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Molecular basis of claudin-17 anion selectivity

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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.

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Abbreviations

TJ:

Tight junction

Cldn:

Claudin

Occl:

Occludin

SEM:

Standard error of the mean

MDCK:

Madin-Darby Canine Kidney

TER:

Transepithelial resistance

wt:

Wildtype

vec:

Vector control

TM:

Transmembrane segment

ECL1 and -2:

Extracellular loop 1 and 2, equivalent to extracellular segment 1 and 2

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Acknowledgments

We appreciate the excellent technical assistance of In-Fah M. Lee and Detlef Sorgenfrei. This study was supported by grants of the Deutsche Forschungsgemeinschaft (DFG FOR 721).

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    Authors and Affiliations

    1. Institute of Clinical Physiology, Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12203, Berlin, Germany

      Marcel P. Conrad, Jörg Piontek, Dorothee Günzel, Michael Fromm & Susanne M. Krug

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    1. Marcel P. Conrad
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    2. Jörg Piontek
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    3. Dorothee Günzel
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    4. Michael Fromm
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    Correspondence to Susanne M. Krug.

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    The authors declare no conflicts of interest.

    Additional information

    M. P. Conrad and J. Piontek contributed equally to the study.

    Electronic supplementary material

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    18_2015_1987_MOESM1_ESM.tiff

    Figure S1. Multiple sequence alignment of ECL1 (left) and ECL2 (right) for Cldn17. 28 orthologous sequences available in UNIPROTKB were aligned using CLUSTAL O (McWilliam et al., Nucleic Acids Res. 2013 Jul; 41(Web Server issue: W597-600.)). Accession number and species is given on the left; residue number is given on top; green, positively charged; red negatively charged; cyan Ser/Thr; *, identical; :, strongly similar properties;., weakly similar properties. The sequences are shown with positions 29 - 81 and 146-164 (ECL1 and ECL2 as defined by uniprot.org). Boxes mark the residues analyzed in this study. Blue boxes mark positions with identical or highly similar residues whereas red boxes mark positions with residues containing different properties (TIFF 2337 kb)

    18_2015_1987_MOESM2_ESM.tif

    Figure S2. Exemplary western blots for other TJ proteins. The blots show exemplary clones of each mutation in comparison to wt Cldn17 and vector control (vec). Detected were Cldn1, Cldn4, occludin, the FLAG-tagged Cldn17 and β-actin as loading control. Expression changes occurred in some, but not all, clones of one mutant. However, these changes had no impact on functional results, as these were similar in all clones of one mutant (TIFF 221 kb)

    18_2015_1987_MOESM3_ESM.tiff

    Figure S3. Exemplary comparison of different clones (E44A). a The blots show exemplary clones of mutation E44A in comparison to wt Cldn17 and a vector control (vec). Detected were Cldn1, Cldn4, occludin, the FLAG-tagged Cldn17 and β-actin as loading control. b The densitometric analysis revealed expression differences which may influence the results of further experiments. However, only a very low mutant expression of 14 % (clone #1) compared to the wt, resulted in PCl/PNa as observed for the vec, while ranges of expression above 43 % to 86 % were comparable to each other c. Also permeabilities for other ions d were only different from the other clones at very low expression levels. Additional expression variations in endogenous TJ protein levels had no effect in the ranges observed. However, to keep clonal variation as low as possible, several clones were analyzed for each mutant and outliers with extreme differences in expressions were excluded (TIFF 3315 kb)

    18_2015_1987_MOESM4_ESM.tiff

    Figure S4. Immunofluorescent staining of exemplary clones. Localization of the 3 × FLAG-tagged mutants of Cldn17 (red) was analyzed using occludin or ZO-1 (green) as TJ marker. All mutant Cldn17 constructs were colocalized (yellow) with the TJ marker, indicating correct insertion into the TJ. Bar = 10 µm (TIFF 16234 kb)

    18_2015_1987_MOESM5_ESM.tiff

    Figure S5. Exemplary images of freeze-fracture electron microscopic samples. Mutation from anion- to cation-selective Cldn17 (mutant K65E) as well as the other mutations of K65 that led to loss of charge selectivity had no effect on TJ ultrastructure. a Vector-transfected control. b wt Cldn17. c Cldn17 K65E. d Cldn17 K65A. e Cldn17 K65R. Bar = 200 nm (TIFF 4714 kb)

    18_2015_1987_MOESM6_ESM.tiff

    Figure S6. Live cell imaging of HEK293 cells transfected with three CFP-tagged constructs. Subcellular localization of a wtCldn17, b Cldn17 Y149A and c Cldn17 H154A (green). Trypan blue (red) marks the cell membrane. All three Cldn17 constructs were expressed and localized within the cell membrane. Enrichment in cell membranes of neighboring cells indicates the ability to trans-interact. Bar 5 µm (TIFF 20632 kb)

    18_2015_1987_MOESM7_ESM.tiff

    Figure S7. Overlay of Cldn17 and Cldn19 homology models. Comparison of Cldn17 homology models based on templates for Cldn15 (PDB: 4P79, backbone as cartoon in green, residues as sticks in magenta) and Cldn19 (PDB: 3X29, backbone as cartoon in cyan, residues as sticks in violet). Both models indicate a similar fold of Cldn17 and similar positions of the residues (sticks) mutated in this study. However, the models differ in the C-terminal half of ECL2, the loop between β1- and β2-strand and the region between β4-strand and TM2. The latter region was not resolved in the Cldn19 crystal, in which Cldn19 was in complex with the C-terminal domain of the Clostridium perfringens enterotoxin [20]. Disulfide bridges in ECL1 are shown as yellow sticks (TIFF 2927 kb)

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    Conrad, M.P., Piontek, J., Günzel, D. et al. Molecular basis of claudin-17 anion selectivity. Cell. Mol. Life Sci. 73, 185–200 (2016). https://doi.org/10.1007/s00018-015-1987-y

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