Figure 1.
Cell surface cleavage of γ-ENaC was assessed biochemically using purified AP and AP Inh. A, a cartoon schematic of the ENaC channel is shown with the α-, β-, and γ-subunits labeled. The protease sensitive tract in γ-ENaC is shown as a gray box and spans residues 139 to 181 [49]. B, cell surface cleavage of ENaC was assessed by western blotting. Total ENaC is shown for control cells (CNTL), AP treated cells, or cell co-treated with AP and AP Inh from whole cell lysates, Whole Cell. The cell surface ENaC was assessed after biotinylation and streptavidin capture, Cell Surface. From the cell surface pool of ENaC the full length (**) and cleaved (*) forms of the channel are observed. Actin was used as a loading control and was not observed in biotinylated samples.
Figure 2.
Folding and activation of serralysins from Pseudmonas aeruginosa and Serratia marcescens.
Protease activities were assessed for both AP and SmP using a fluorescently-conjugated casein substrate, A, or a fluorescent metalloprotease peptide substrate, B. Addition of 2 mM Ca2+ to the refolding buffers resulted in robust folding and activation of both AP and SmP, as measured by protease activity. C, protease activity was assessed immediately after refolding or following a 24 hour incubation at 37°C using the metalloprotease peptide substrate. Buffer controls are subtracted from all samples and the activities are normalized to AP, A, B, or protease activity measured immediately after refolding, C. Data shown are mean ± standard deviation from n = 6 experiments. P<0.001, *.
Figure 3.
Binding and inhibition of AP Inh to AP and SmP.
Inhibition of protease activity was evaluated using purified AP and the Pseudomonas aeruginosa AprI inhibitor. A, analytical gel filtration was used to assess complex formation between the refolded AP and the purified inhibitor. Representative chromatograms are shown for protease, black line, the inhibitor, dark gray line, and the complex, light gray line. B, activity assays were performed using the fluorescent peptide substrate for AP. C, analytical gel filtration was used to assess complex formation between the refolded SmP and the purified inhibitor. Representative chromatograms are shown for the protease, black line, the inhibitor, dark gray line, and the complex, light gray line. D, activity assays were performed using the fluorescent peptide substrate for AP. Data shown are mean ± standard deviation for n = 5 experiments. P<0.001, *; n.s., not significant.
Figure 4.
ENaC regulation in mCCD cells by the proteases and the AP inhibitor.
The activation of ENaC was assessed using mCCD cells by recording short circuit currents in Ussing chambers. A, a representative trace showing the activation of ENaC in mCCD cells is shown. B, a representative trace of AP treatment after AP Inh pretreatment is shown. C, quantification of the relative currents seen after treatment with either AP or AP and AP Inh for mCCD cells are shown. Basal currents are normalized to 100%. D, a representative trace showing the activation of ENaC by SmP in mCCD cells is shown. E, a representative trace of SmP after AP Inh pretreatment is shown. F, quantification of the relative currents seen after treatment with either SmP or SmP and AP Inh for mCCD cells is shown. G, the kinetics of ENaC activation are shown under conditions of maximal stimulation using trypsin, closed circles, AP, closed triangles, or SmP, open circles. Time constants of ENaC activation are shown for each protease. Serial addition of proteases and inhibitors is shown by the bars above the current trace and labelled. The period in the recording where the apical chamber was washed to remove the serine protease inhibitor, camostat, is indicated with a W. Data shown are representative of at least five experiments. P<0.004, *.
Figure 5.
ENaC regulation in HBE cells by the proteases and the AP inhibitor.
The activation of ENaC was assessed in HBE cells by recording short circuit currents. A, a representative trace showing the activation of ENaC in HBE cells is shown. B, a representative trace of AP treatment after AP Inh pretreatment is shown. C, quantification of the relative currents seen after treatment with either AP or AP and AP Inh for HBE cells are shown (n = 9). Basal currents are normalized to 100%. D, a representative trace showing the activation of ENaC by SmP in HBE cells is shown. E, a representative trace of SmP after AP Inh pretreatment is shown. F, quantification of the relative currents seen after treatment with either SmP or SmP and AP Inh for HBE cells is shown (n = 5). G, the kinetics of ENaC activation are shown under conditions of maximal stimulation using trypsin, closed circles, AP, closed tringles, or SmP, open circles. Time constants of ENaC activation are shown for each protease. Serial addition of proteases and inhibitors is shown by the bars above the current trace and labelled. The period in the recording where the apical chamber was washed to remove the serine protease inhibitor, camostat, is indicated with a W. Data shown are representative of at least seven experiments. P<0.001, *.