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To investigate the physiological role of a kidney-specific chloride channel (CIC-Kl), we sought to determine its exact localization by immunohistochemistry and its functional regulation using Xenopus oocyte expression system. The antiserum specifically recognized a 70-kD protein in SDS-PAGE of membrane protein from rat inner medulla and an in vitro translated CiC-K1 protein. Immunohistochemistry revealed that ClC-K1 was exclusively localized to the thin limb of Henle's loop in rat inner medulla. In comparison with the immunostaining with anti-aquaporin-CHIP antibody that only stains the descending thin limb of Henle's loop (tDL), ClC-K1 was found to be localized only in the ascending limb (tAL) which has the highest chloride permeability among nephron segments. Immunoelectron microscopy confirmed that the staining of ClC-K1 in tAL was observed in the region of both apical and basolateral plasma membranes. Expressed chloride current in Xenopus oocytes by ClC-K1 cRNA was regulated by extracellular pH and extracellular calcium. Furosemide inhibited the expressed current (Ki = 100 ,uM), whereas N-ethyl-maleimide stimulated the current. These functional characteristics were consistent with the in vitro perfusion studies of chloride transport in tAL. The localization and the functional characteristics described here indicate that ClC-K1 is responsible for the transepithelial chloride transport in tAL. (J. Clin. Invest. 1995. 95:104-113.)

Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl(-) ion pump. For efficient Cl(-) transport, the existence of Cl(-)-binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl(-) to anion-free NpHR. The binding affinity was affected in mutants in which certain EC residues had been replaced; this finding revealed the importance of Arg123. On the other hand, mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants, L2-photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl(-) to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.

Tachycitin, a 73-residue polypeptide having antimicrobial activity is present in the hemocyte of horseshoe crab (Tachypleus tridentatus). The first three-dimensional structure of invertebrate chitin-binding protein was determined for tachycitin using two-dimensional nuclear magnetic resonance spectroscopy. The measurements indicate that the structure of tachycitin is largely divided into N-and C-terminal domains; the former comprises a three-stranded ␤-sheet and the latter a two-stranded ␤-sheet following a short helical turn. The latter structural motif shares a significant tertiary structural similarity with the chitin-binding domain of plant chitin-binding protein. This result is thought to provide faithful experimental evidence to the recent hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process.An invertebrate chitin-binding protein named tachycitin is recently found to be a member of the primordial elements of innate immune defense against bacterial and fungal infections (1-5). The antimicrobial activity is initially identified for chitin-binding proteins extracted from plants (6 -7), which commonly comprise single or multiple copies of the chitin-binding domain. The plant chitin-binding domain is mostly composed of 30 -43 residues including eight cysteines, three aromatic residues, and glycines and is frequently referred to as a hevein domain (8). It has been well demonstrated that this domain is indispensable for the antimicrobial activity and exhibits a significant conservation in primary sequence (Ͼ40%) and in three-dimensional (3D) 1 structure (9 -12). Although this advanced knowledge has been provided for the plant chitin-binding proteins, less is known for the invertebrate chitin-binding proteins including tachycitin (1,(13)(14)(15)(16). Kawabata et al. (1) identified that tachycitin is a 73-residues chitin-binding protein having antimicrobial activity. They also revealed that tachycitin consists of five intramolecular disulfide bridges; the connected Cys pairs are 6 -33, 12-30, 24 -61, 25-68, and 40 -53. For invertebrates, the chitin-binding domain was assumed to comprise about 65 residues (17) involving a high percentage of cysteine and aromatic residues in a similar manner to the plant chitin-binding domain. On the basis of such similarity between plant and invertebrate chitin-binding proteins, Shen and Jacobs-Lorena (17) proposed a hypothesis that they are correlated by a rare evolutional process, convergent evolution, i.e. proteins from different origins develop to construct the same active site structure to acquire the same function. However, complete lack of 3D-structural information of invertebrate chitin-binding protein obscures the evolutional relationship between invertebrate and plant chitin-binding proteins. The present study determines the solution structure of tachycitin using NMR spectroscopy, which provides the first 3D structural information of invertebrate chitin-binding protein. EXPERIMENTAL PROCEDURESAn invertebrate...

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