Project A3, 3^rd cohort:

Functional-structural relationship of paracellular channels formed by claudin-10b and claudin-15

State of the art: Claudins are tetraspan membrane proteins of the bicellular tight junction (TJ) that form a paracellular barrier against solutes and water passage across epithelia. However, a subset of claudins not only build a barrier against large molecules but also form paracellular channels through the tight junction. Of these, claudin-10b and claudin-15 form cation-selective homo-polymeric channels. They differ in permeability for various cations depending on their re-spective size and hydration shell (Eisenman sequence) as well as for water. These differences result in specific transport functions, for instance in kidney tubules mutations of claudin-10b lead to diseases caused by altered reabsorption, and in small intestine loss of claudin-15 leads to malabsorption. The channel structure of claudins is yet unknown, although models of their molecular architecture have been suggested based on the claudin-15 crystal structure. The amino acids of the extracellular segments that are widely conserved between claudin-10b and claudin-15 have been shown to be critical for charge selectivity.
Aims: Clarifying the molecular determinants of cation and water channel function for claudin-10b and claudin-15.
Hypotheses: The permeability properties of claudin-10b and of claudin-15 channels are determined by specific sequence differences in their extracellular segments.
Methods: Mutagenesis, culture of epithelial cell clones, transfection, immunostaining, confocal and STED microscopy, dilution potentials, water transport, and structural bioinformatics.
Thesis project: We will study molecular determinants regulating the function of claudin-10b and -15 channels. Comparison of molecular models of claudin-10b and -15 channels are used to select regions and individual pore-lining amino acid residues in the extracellular domain that differ between both. Candidate chimeric mutations predicted to convert one claudin functionally into the other claudin (Eisenman sequence, water permeability) will be designed, generated by site-directed mutagenesis and stably expressed in epithelial cell lines with different claudin back-ground (MDCK-C7 with, quinKO MDCKII without endogenous TJs). Cell membrane transport and TJ incorporation will be tested by immunostaining (claudin mutant, TJ markers), confocal and STED microscopy. Mutants lacking TJ localization provide information about claudin polymer assembly. Mutants with TJ localization will be further analyzed functionally: Barrier formation against small and large molecule permeation by tracer flux assays; selective ion channel formation by electrophysiology and water transport assays. Iterative combination of (i) channel structure modelling, (ii) candidate mutant selection, (iii) mutant generation/testing, and (iv) evaluation concerning model consistency will be performed.

3^rd cohort PhD doctoral student

Alina Handreg

2^nd cohort PhD doctoral student

Fabián Martínez-Perafán

1^st cohort PhD doctoral student

Carlos Ayala-Torres
29.04.21: Doctoral examination passed, Dr. rer. nat. (PhD), Freie Universität Berlin, magna cum laude

• Ayala-Torres C, Krug SM, Rosenthal R*, Fromm M* (*shared last authorship) (2021) Angulin-1 (LSR) affects paracellular water transport, however only in tight epithelial cells. Int. J. Mol. Sci. 22: 7827 (25 pages). doi: 10.3390/ijms22157827 (IF 6.2)

• Ayala-Torres C, Krug SM, Schulzke JD, Rosenthal R*, Fromm M* (*shared last authorship) (2019) Tricellulin effect on paracellular water transport. Int. J. Mol. Sci. 20 (22): 5700 (15 pages) [PubMed] [WebPage] [PDF] (IF 4.6)

• Rosenthal R, Günzel D, Piontek J, Krug SM, Ayala-Torres C, Hempel C, Theune D, Fromm M (2020) Claudin-15 forms a water channel through the tight junction with distinct function compared to claudin-2. Acta Physiol. 228(1): e13334 (15 pages) [PubMed] [WebPage] [PDF]  (°IF 6.3)