Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition

Department of Biochemistry, University of Texas Health Science Center at Tyler, TX, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 03/2011; 1808(7):1818-26. DOI: 10.1016/j.bbamem.2011.03.004
Source: PubMed


External Na(+) self-inhibition is an intrinsic feature of epithelial sodium channels (ENaC). Cpt-cAMP regulates heterologous guinea pig but not rat αβγ ENaC in a ligand-gated manner. We hypothesized that cpt-cAMP may eliminate the self-inhibition of human ENaC thereby open channels. Regulation of self-inhibition by this compound in oocytes was analyzed using the two-electrode voltage clamp and Ussing chamber setups. External cpt-cAMP stimulated human but not rat and murine αβγ ENaC in a dose- and external Na(+) concentration-dependent fashion. Intriguingly, cpt-cAMP activated human δβγ more potently than αβγ channels, suggesting that structural diversity in ectoloop between human α, δ, and those ENaC of other species determines the stimulating effects of cpt-cAMP. Cpt-cAMP increased the ratio of stationary and maximal currents. Mutants having abolished self-inhibition (β(ΔV348) and γ(H233R)) almost completely eliminated cpt-cAMP mediated activation of ENaC. On the other hand, mutants both enhancing self-inhibition and elevating cpt-cAMP sensitivity increased the stimulating effects of the compound. This compound, however, could not activate already fully opened channels, e.g., degenerin mutation (αβ(S520C)γ) and the proteolytically cleaved ENaC by plasmin. Cpt-cAMP activated native ENaC to the same extent as that for heterologous ENaC in human lung epithelial cells. Our data demonstrate that cpt-cAMP, a broadly used PKA activator, stimulates human αβγ and δβγ ENaC channels by relieving self-inhibition.

Download full-text


Available from: Hong-Long Ji, Feb 17, 2014
  • Source
    • "For example, cpt-cGMP activates human but not murine ENaC activity [22]. The responses to cpt-cAMP are different between human and murine ENaC, too [26]. It is conceivable that ketamine may reduce ENaC activity in human lungs but may not alter rat counterpart. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ketamine is a broadly used anaesthetic for analgosedation. Accumulating clinical evidence shows that ketamine causes pulmonary edema with unknown mechanisms. We measured the effects of ketamine on alveolar fluid clearance in human lung lobes ex vivo. Our results showed that intratracheal instillation of ketamine markedly decreased the reabsorption of 5% bovine serum albumin instillate. In the presence of amiloride (a specific ENaC blocker), fluid resolution was not further decreased, suggesting that ketamine could decrease amiloride-sensitive fraction of AFC associated with ENaC. Moreover, we measured the regulation of amiloride-sensitive currents by ketamine in A549 cells using whole-cell patch clamp mode. Our results suggested that ketamine decreased amiloride-sensitive Na+ currents (ENaC activity) in a dose-dependent fashion. These data demonstrate that reduction in lung ENaC activity and lung fluid clearance following administration of ketamine may be the crucial step of the pathogenesis of resultant pulmonary edema.
    Full-text · Article · Oct 2011 · BioMed Research International
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Salt absorption via alveolar epithelial Na(+) channels (ENaC) is a critical step for maintaining an airspace free of flooding. Previously, we found that 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate-Na (CPT-cGMP) activated native and heterologous ENaC. To investigate the potential pharmacological relevance, we applied this compound intratracheally to human lungs and found that ex vivo alveolar fluid clearance was increased significantly. Furthermore, this compound eliminated self-inhibition in human lung H441 cells and in oocytes expressing human αβγ but not δβγ channels. To further elucidate this novel mechanism, we constructed mutants abolishing (β(ΔV348) and γ(H233R)) or augmenting (α(Y458A) and γ(M432G)) self-inhibition. The mutants eliminating self-inhibition lost their responses to CPT-cGMP, whereas those enhancing self-inhibition facilitated the stimulatory effects of this compound. CPT-cGMP was unable to activate a high P(o) mutant (β(S520C)) and plasmin proteolytically cleaved channels. Our data suggest that elimination of self-inhibition of αβγ ENaC may be a novel mechanism for CPT-cGMP to stimulate salt reabsorption in human lungs.
    Full-text · Article · May 2011 · American Journal of Respiratory Cell and Molecular Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Extracellular Zn2+ activates the epithelial Na+ channel (ENaC) by relieving Na+ self-inhibition. However, a biphasic Zn2+ dose response was observed, suggesting that Zn2+ has dual effects on the channel (i.e. activating and inhibitory). To investigate the structural basis for this biphasic effect of Zn2+, we examined the effects of mutating the 10 extracellular His residues of mouse γENaC. Four mutations within the finger subdomain (γH193A, γH200A, γH202A, and γH239A) significantly reduced the maximal Zn2+ activation of the channel. Whereas γH193A, γH200A, and γH202A reduced the apparent affinity of the Zn2+ activating site, γH239A diminished Na+ self-inhibition and thus concealed the activating effects of Zn2+. Mutation of a His residue within the palm subdomain (γH88A) abolished the low-affinity Zn2+ inhibitory effect. Based on structural homology with acid-sensing ion channel 1, γAsp516 was predicted to be in close proximity to γHis88. Ala substitution of the residue (γD516A) blunted the inhibitory effect of Zn2+. Our results suggest that external Zn2+ regulates ENaC activity by binding to multiple extracellular sites within the γ-subunit, including (i) a high-affinity stimulatory site within the finger subdomain involving His193, His200, and His202 and (ii) a low-affinity Zn2+ inhibitory site within the palm subdomain that includes His88 and Asp516.
    Preview · Article · Aug 2012 · Journal of Biological Chemistry
Show more