Hydration changes implicated in the remarkable temperature-dependent membrane permeation of cyclosporin A

Cyclosporin A is a cyclic peptide believed to exist as multiple conformers in aqueous solution. Two major conformations, distinguished by a single cis :trans isomerization and the presence of four either intramolecular or inte rmolecular hydrogen bonds, have been confirmed depending on whether CsA is characterized in organic solvents or bound in aqueous complex with cyclophi lin. The relationship between CsA conformation and its ability to penetrate biological membranes is currently unknown. Using Caco-2 cell monolayers, w e documented a remarkable increase (more than 2 orders of magnitude) in the membrane permeation of the peptide as temperature was increased from 5 to 37 degrees C. The solubility of CsA was 72 mu M at 5 degrees C, but decreas ed by more than an order of magnitude at 37 degrees C. Moreover, CsA partit ioned into non-hydrogen bond donating solvents linearly as a function of in creasing temperature, suggestive of a significant conformational change. Ho wever, while NMR spectra of CsA confirmed the previously predicted presence of multiple conformers in aqueous solution, the equilibrium between the tw o major species was not affected by changes in temperature. These NMR data indicated that the observed temperature-dependent changes in the membrane p ermeability of CsA do not originate from changes in the peptide backbone co nformation. Sedimentation equilibrium analysis revealed that CsA behaves in a highly nonideal manner over the temperature range tested. We interpret t his behavior as a change in the hydration state with a smaller (or weaker) hydration shell surrounding the peptide at higher temperatures. Such a chan ge would result in lower peptide desolvation energy, thereby promoting part itioning into cellular membranes. We contend that changes in membrane penet ration result from alterations in the hydration state of CsA and are not re lated to the interconversion of the defined conformations.