Anion Transporters DOI: 10.1002/anie.200504489 Regulating Supramolecular Function in Membranes: Calixarenes that Enable or Inhibit Transmembrane Cl  Transport** JenniferL.Seganish,PaulV.Santacroce, KevanJ.Salimian,JamesC.Fettinger,PeterZavalij,and JefferyT.Davis* Self-assembly is an attractive strategy for building synthetic ion channels. [1] Solid-state structures, when combined with functional data, can provide clues about how self-assembly regulates transmembrane ion transport. [2,3] We show that modest changes in the primary structure of a calix[4]arene amide lead to dramatic differences in the transport of chloride ions across phospholipid membranes. Comparison of solid- state structures for partial-cone calix[4]arene (paco-H) 1 and its para-substituted analogue (paco-tBu) 2, suggested that self-association and Cl  ion transport activity might be controlled by the conformation of the side chain on the inverted arene of the partial cone. Using this structural information as a basis for probing function, we describe a novel regulatory system, wherein the inactive 2 inhibits transmembrane transport of Cl  ions by partial-cone 1. This last finding is timely, given the interest in regulating the activity of synthetic transporters using external stimuli such as light, temperature, voltage, or ligand-gating to turn ion transport on or off. [4] Various “small molecules” transport Cl  ions across phospholipid membranes. These compounds range from ion carriers such as prodigiosins and cholapods, [5,6] to sterols and peptides that self-assemble in the membrane. [7,8] Our contri- butions to the field have involved calix[4]arenes [9] —scaffolds previously used for synthetic cation channels. [10] We found that a 1,3-alt-calix[4]arene tetraamide and an acylic analogue could transport Cl  ions across liposomal membranes. [11,12] Solid-state structures revealed that amide NH···Cl  hydrogen bonds mediated self-assembly of the 1,3-alt-calix[4]arene tetraamide and voltage clamp experiments confirmed forma- tion of stable ion channels in phospholipid and cell mem- branes. [11] We now compare the structure and function of 1 and 2, both prepared from conformationally fixed esters. [13] Proton NMR titrations showed that both calixarene tetraamides bind tetrabutylammonium chloride weakly in CDCl 3 , with similar values (K a = 10–20 m 1 ). [14,15] Although both compounds bind Cl  ions in organic solution, only 1 transports Cl  ions across phospholipid membranes. Figure 1 shows Cl  transport activ- ity in the presence of 1 or 2 in dipalmitoyl phosphatidylcho- line (DPPC) liposomes at 43 8C. Liposomes (100 nm) con- taining 1 mm of the Cl  -sensitive dye, lucigenin, [16] and 100 mm NaNO 3 were suspended in a solution of 100 mm NaNO 3 and 10 mm sodium phosphate (pH 6.4). Calixarenes 1 or 2 were added to the liposome suspension to give a 2:100 calixarene:lipid ratio. Evidence for transmembrane transport of Cl  ions was obtained from the quenching of lucigenin)s fluorescence after NaCl was added to give an extravesicular [*] J. L. Seganish, Dr. P. V. Santacroce, K. J. Salimian, Dr. J. C. Fettinger, Dr. P. Zavalij, Prof. J. T. Davis Department of Chemistry and Biochemistry University of Maryland College Park, MD 20742 (USA) Fax: (+ 1)301-314-9121 E-mail: jdavis@umd.edu [**] We thank the U.S. Department of Energy for financial support. K.J.S. thanks the University of Maryland’s Rollinson Scholars program and HHMI program for fellowships. We thank Frank Kotch and Vladimir Sidorov for discussions, and Prof. Marco Colombini for a preprint of reference [23] before its publication. Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. Communications 3334 # 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2006, 45, 3334 –3338