased single-channel currents, the MTSAC-treated channels also showed a pattern of steps in the current recordings on the time scale of the carbamate bond isomerization. Moreover, the pattern and size of these steps were sensitive to the location of the thiol-reactive site in relation to the channel entrance. Thus, MTSAC may prove useful as a reagent for establishing the proximity to the pore in studies of ion channel proteins of unknown structure.

Functional studies of the rabbit intestinal Na+/glucose carrier (SGLT1) expressed in COS-7 cells: evaluation of the mutant A166C indicates this region is important for Na+-activation of the carrier

“…In our laboratory we have initiated a systematic cysteinescanning mutagenesis project of the rabbit intestinal Na + \glucose carrier. The technique of cysteine-scanning mutagenesis followed by chemical modification has previously been employed to investigate the function, secondary structure and topology of cloned ion channels, or receptors [11][12][13][14][15][16]. We have obtained a series of single-cysteine-substituted SGLT1 mutants, some of which display functional characteristics distinct from those of wild-type SGLT1.…”

New sulfurated derivatives of cinnamic acids and rosmaricine as inhibitors of STAT3 and NF-κB transcription factors

“…Six sulfurated moieties were previously described, and were prepared accordingly: 1 59 , 3 60 , 5 61 , 6 62 , 9 63 , 11 63 . The alcohols 2 and 4 were synthesised according to Scheme 1 .…”

“…The known sulfurated starting compounds 1 59 , 3 60 , 5 61 , 6 62 , 9 63 , 11 63 , and the ferulic and caffeic acid methyl esters 13 64 and 20 65 have been synthesised according to the literature procedures.…”

Methanethiosulfonate derivatives as ligands of the STAT3-SH2 domain

“…A 1 N solution of dicyclohexylcarbodiimide (DCC, 4.22 ml) in CH 2 Cl 2 was added to a solution of S-(2-hydroxyethyl) methanesulfonothioate 25 ( 9 ; 3.84 mmol), 4-dimethylaminopyridine (DMAP, 0.18 mmol), and sulindac or acetyl salicylic acid (3.84 mmol) in CH 2 Cl 2 (67 ml), and the mixture was stirred for 1.5 h at room temperature, under nitrogen. At the end of the reaction, the dicyclohexylurea (DCU) was filtered and the solution was extracted successively with a solution of 1 N HCl, afterward with water, then with a saturated solution of NaHCO 3 and water.…”