Characterization of a novel splice variant of δ ENaC subunit in human lungs

Abstract

Salt absorption via apical epithelial sodium channels (ENaC) is a critical rate-limiting process in maintaining airway and lung lining fluid at the physiological level. ␦ ENaC (termed ␦1 in this article) has been detected in human lung epithelial cells in addition to ␣, ␤, and ␥ subunits (Ji HL, Su XF, Kedar S, Li J, Barbry P, Smith PR, Matalon S, Benos DJ. J Biol Chem 281: [8233][8234][8235][8236][8237][8238][8239][8240][8241] 2006; Nie HG, Chen L, Han DY, Li J, Song WF, Wei SP, Fang XH, Gu X, Matalon S, Ji HL, J Physiol 587: [2663][2664][2665][2666][2667][2668][2669][2670][2671][2672][2673][2674][2675][2676] 2009) and may contribute to the differences in the biophysical properties of amiloride-inhibitable cation channels in pulmonary epithelial cells. Here we cloned a splicing variant of the ␦1 ENaC, namely, ␦2 ENaC in human bronchoalveolar epithelial cells (16HBEo). ␦2 ENaC possesses 66 extra amino acids attached to the distal amino terminal tail of the ␦1 ENaC. ␦2 ENaC was expressed in both alveolar type I and II cells of human lungs as revealed by in situ hybridization and real-time RT-PCR. To characterize the biophysical and pharmacological features of the splicing variant, we injected Xenopus oocytes with human ENaC cRNAs and measured whole cell and single channel currents of ␦1␤␥, ␦2␤␥, and ␣␤␥ channels. Oocytes injected with ␦2␤␥ cRNAs exhibited whole cell currents significantly greater than those expressing ␦1␤␥ and ␣␤␥ channels. Single channel activity, unitary conductance, and open probability of ␦2␤␥ channels were significantly greater compared with ␦1␤␥ and ␣␤␥ channels. In addition, ␦2␤␥ and ␦1␤␥ channels displayed significant differences in apparent Na ϩ affinity, dissociation constant for amiloride (Ki amil ), the EC50 for capsazepine activation, and gating kinetics by protons. Channels comprising of this novel splice variant may contribute to the diversities of native epithelial Na ϩ channels.biophysics; electrophysiology; epithelial sodium channels; in situ hybridization; splicing variant THE LINING FLUID ALONG airway and alveolar epithelial surface is precisely regulated by apical and basolateral ion transport systems. The epithelial sodium channels (ENaC) are the main conduits for the entry of sodium ions (Na ϩ ) from the luminal fluid into the cell interiors (12,15,34). Native lung amilorideinhibitable cation channels have been functionally classified into three groups: highly selective for Na ϩ over other cations, moderate, and nonselective channels (12,15,34). The highly selective cation channels have been thought to consist of heteromultimeric ␣␤␥ ENaC subunits (6). The molecular basis of the other two groups, however, remains in dispute. Channels comprised of ␣ ENaC alone and channels consisting of the ␣␤␥ three subunits expressed in nonpolarized epithelial cells behave as nonselective phenotypes (23,33,56). Recently another ␦ ENaC variant was detected in the human brain and was thought to act as a pore-forming subunit (44). The distribution of ␦ ENaC in other nonepithelial tissues, suc...

Novel concepts of acute lung injury and alveolar-capillary barrier dysfunction

“…Of note, children with genetic deletion of genes encoding for ␦ ENaC are prone to respiratory infections and nasal congestion during winter (113). Most recently, a novel splice variant of ␦ ENaC, termed ␦2 ENaC, has been characterized, which displays divergent features from the ␦1 counterpart in biophysics and pharmacology regulation (274).…”

Receptor for Advanced Glycation End-Products Regulates Lung Fluid Balance via Protein Kinase C–gp91phox Signaling to Epithelial Sodium Channels

“…The a subunit is abundantly expressed at the cell membrane, and is capable of forming amiloride-sensitive current alone, or alongside b-and g-ENaC subunits (14,15). A d subunit has recently been characterized in the human lung, albeit the functional role of this subunit remains unclear (16,17). Together, these subunits play an important role in reabsorbing sodium from the epithelial lining fluid (ELF), which is then extruded transcellularly via the basolaterally located Na…”

Alveolar Epithelium and Fluid Transport

“…Finally, there is a newly cloned δ subunit of ENaC that has been identified in human lungs and may play a role in fluid balance in the lung. 33 …”