Division of Nephrology and Hypertension and The Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
J Biol Chem. 2013 Aug 2;288(31):22790-7. doi: 10.1074/jbc.M113.484238. Epub 2013 Jun 12.
In tight junctions, both claudin-2 and claudin-10b form paracellular cation-selective pores by the interaction of the first ECL 1 with permeating ions. We hypothesized that a highly conserved aromatic residue near the pore selectivity filter of claudins contributes to cation selectivity by cation-π interaction with the permeating cation. To test this, we generated MDCK I Tet-off cells stably transfected with claudin-2 Tyr(67) mutants. The Y67L mutant showed reduced cation selectivity compared with wild-type claudin-2 due to a decrease in Na(+) permeability, without affecting the Cl(-) permeability. The Y67A mutant enlarged the pore size and further decreased the charge selectivity due to an increase in Cl(-) permeability. The Y67F mutant restored the Na(+) permeability, Cl(-) permeability, and pore size back to wild-type. The accessibility of Y67C to methanethiosulfonate modification indicated that its side chain faces the lumen of the pore. In claudin-10b, the F66L mutant reduced cation selectivity, and the F66A mutant lost pore conductance. We conclude that the conserved aromatic residue near the cation pore domain of claudins contributes to cation selectivity by a dual role of cation-π interaction and a luminal steric effect. Our findings provide new insight into how ion selectivity is achieved in the paracellular pore.
在紧密连接中，claudin-2 和 claudin-10b 通过第一 ECL1 与渗透离子的相互作用形成细胞旁阳离子选择性孔道。我们假设 claudin 孔选择性过滤器附近的高度保守的芳族残基通过与渗透阳离子的阳离子-π 相互作用有助于阳离子选择性。为了验证这一点，我们生成了稳定转染 claudin-2 Tyr(67)突变体的 MDCK I Tet-off 细胞。由于 Na+通透性降低，Y67L 突变体与野生型 claudin-2 相比表现出降低的阳离子选择性，而不影响 Cl-通透性。Y67A 突变体增大了孔径并进一步降低了电荷选择性，因为 Cl-通透性增加。Y67F 突变体将 Na+通透性、Cl-通透性和孔径恢复为野生型。Y67C 对甲硫磺酸酯修饰的可及性表明其侧链面向孔的内腔。在 claudin-10b 中，F66L 突变体降低了阳离子选择性，而 F66A 突变体丧失了孔电导。我们得出结论，claudin 阳离子孔域附近的保守芳族残基通过阳离子-π 相互作用和内腔空间位阻的双重作用有助于阳离子选择性。我们的发现为离子选择性如何在细胞旁孔中实现提供了新的见解。
