Biophys J, April 2001, p. 1810-1818, Vol. 80, No. 4

Covalently Linked Gramicidin Channels: Effects of Linker Hydrophobicity and Alkaline Metals on Different Stereoisomers

Kathryn M. Armstrong,^* Edward P. Quigley,^* Paulene Quigley, David S. Crumrine, and Samuel Cukierman^*

 ^*Department of Physiology, Loyola University Medical School, and  Department of Chemistry, Loyola University Chicago, Maywood, Illinois, 60153 USA

The direct role of the dioxolane group on the gating and single-channel conductance of different stereoisomers of the dioxolane-linked gramicidin A (gA) channels reconstituted in planar lipid bilayers was investigated. Four different covalently linked gA dimers were synthesized. In two of them, the linker was the conventional dioxolane described previously (SS and RR channels). Two gAs were covalently linked with a novel modified dioxolane group containing a retinal attachment (ret-SS and ret-RR gA dimers). These proteins also formed ion channels in lipid bilayers and were selective for monovalent cations. The presence of the bulky and hydrophobic retinal group immobilizes the dioxolane linker in the bilayer core preventing its rotation into the hydrophilic lumen of the pore. In 1 M HCl the gating kinetics of the SS or RR dimers were indistinguishable from their retinal counterparts; the dwell-time distributions of the open and closed states in the SS and ret-SS were basically the same. In particular, the inactivation of the RR was not prevented by the presence of the retinal group. It is concluded that neither the fast closing events in the SS or RR dimers nor the inactivation of the RR are likely to be a functional consequence of the flipping of the dioxolane inside the pore of the channel. On the other hand, the inactivation of the RR dimer was entirely eliminated when alkaline metals (Cs⁺ or K⁺) were the permeating cations in the channel. In fact, the open state of the RR channel became extremely stable, and the gating characteristics of both the SS and RR channels were different from what was seen before with permeating protons. As in HCl, the presence of a retinal in the dioxolane linker did not affect the gating behavior of the SS and RR in Cs⁺- or K⁺-containing solutions. Alternative hypotheses concerning the gating of linked gA dimers are discussed.

Biophys J, April 2001, p. 1810-1818, Vol. 80, No. 4
© 2001 by the Biophysical Society   0006-3495/01/04/1810/09  $2.00

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