title: Interregulation of proton-gated Na(+) channel 3 and cystic fibrosis transmembrane conductance regulator.
abstract: Proton-gated Na(+) channels (ASIC) are new members of the epithelial sodium channel/degenerin gene family. ASIC3 mRNA has been detected in the homogenate of pulmonary tissues. However, whether ASIC3 is expressed in the apical membranes of lung epithelial cells and whether it regulates cystic fibrosis transmembrane conductance regulator (CFTR) function are not known at the present time. Using reverse transcription-PCR, we found that the ASIC3 mRNA was expressed in the human airway mucosal gland (Calu-3) and human airway epithelial (16HBE14o) cells. Indirect immunofluorescence microscopy revealed that ASIC3 was co-segregated with CFTR in the apical membranes of Calu-3 cells. Proton-gated, amiloride-sensitive short circuit Na(+) currents were recorded across Calu-3 monolayers mounted in an Ussing chamber. In whole-cell patch clamp studies, activation of CFTR channels with cAMP reduced proton-gated Na(+) current in Calu-3 cells from -154 +/- 28 to -33 +/- 16 pA (n = 5, p < 0.05) at -100 mV. On the other hand, cAMP-activated CFTR activity was significantly inhibited following constitutive activation of putative ASIC3 at pH 6.0. Immunoassays showed that both ASIC3 and CFTR proteins were expressed and co-immunoprecipitated mutually in Calu-3 cells. Similar results were obtained in human embryonic kidney 293T cells following transient co-transfection of ASIC3 and CFTR. Our results indicate that putative CFTR and ASIC3 channels functionally interact with each other, possibly via an intermolecular association. Because acidic luminal fluid in the cystic fibrosis airway and lung tends to stimulate ASIC3 channel expression and activity, the interaction of ASIC3 and CFTR may contribute to defective salt and fluid transepithelial transport in the cystic fibrotic pulmonary system.
authors.affiliation: Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama 35205, USA.
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Su
authors.forename: Qingnan
authors.initials: Q
authors.lastname: Li
authors.forename: Kedar
authors.initials: K
authors.lastname: Shrestha
authors.forename: Estelle
authors.initials: E
authors.lastname: Cormet-Boyaka
authors.forename: Lan
authors.initials: L
authors.lastname: Chen
authors.forename: Peter R
authors.initials: PR
authors.lastname: Smith
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Sorscher
authors.forename: Dale J
authors.initials: DJ
authors.lastname: Benos
authors.forename: Sadis
authors.initials: S
authors.lastname: Matalon
authors.forename: Hong-Long
authors.initials: HL
authors.lastname: Ji
mesh_terms: D062565:Acid Sensing Ion Channels; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation; D006801:Humans; D008168:Lung / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000502:physiology*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011522:Protons; D012333:RNA, Messenger / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology
chemical_list: C112231:ASIC3 protein, human; D062565:Acid Sensing Ion Channels; C505032:CFTR protein, human; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011522:Protons; D012333:RNA, Messenger; D015222:Sodium Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP