Molecular basis of claudin-17 anion selectivity

Abstract

Claudin-17 is a paracellular channel-forming tight junction protein. Unlike the cation channels claudin-2 and -15, claudin-17 forms a distinct anion-selective channel. Aim of this study was to determine the molecular basis of channel formation and charge selectivity of this protein. To achieve this, residues located in the extracellular loops (ECL) 1 and 2 of claudin-17 were substituted, preferably those whose charges differed in claudin-17 and in claudin-2 or -15. The respective mutants were stably expressed in MDCK C7 cells and their ability to form charge-selective channels was analyzed by measuring ion permeabilities and transepithelial electrical resistance. The functional data were combined with homology modeling of the claudin-17 protomer using the structure of claudin-15 as template. In ECL1, K65, R31, E48, and E44 were found to be stronger involved in Cldn17 channel function than the clustered R45, R56, R59, and R61. For K65, not only charge but also stereochemical properties were crucial for formation of the anion-selective channel. In ECL2, both Y149 and H154 were found to contribute to constitution of the anion channel in a distinct manner. In conclusion, we provide insight into the molecular mechanism of the formation of charge- and size-selective paracellular ion channels. In detail, we propose a hydrophilic furrow in the claudin-17 protomer spanning from a gap between the ends of TM2 and TM3 along R31, E48, and Y67 to a gap between K65 and S68 lining the anion channel.

Polar and charged extracellular residues conserved among barrier‐forming claudins contribute to tight junction strand formation

“…A). Intramolecular electrostatic interaction between Lys and Asp at these positions might contribute to barrier versus channel formation . Alternatively, these positions might interact in either cis or trans .…”

“…The strands were strongly associated with the PF, whereas, on the EF, mainly particle‐free grooves were observed. The ultrastructure of the TJ strands in these MDCK‐C7 lines was similar to those of other MDCK‐C7 lines described previously . However, owing to variations within the specimens, the effects of the S53E/K65D substitution on detailed aspects of the TJ ultrastructure could not be evaluated.…”

“…K65 of Cldn1 corresponds to the charge selectivity filter of Cldn2 (D65) and of Cldn17 (K65) . D65N disturbs the charge selectivity of Cldn2 channels and K65E disturbs that of Cldn17 .…”

Developmental Expression of Claudins in the Mammary Gland

“…1b). In particular, claudins-2, −10b, and −15 have been shown to mediate cation permeation, while claudins-10a and -17 mediate anion permeation [6, 7]. Claudin-2 also allows water to permeate the epithelial barrier [6].…”

“…In particular, claudins-2, −10b, and −15 have been shown to mediate cation permeation, while claudins-10a and -17 mediate anion permeation [6, 7]. Claudin-2 also allows water to permeate the epithelial barrier [6]. The crystal structure of pore forming claudin-15 has recently been published [8] showing that the four transmembrane (TM) segments of the molecules form alpha-helices while extracellular loop 1 (ECL1), involved in intercellular interactions, possesses four beta-pleated sheets and the smaller extracellular loop 2 (ECL2) has one (Fig.…”