TRPV4 and purinergic receptor signalling pathways are separately linked in airway epithelia to CFTR and TMEM16A chloride channels

Abstract

Key points Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+‐permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca2+ elevation by TRPV4 stimulation leads to CFTR channel activation. Abstract TMEM16A is a Ca2+‐activated Cl− channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short‐circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca2+‐permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4 stimulation induced activation of the CFTR Cl− channel. Our study reveals the existence of separate Ca2+ signalling pathways linked to different Cl− secretory processes.

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

“…Similarly, Eact or GSK1016790 did not increase whole cell currents in patch clamp experiments ( Figure 2D,E). This may correspond to the results of a previous report, demonstrating Eact as an activator of Ca 2+ permeable TRPV4 channels, rather than a direct activator of TMEM16A [16]. Although TRPV4 is expressed in HT 29 cells, the rise in intracellular Ca 2+ induced by Eact was insignificant, particularly when compared with the effect of ATP on intracellular Ca 2+ ([Ca 2+ ] i ) ( Figure 2H,I).…”

“…Similar differential activation of endogenous vs. exogenous TMEM16A was observed for the compound Eact. Eact has been reported as an activator of TMEM16A [15], but was suggested recently to activate TMEM16A indirectly by inducing Ca 2+ influx through activation of TRPV1/4 [16,45]. Currently, Eact is one of the few accessible TMEM16A activators and therefore deserved further analysis: (i) Although TRPV4 is expressed in HT29 cells, Eact and the TRPV4-activator GSK1016790 did not activate TMEM16A.…”

“…Similarly, quite different molecules were reported to activate TMEM16A [2], with controversial reports on their efficacy and mechanism of action. Some molecules, such as the putative TMEM16A-activator Eact, were suspected to increase intracellular Ca 2+ rather than directly activating TMEM16A [15,16].…”

“…Thus, TMEM16A modulates its own local environment in a way so that it is activated through locally concentrated Ca 2+ release [24]. In many previous studies as well as ongoing work, inhibitors and activators of TMEM16A are used, although they may also affect other TMEM16 paralogs or may exert additional effects on Ca 2+ regulating proteins [16,25]. Here we confirm the role of TMEM16A for intracellular Ca 2+ signaling and show for a number of compounds that they also affect TMEM16F, a paralog of TMEM16A.…”

TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis

“…To our knowledge, this is the first report of the discovery and efficacy of TMEM16A potentiator compounds. Compounds initially reported to be direct modulators of the channel (e.g., E act ) (38) have subsequently been shown to act indirectly via elevation of [Ca 21 ] i (39,40), negating their utility for validating CaCC function due to the numerous other cellular effects elevation of [Ca 21 ] i will trigger (e.g., goblet cell exocytosis and bronchospasm). In contrast, ETX001 potentiates the opening of the TMEM16A channel (i.e., it enhances the magnitude of the current in the presence of physiologically regulated [Ca 21 ] i ), but ETX001 does not…”