Cyclic AMP and its catalogs, particularly the cell permeably compounds, are designed to modulate cAMP-PKA signal pathway. Native ENaC channels were found to be activated by cell permeable cpt-cAMP in pneumocytes [24
], endometrium [55
], cortical collecting epithelium [56
], and peripheral blood lymphocytes [25
]. Heterologous ENaC activity was also augmented by cpt-cAMP in MDCK and oocytes [23
]. The following evidence provides a novel mechanism for cpt-cAMP mediated acute activation of both heterologous and native human ENaC channels. First, external but not internal cpt-cAMP activates ENaC in seconds in a dose-dependent and reversible manner. Second, the specific chlorophenylthio (cpt) groups are required but not other more specific PKA activators with more potency in cell permeability [12
]. Third, cpt-cAMP may not recruit new channel proteins to increase electrically detectable channel density at cell surface, as the stimulatory effects were also observed in outside-out patches, a cell-free model without functional machinery for exocytosis, by our and other groups [12
]. Forth, mutations both enhancing and eliminating self-inhibition affect the effects of cpt-cAMP on ENaC. Fifth, self-inhibition modifying maneuvers, including proteolytic cleavage and thiol modification, prevent the cpt-cAMP-mediated activation.
So far, the identified critical amino acid residues in each subunit, which either boost (i.e.,
) or abolish (i.e.,
) self-inhibition, reside in extracellular finger, thumb, and palm domains and are the superficial domains of complexes. The location of these domains determines the susceptibility to cpt-cAMP. We cannot, however, conclude that cpt-cAMP directly modifies these self-inhibition domains, for the fact that external binding sites for H+
ions in crystallized structure of ENaC/DEG proteins [5
], which also interrupt self-inhibition, locate differently. Because the unitary conductance was not altered by cpt-cAMP, the Na+
conduction sites of ENaC seem not be modified. Self-inhibition appears initiated from external finger and thumb domains, palm domains are involved in secondarily conducting the signal of Na+
concentration. The three-dimensional structural alteration of palm domains may subsequently result in the process of inactivation of channel gating. Eventually channel activity is reduced. Although we do not know if the self-inhibition modifying reagents augment the Na+
binding and gating processes, which has not been understood yet so far, it is conceivable to infer that cpt-cAMP and other compounds may be capable of down-regulating the transition of Na+
signal and resultant gating kinetics.
Few channel proteins, including cyclic nucleotide-gated channels (CNG), contain intracellular nucleotide-binding domains (NBD) [59
]. It is worthy to note that these NBD domains do not require for specific structures of nucleotides, even the parental compounds, e.g.
, cAMP and cGMP, can bind to the channel proteins [59
]. Requirement of specific cpt
groups in cAMP analogs to activate ENaC implys that cpt-cAMP may not dock into an external site with similar three-dimensional structure to NBD domains. Further studies are required to identify the docking site for cpt-cAMP and other analogs.
Domains in the extracellular loop in human δ ENaC facilitate activation of ENaC by cpt-cAMP via unknown mechanisms. These domains may form a more efficient pocket for cpt-cAMP binding, but our dose-response data do not support this notion. The estimated EC50 value of δβγ ENaC is apparently much greater than those for αβγ and δαβγ channels (). Diversities in extracellular loop may also contribute to the species-dependent responses to cpt-cAMP.
The relief of self-inhibition by cpt-cAMP contributes to the acute effects, in addition to the well-known cAMP-PKA signal pathway. Although cpt-cAMP is not a natural endogenous cellular signal molecule, and the EC50
values are much greater than that for activating PKA, without doubt, it can be delivered into epithelial organs, such as alveolar spaces, as a potent pharmaceutical maneuver to acutely release edematous disorders. The clinical relevance, in fact, has been implicated in the up-regulation of ENaC in autosomal recessive pseudohypoaldosteronism type 1 (PHA-1) [25
] and edematous lung injury [24