Expression of cyclic nucleotide-gated cation channels in airway epithelial cells

Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with halphaCNC1. Whole cell currents stimulated by 8-Br-cGMP in HK293 cells transfected with halphaCNC1 or A549 cells are carried by inward sodium and outward potassium with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected with halphaCNC1 are inhibited by l-cis-diltiazem with an IC(50) of 154 microm, by 2',4'-dichlorobenzamil with an IC(50) of 50 microm and by amiloride with an IC(50) of 133 microm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an IC(50) of 87 microm, by 2'4'-dichlorobenzamil with an IC(50) of 38 microm and by amiloride with an IC(50) of 32 microm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both alphaCNC1 and betaCNC subunits are present in A549 cells and the presence of the betaCNC subunit, may as previously reported, increase the affinity of these channel blockers compared to the halphaCNC1 subunit alone. The mRNA of two other isoforms of this channel, CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC(50) of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption via cyclic nucleotide-gated cation channels in airway cells.