RESUMO
The channel-forming properties of two analogs of gramicidin, gramicidin-ethylenediamine (gram-EDA), and gramicidin-N,N-dimethylethylenediamine (gram-DMEDA) were studied in planar lipid bilayers, using protons as the permeant ion. These peptides have positively charged amino groups tethered to their C-terminal ends via a linker containing a carbamate group. Gram-DMEDA has two extra methyl groups attached to the terminal amino group, making it a bulkier derivative. The carbamate groups undergo thermal cis-trans isomerization on the 10-100-ms time scale. The conductance behavior of gram-EDA is found to be markedly voltage dependent, whereas the behavior of gram-DMEDA is not. In addition, voltage affects the cis-trans ratios of the carbamate groups of gram-EDA, but not those of gram-DMEDA. A model is proposed to account for these observations, in which voltage can promote the binding of the terminal amino group of gram-EDA to the pore in a "ball-and-chain" fashion. The bulkiness of the gram-DMEDA derivative prevents this binding.
Assuntos
Gramicidina/química , Canais Iônicos/química , Sequência de Aminoácidos , Carbamatos/química , Carbamatos/metabolismo , Cloretos/metabolismo , Condutividade Elétrica , Eletrofisiologia , Gramicidina/metabolismo , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Isomerismo , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , PrótonsRESUMO
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.