Comprehensive Cysteine-scanning Mutagenesis Reveals Claudin-2 Pore-lining Residues with Different Intrapore Locations

Abstract

Background:The composition of the claudin paracellular pore region is incompletely known. Results: Cysteine-scanning mutagenesis of the first extracellular domain of claudin-2 was used to identify all pore-lining residues and their intrapore locations. Conclusion: This study maps out the claudin-2 pore region. Significance: This advances understanding of the structure-function relationship of claudin pores.

Cell Biology of Tight Junction Barrier Regulation and Mucosal Disease

“…The first extracellular loop (ECL1) of claudin proteins contains several conserved sequences, and most claudins also include a carboxy-terminal PDZ-binding sequence that interacts with the PDZ1 domain of ZO proteins (Itoh et al 1999). It is well-documented that many claudin proteins form paracellular pores, and that the charge selectivity of these pores is defined by amino acids within ECL1 (Colegio et al 2003; Li et al 2014; Weber et al 2015). The relationship of this to health and disease was initially shown by genetic linkage of para-cellin-1, or claudin-16, to the autosomal recessive disease of renal hypomagnesemia with hypercalciuria and nephrocalcinosis (Simon et al 1999).…”

“…Further, whereas the MLCK inhibitor PIK can reverse TNF-induced barrier loss, it is ineffective against IL-13-induced barrier loss (Weber et al 2010). The latter is explained by the observation that IL-13 augments paracellular permeability by increasing claudin-2 expression (Heller et al 2005; Weber et al 2010), which has been shown to create paracellular cation-selective pores (Furuse et al 2001; Amasheh et al 2002; Li et al 2014; Weber et al 2015). Intriguingly, claudin-2 is selectively upregulated by IL-13 (Weber et al 2010), whereas other stimuli regulate expression of multiple tight junction-related proteins.…”

“…Detailed analyses have shown that claudin-2 expression increases the density of cation-selective, paracellular pores with a diameter of 7-8 Å (Van Itallie et al 2008; Li et al 2014). Development of a novel paracellular patch clamp technique has recently allowed biophysical analyses demonstrating that these pores are actively gated and display a flickering behavior, with unstable open and closed conformations and a third, stable, closed conformation (Weber et al 2015).…”

The mucosal barrier at a glance

“…Claudin-2 pores also permit the flux of water through a common pore (Muto et al, 2010;Rosenthal et al, 2016Rosenthal et al, , 2010. The specificity for Na + is governed by the first of two extracellular loops of claudin-2 that are adjoined to four transmembrane domains with intracellular N-and C-termini Li et al, 2014;Suzuki et al, 2014;Yu et al, 2009). Pore-forming claudins define the charge and size selectivity of the high-capacity pore pathway Colegio et al, 2002;Shen et al, 2011;Turner, 2009;Van Itallie et al, 2008;Weber et al, 2010).…”

Claudin-2 as a mediator of leaky gut barrier during intestinal inflammation

“…13,14 Additional porelining residues were identified in a study using cysteine-scanning mutagenesis and thiol group modification including, from the narrowest to the widest part of the pore, S68, S47, T62/I66, T56, T32/G45, and M52. 15 Pore-lining residues from D65 to S68 in the ECL1 are believed to be the most important ones for the paracellular functions of claudin-2, and the negatively charged D65 is already known to be determinative for cation selectivity. 11,12 Additionally, through electrostatic interactions with D65 12 and weaker interactions with negatively charged p-electrons of the Y67 aromatic residue 16 , cations are able to permeate the pore in a dehydrated or partially dehydrated state.…”