Stauffer BB , Cui G , Cottrill KA , Infield DT , McCarty NA .

T32 GM008602 NIGMS NIH HHS

                chronic obstructive pulmonary disease

	Figure 1. Inhibition results from enzymatic activity of SMase: (a) WT-human-CFTR currents elicited with 0.1 mM IBMX and a voltage step to −60 mV were inhibited by 2 μg/mL WT SMase but not by the inactive H322A mutant (n = 6). (b) Example traces show that inhibition rate was proportional to SMase concentration. For display, currents were normalized to the amplitude 1 minute prior to application of enzyme. SMase was applied from 0–10 minutes. Sensitivity to GlyH-101 applied at the end of the experiment shows that remaining currents represented uninhibited CFTR channels. (c) Summary data showing that fractional inhibition at 10 minutes was proportional to SMase concentration (n = 4–6).
	Figure 2. Inhibition is not membrane delimited: (a) CFTR currents recorded in the cell-attached configuration after activation with 0.2 mM IBMX were inhibited by 10 μg/ml SMase in the recording chamber suggesting activation of a signaling pathway that impinged upon the channels in the patch. (b) CFTR channels activated with 25 U PKA (expected activation of ~50% max)36, and recorded in the excised inside-out patch configuration were insensitive to 10 μg/mL SMase backfilled into the recording pipette. (c) Summary data showing fractional inhibition of currents in each configuration (p = 0.0021, unpaired t-test).
	Figure 3. Channel internalization does not underlie SMase-mediated inhibition of CFTR: (a) Molecular model of the exGFP-CFTR protein showing the GFP tag on the extracellular side of the plasma membrane (represented in grey). (b) Fluorescence measured from oocytes expressing exGFP-CFTR was sensitive to a drop in extracellular pH. Representative background-subtracted voltage from the photomultiplier tube during a brief exposure to pH 5.5 recording solution is shown. (c) Treatment with 10 μg/mL SMase for 10 minutes led to inhibition of currents (p = 0.0012) while the pH-dependent fluorescence change was not significantly affected (p = 0.4437, paired t-test). (d) In a cell-ELISA experiment, treatment with 10 μg/mL SMase for 10 minutes did not change the accessibility of the extracellular GFP on exGFP-CFTR to an anti-GFP antibody. Uninjected cells and cells expressing inGFP-CFTR showed minimal signal (n = 3).
	Figure 4. Inhibition is inversely proportional to the level of cAMP-mediated activation of CFTR: (a) An example trace shows the graded nature of CFTR channel function where total cellular current increases with increasing IBMX concentration. Maximum current was defined as that which was elicited by 1 mM IBMX + 50 μM FSK. (b) Summary data show the fractional current elicited with each activation condition. (c) Summary traces show the distinct kinetics associated with inhibition of channels by 1 μg/mL SMase following activation with 0.01, 0.1, or 1.0 mM IBMX (n = 4–5). (d) Summary data showing that fractional inhibition at 10 minutes was dependent upon activation condition (p = 0.0003, 1-way ANOVA).
	Figure 5. Increased channel activity imparts resistance to SMase-mediated inhibition: (a,b) The ATP hydrolysis-deficient K1250A-CFTR mutant was resistant to inhibition by 1 μg/mL SMase (p < 0.0001, unpaired t-test). (c,d) The phosphorylation-independent variant split-ΔR-CFTR was less sensitive to inhibition by SMase than was WT-CFTR in the presence of 0.1 mM IBMX (p = 0.034, unpaired t-test). (e,f) Potentiation by VX-770 protected CFTR from inhibition by SMase (p = 0.019, unpaired t-test).
	Figure 6. Mutations that disrupt channel pore gating had differential effects on sensitivity to SMase-mediated inhibition: (a,b) The R117H mutation, which disrupts both pore stability and NBD-dimer stability, increased sensitivity of CFTR to inhibition by SMase (p = 0.0057, unpaired t-test). (c–f) The D110R and R347D variants of CFTR were inhibited similarly to WT channels (p = 0.348 and p = 0.147, respectively, unpaired t-test).
	Figure 7. SMase pretreatment diminishes the effect of VX-770: (a) Application of VX-770 to WT-CFTR channels activated with 0.1 mM IBMX led to a 10.6 ± 3.6-fold increase in current in 2 minutes. (b) An example trace showing that application of 1 μM VX-770 led to a fractional recovery of only 0.01 ± 0.01 when applied after currents were inhibited by SMase. (c) Expansion of the area denoted by the grey box in part B shows details of VX-770-mediated potentiation kinetics. (d) Summary data showing that the effect of VX-770 on CFTR currents was diminished following inhibition by SMase as compared to IBMX-stimulated currents alone (p = 0.007, unpaired t-test): IPI is the current elicited by VX-770 + IBMX, II = is the initial current elicited by 0.1 mM IBMX.
	Figure 8. Basolateral treatment with SMase diminishes transepithelial currents in HBEs: (a) An example trace showing that pretreatment with basolateral WT SMase diminishes FSK-stimulated transepithelial currents in HBEs expressing WT CFTR. Scale bars: 10 μA/cm2, 10 mins. (b) Summary data showing significant difference in current amplitudes following basolateral WT SMase (p < 0.0001 for FSK and SMase treatments, 2-way ANOVA; asterisks indicate p < 0.05 for subsequent pairwise t-test analyses). Inset-Fractional inhibition is significantly higher in the presence of 100 nM FSK than 100 μM FSK (p = 0.0016, t-test). (c) An example trace showing that pretreatment of temperature-corrected CFHBEs with basolateral SMase leads to a similar reduction in maximal current. Scale bars: 2 μA/cm2, 20 mins. (d) Summary data showing that basolateral treatment with WT SMase reduces currents elicited by 100 μM FSK + 1 μM VX-770 (NHBE p = 0.04, CFHBE-27 °C p = 0.02; t-test).

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