Location, location, location: lessons from airway epithelial anion channels

Keeping our lungs healthy is a challenging job as every breath we take exposes our airways to myriad pathogenic organisms. The conducting airways have evolved a sophisticated system that prevents this by first capturing the nasty bugs in a sea of mucus and then propelling the entrapped microorganisms upwards towards the mouth, where they are either swallowed or spat out. The removal of the bug-laden mucus by mucociliary clearance (MCC) is a key process and the first line of defence against inhaled pathogens (Saint-Criq & Gray, 2017). Reduction in MCC leads to airway disease such as cystic fibrosis (CF). Here, dysfunction in the apically located CFTR chloride channel causes a severe reduction in the airways’ surface hydration. This makes the mucus very sticky and difficult to move, resulting in MCC failure and eventual chronic lung-destroying infections. While CFTR is clearly essential for MCC, there are other chloride channels that provide a partial back-up to CFTR. The most well studied is the calcium-activated chloride channel (CaCC), which is switched on by a rise in cytosolic calcium, conventionally caused by G-protein-coupled receptor agonists such as nucleotides and acetylcholine (Saint-Criq & Gray, 2017). For many years the identity of the airway CaCC was unknown until three papers in 2008 discovered that the TMEM16A gene encodes a CaCC which is expressed in airway epithelial cells, as well as many other cell types (Kunzelmann et al. 2019). These papers were the catalyst that led to a much better understanding of how the channel works at an atomic level as well as furthering our understanding of the physiological function of TMEM16A in diverse tissues, including epithelial, sensory and muscle (Kunzelmann et al. 2019). Not surprisingly, scientists realised the potential of TMEM16A as a target for treating important human disease such as CF, asthma, hypertension and gastrointestinal motility disorders. Several groups have reported the identification of ‘specific’ small molecule activators and potentiators, as well as a plethora of putative inhibitors of TMEM16A (Kunzelmann et al. 2019). One such TMEM16A modulator, known as Eact, was identified by the Verkman group back in 2011. This compound was shown to stimulate TMEM16A without changing cytosolic calcium and therefore thought to be a bone fide TMEM16A activator. Since then, several groups have reported conflicting results, either that Eact failed to activate TMEM16A, or that it activated TMEM16A, but this was due to an Eact-induced increase in cytosolic calcium. The paper in this issue of The Journal of Physiology by Genovese et al. (2019) has shed new light on the ‘Eact/TMEM16A conundrum’, and provides a very nice physiological explanation for these apparent discrepancies. The authors found that the response to Eact was cell model dependent. In cell lines expressing TMEM16A, Eact activated the channel. However, in fully differentiated airway epithelial cultures, Eact failed to activate TMEM16A, even though UTP did, with the latter response due to an increase in cytosolic calcium. Paradoxically, in these primary airway cultures, Eact did cause a sustained rise in intracellular calcium, but this only occurred in a small subset of cells, distinct from those that responded to UTP. Importantly, the Eact-induced increase in calcium was driven by the activation of the calcium influx channel, TRPV4. Satisfyingly, the reason why the Eact-induced increase in calcium in the primary airway cultures did not lead to TMEM16A activation was because TRPV4 and TMEM16A were expressed in completely different cell types. Even more surprising was the fact that Eact increased transepithelial short-circuit current due to CFTR activation, and this occurred through a TRPV4-dependent increase in cytosolic calcium. This novel result represents the first demonstration of a functional coupling between CFTR and TRPV4 in the airways. While this intriguing paper by Genovese has clearly shed new light on TMEM16A and CFTR regulation in the airways, some important questions remain. Which cell type(s) co-express TRPV4 and CFTR in the airways both in vitro and in vivo? Recent single cell RNA sequencing work has suggested that the majority of CFTR in the airways is expressed in a very specific, but minor, cell type termed the ionocyte (Plasschaert et al. 2018). It will therefore be important to establish if TRPV4 is expressed in ionocytes, and if so, whether TRPV4 activation leads to a calcium-dependent increase in CFTR activity. Indeed, how a TRPV4-mediated increase in calcium leads to CFTR activation requires further work. This is especially important because current models of CFTR regulation have somewhat ignored the role of intracellular calcium, despite evidence to the contrary (Billet & Hanrahan, 2013). What is the ‘physiological’ regulator of TRPV4 in the airways and can this be harnessed to improve CFTR activity in diseases involving CFTR dysfunction such as CF and chronic obstructive pulmonary disease (COPD)? Furthermore, are TRPV4 and CFTR co-localised in other epithelial tissues? This is an interesting point since TRPV4 is attracting considerable attention in other tissues, such as the gastrointestinal tract and neuro-muscular systems. Finally, because TMEM16A has so many important physiological roles in the body, there is a real need to find a specific TMEM16A activator. In the case of the airways, the majority of TMEM16A is located in mucus-secreting cells (Genovese et al. 2019), and its expression is casually associated with mucus cell hyperplasia and mucus secretion. A specific TMEM16A activator would therefore be a valuable tool to help decide if TMEM16A activation would be beneficial, or harmful, as a novel treatment for chronic airway diseases such as CF, COPD or asthma (Kunzelmann et al. 2019). Using the multi-methodological approach taken by Genovese et al. and combining these with functional read-outs of airway hydration and MCC should enable answers to these important questions. These are urgently needed for the future development