ENV59-GP3 inhibition of recombinant human KCa3.1
channels stably expressed in HEK293 cells
Figure 4D-E summarizes the results for ENV59-GP3 with the curve
representing fits to the Hill equation, revealing IC50value of 43µM for ENV59-GP3. To confirm that the inhibiting effect of
ENV59-GP3 peptide represent an interaction with the
KCa3.1 channel and not an indirect effect, we evaluated
the peptide’s effect on the recombinant hKCa3.1 stably
over-expressed in HEK293 cells (Figure 5 A-D), a cell type which does
not naturally express any calcium activated potassium channels. As
observed in the previous experiment with THP-1 cells, after the initial
transient rise in activity, the KCa3.1 current
stabilized. Initially we blocked the current by using Charybdotoxin-a
very potent KCa3.1 blocker [26]. The ENV59-GP3
peptide exert a qualitatively similar reversible inhibition, whereas the
control peptide Flu ISU did not show any effect, in accordance with its
lack of effect in the [Ca2+] assay (Figure 5 C-D).
Finally, the NS309 activator was applied yielding the expected specific
increase in KCa3.1 current as also observed for the
THP-1 channel.