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.

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

“…More recently, ␦ ENaC, widely distributed throughout the brain and activated by protons, has been proposed to act as a pH sensor and to play a role in learning and memory in the human brain (53). Proton sensitivity of ␦␤␥ ENaC may be an important mechanism for integrating external ischemic signals in inflamed and hypoxic tissues (24,41). Our results indicate that ␦␤␥ ENaC could serve as a pH sensor to respond to noxious acidic aspiration.…”

Acid-sensing Ion Channels in Sensory Perception

“…The activation of ASIC1a during the metabolic acidosis accompanying experimental stroke has been proposed to contribute to neuronal death associated with brain ischemia (48). An interaction between ASIC3 and CFTR has also been described in airway epithelial cells (49), suggesting a putative unexpected functional link between these channels. The physiological relevance of most of these interactions, however, remains to be clarified.…”

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na+ Self-Inhibition of Epithelial Na+ Channels

“…To elicit self-inhibition, oocytes were held at 260 mV continuously while the bath solutions were switched quickly from ND1 and ND96 solutions, controlled with a SF-77B Perfusion Fast-Step (Warner Ins., Hamden, CT) fluidic exchange system and software pCLAMP as previously described (32,33). CPT-cGMP (AXXORA, LLC, San Diego, CA) stock solution (50 mM) was prepared in water and stored at 220 8 C. MTSET (Toronto Research Chemicals, Downsview, ON, Canada) was freshly prepared every 10 minutes.…”