Rier (1). Each claudin-2 and claudin-10b can type paracellular cation pores
Rier (1). Each claudin-2 and claudin-10b can type paracellular cation pores with PNa PCl of six to 8 (24). The pore diameter of claudin-2 is estimated to be six.5.five (two, 5). The key determinant of claudin-2 ion charge selectivity is definitely an aspartate residue in ECL1 (Asp65) (2, six). When all 3 negatively charged residues in the claudin-2 ECL 1, such as Asp65, have been mutated to neutral amino acids, the pore became less cat- Thiswork was supported by National Institutes of Overall health Grants R01DK062283 and U01GM094627 (to A. S. L. Y.). 1 To whom correspondence must be addressed: The Kidney Inst., University of Kansas Medical Ctr., 3901 Rainbow Blvd., Mail Quit 3018, Kansas City, KS 66160. Tel.: 913-588-9252; 913-588-9251; E-mail: ayukumc.edu.ion-selective. Even so, it remained four instances more selective to Na than to Cl (two). This observation led us to postulate that other mechanisms may perhaps also play a mAChR5 list function in cation selectivity which include cation interaction with polar residues (e.g. carbonyl oxygen, as is the case inside the KcsA potassium channel (7)), or cationinteractions. The latter possibility prompted us to search for a conserved aromatic residue near Asp65 and Ile66, exactly where the cation-selective filter is located (two, 8). We discovered position 67 of claudin-2 and position 66 of claudin-10b to possess an aromatic residue that may be highly conserved in all the classic claudins (tyrosine in claudin-2 and phenylalanine in claudin-10b). The objective of this study was to assess the function of this aromatic residue in cation pore-forming claudins. We hypothesized that Tyr67 (claudin-2) and Phe66 (claudin10b) may perhaps interact with permeating cations by way of cationinteraction. Cation- interaction is defined as the interaction among positively charged molecules and negatively charged electrons on the benzene ring on the aromatic amino acid side chain. Cation- interaction has been identified within the nicotinic receptor ligand binding web-site (9) too as in the binding web site for tetraethylammonium in potassium channel (10). To test whether or not Tyr67 or Phe66 interacts using the permeating cations by way of cation- interaction, we mutated this aromatic residue to leucine, a bulky and IL-5 site hydrophobic residue without the need of the benzene ring. By eliminating the cation- interaction, both claudin-2 Y67L and claudin-10b F66L have been predicted to become significantly less cation-selective than its respective wild-type protein. The aromatic residue might also have a function via its steric impact. Its bulky benzene group could have a mechanical impact to modulate protein conformation and therefore function. In the ATPsensitive K channel, a pore-lining phenylalanine gates the channel by steric hindrance (11), and in the KcsA channel, activation and inactivation are mechanically coupled by a phenylalanine residue (12). To test no matter whether the conserved aromatic residue exerts a steric effect, we substituted Tyr67 (claudin-2) or Phe66 (claudin-10b) with an alanine, a smaller hydrophobic residue. Our findings recommend that the conserved aromatic residue confers cation selectivity in cation pore-forming claudins by interacting using the permeating cation both by way of cation- interaction and by restricting the pore size by means of its steric impact.VOLUME 288 Number 31 AUGUST two,22790 JOURNAL OF BIOLOGICAL CHEMISTRYConserved Aromatic Residue in Cation Pore-forming ClaudinsEXPERIMENTAL PROCEDURESGeneration and Screening of MDCK I Tet-off Claudin-2 and Claudin-10b Cell Lines–MDCK I Tet-off cells expressing wildtype claudin-2, wild-type claudin-10b, claudin-2 mut.
