Étude moléculaire des mécanismes d’action
de potentiateurs du canal CFTR sur le canal KCa3.1

Airway epithelial cells are the site of Cl- secretion through CFTR. Cystic fibrosis is a fatal genetic disease caused by mutations in CFTR. The most frequent mutation in North America (∆F508) results in impaired maturation and altered channel gating of the protein. In the last years, several small molecules were identified by high throughput screening that could restore mutated CFTR function. Compounds addressing CFTR gating defects are referred to as potentiators. The basolateral K+ channel KCa3.1 has been documented to play a prominent role in establishing a suitable driving force for CFTR-mediated Clsecretion in airway epithelial cells. It has been shown, for example, that the application of 1-EBIO on T84 monolayers results in a sustained increase of Clsecretion and that this current can be reversed by application of CTX, a KCa3.1 inhibitor (Devor et al., 1996). Thus, in a global approach of transepithelial transport, the research for physiologically relevant CFTR potentiators should also consider their effects on the KCa3.1 channel. Electrophysiological patch clamp measurements and channel structural modification by site directed mutagenesis were used to characterize the action of CFTR potentiators on KCa3.1 and study their molecular mode of action. In this work we present results on the effects on KCa3.1 of several CFTR potentiators of different structures. We observed that the CFTR potentiators genistein, curcumin, SF-03 and VRT-532 could inhibit KCa3.1 activity at concentrations known to activate CFTR. Our results suggest that SF- 03 could act indirectly on KCa3.1 through a mechanism involving an accessory protein. Curcumin would also have an indirect inhibitory effect, probably mediated by the plasma membrane, as documented for other ion channels. A detailed study of VRT-532 revealed that this molecule has access to its binding site in a state independent manner, and is poorly effective on the V282G mutant of KCa3.1, which is constitutively active. These results suggest that VRT-532 could act through the CaM/KCa3.1 complex and require the presence of Ca2+ to inhibit channel activity. In contrast, CBIQ, another CFTR potentiator, succeeded to activate KCa3.1. Our results in single channel show that CBIQ vii destabilizes a non conducting state of the channel. We also showed that this molecule increases the apparent Ca2+ affinity as well as the channel open probability, even in saturating Ca2+ conditions. Experiences in which Ba2+ was used as a probe were also performed to determine if the action mechanism of CBIQ involves an effect on the selectivity filter. Our results showed that Ba2+ could displace CBIQ from its interacting site, suggesting that the increases in channel activity induced by CBIQ could result from a change in the energetics of the channel at the level of the selectivity filter. On the basis of our results, we conclude that CBIQ, a CFTR potentiator, could activate KCa3.1 by destabilizing a non conducting state of the channel, probably through an action near the selectivity filter region. Also, CFTR potentiators having an inhibitory effect on KCa3.1 are likely to act through the plasmic membrane, the CaM/KCa3.1 interaction or an accessory protein of the channel. In a perspective of future treatments for CF, our results indicate that CBIQ could be an efficient potentiator since this product stimulates KCa3.1 as well as CFTR. Conversly, the VRT-532 and SF-03 could be less efficient than on CFTR alone, due to their inhibition of KCa3.1.

Keyphrases: drug screening, CBIQ, calcium-activated potassium channel, cystic fibrosis