Molecular Mechanisms That Underlie The [Cl-]I-Sensitive Kinases-Mediated Regulation of Cftr Anion Selectivity

Abstract

MoleculAr MechAnisMs ThAT underlie The [cl-]i-sensiTive KinAses-MediATed regulATion of cfTr Anion selecTiviTy I. Jun; J. Jang; J. Jung; Y. Kim; and M.G. Lee Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Korea; and Department of Hematology, Yonsei University College of Medicine, Seoul 120-752, Korea Introduction: CFTR is cAMP activated anion channel which secretes chloride and bicarbonate in airway, exocrine pancreas, intestine, and genitourinary systems. Human pancreas secretes pancreatic juice which contains as much as 140 mM bicarbonate (HCO3-) via CFTR. Recently, we have shown that [Cl-]i-sensitive activation of WNK1OSR1/SPAK pathway plays a critical role in pancreatic HCO3secretion by increasing the bicarbonate permeability (PHCO3/PCl) of CFTR. However, how [Cl-]i-sensitive kinases modulate PHCO3/ PCl of CFTR remains elusive. Material and Methods: Overexpression and knockdown of each kinase in HEK293T cells were performed. Using patch clamp, we measured permeability of bicarbonate and halide ions. We did pulldown assay between truncated WNK1 and CFTR at 150mM and 0mM Clconcentrations. Results: WNK1 affects permeability of other anions as well as bicarbonate in patch clamp recordings. Especially, the interval of relative permeabilities (Px/PCl) between each anion was greatly narrowed by WNK1. Consequently, WNK1 increased the dielectric constant of the hypothetical selectivity filter of CFTR. And we measured the pore size of CFTR at each state. WNK1 makes CFTR pore size larger. We figured out that WNK1 kinase domain binds to CFTR and truncated forms of WNK1 respond to low Clconcentrations. Conclusions: These findings suggest that WNK1 increases the bicarbonate permeability of CFTR by modulating the polarizability of anion selectivity filter and provide insight into the fundamental question of how ion selectivity of anion channels can be regulated by cytosolic signaling at the molecular level. Furthermore, WNK1 senses low [Cl-]i, then kinase domain is exposed and binds to CFTR.