Joana Almaça

TMEM16 proteins produce volume regulated chloride currents that are reduced in mice lacking TMEM16A

All vertebrate cells regulate their cell volume by activating chloride channels of unknown molecular identity, thereby activating regulatory volume decrease. We show that the Ca2+-activated Cl− channel TMEM16A together with other TMEM16 proteins are activated by cell swelling through an autocrine mechanism that involves ATP release and binding to purinergic P2Y2 receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca2+ and a Ca2+-independent mechanism engaging extracellular-regulated protein kinases (ERK1/2). The ability of epithelial cells to activate a Cl− conductance upon cell swelling, and to decrease their cell volume (regulatory volume decrease) was dependent on TMEM16 proteins. Activation of ICl,swell was reduced in the colonic epithelium and in salivary acinar cells from mice lacking expression of TMEM16A. Thus TMEM16 proteins appear to be a crucial component of epithelial volume-regulated Cl− channels and may also have a function during proliferation and apoptotic cell death.

ER-localized bestrophin 1 activates Ca2+-dependent ion channels TMEM16A and SK4 possibly by acting as a counterion channel.

Bestrophins form Ca2+-activated Cl− channels and regulate intracellular Ca2+ signaling. We demonstrate that bestrophin 1 is localized in the endoplasmic reticulum (ER), where it interacts with stromal interacting molecule 1, the ER-Ca2+ sensor. Intracellular Ca2+ transients elicited by stimulation of purinergic P2Y2 receptors in HEK293 cells were augmented by hBest1. The p21-activated protein kinase Pak2 was found to phosphorylate hBest1, thereby enhancing Ca2+ signaling and activation of Ca2+-dependent Cl− (TMEM16A) and K+ (SK4) channels. Lack of bestrophin 1 expression in respiratory epithelial cells of mBest1 knockout mice caused expansion of ER cisterns and induced Ca2+ deposits. hBest1 is, therefore, important for Ca2+ handling of the ER store and may resemble the long-suspected counterion channel to balance transient membrane potentials occurring through inositol triphosphate (IP3)-induced Ca2+ release and store refill. Thus, bestrophin 1 regulates compartmentalized Ca2+ signaling that plays an essential role in Best macular dystrophy, inflammatory diseases such as cystic fibrosis, as well as proliferation.

AMPK controls epithelial Na+ channels through Nedd4-2 and causes an epithelial phenotype when mutated

The metabolic sensor adenosine-monophosphate-activated kinase (AMPK) detects the cellular energy status and adjusts metabolic activity according to the cytosolic AMP to ATP ratio. Na+ absorption by epithelial Na+ channels (ENaC) is a highly energy-consuming process that is inhibited by AMPK. We show that the catalytic subunit α1 of AMPK inhibits ENaC in epithelial tissues from airways, kidney, and colon and that AMPK regulation of ENaC is absent in AMPKα1−/− mice. These mice demonstrate enhanced electrogenic Na+ absorption that leads to subtle changes in intestinal and renal function and may also affect Na+ absorption and mucociliary clearance in the airways. We demonstrate that AMPK uses the ubiquitin ligase Nedd4-2 to inhibit ENaC by increasing ubiquitination and endocytosis of ENaC. Thus, enhanced expression of epithelial Na+ channels was detected in colon, airways, and kidney of AMPKα1−/− mice. Therefore, AMPKα1 is a physiologically important regulator of electrogenic Na+ absorption and may provide a novel pharmacological target for controlling epithelial Na+ transport.

High-Content siRNA Screen Reveals Global ENaC Regulators and Potential Cystic Fibrosis Therapy Targets

Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na+ and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.

Regulation of the Epithelial Na+ Channel by the Protein Kinase CK2

CK2 is a ubiquitous, pleiotropic, and constitutively active Ser/Thr protein kinase that controls protein expression, cell signaling, and ion channel activity. Phosphorylation sites for CK2 are located in the C terminus of both β- and γ-subunits of the epithelial Na+ channel (ENaC). We examined the role of CK2 on the regulation of both endogenous ENaC in native murine epithelia and in Xenopus oocytes expressing rENaC. In Ussing chamber experiments with mouse airways, colon, and cultured M1-collecting duct cells, amiloride-sensitive Na+ transport was inhibited dose-dependently by the selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). In oocytes, ENaC currents were also inhibited by TBB and by the structurally unrelated inhibitors heparin and poly(E:Y). Expression of a trimeric channel lacking both CK2 sites (αβS631AγT599A) produced a largely attenuated amiloride-sensitive whole cell conductance and rendered the mutant channel insensitive to CK2. In Xenopus oocytes, CK2 was translocated to the cell membrane upon expression of wt-ENaC but not of αβS631AγT599A-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of αβγ-ENaC. Channels lacking the Nedd4-2 binding motif in β-ENaC (R561X, Y618A) no longer required the CK2 site for channel activity and siRNA-knockdown of Nedd4-2 eliminated the effects of TBB. This implies a role for CK2 in inhibiting the Nedd4-2 pathway. We propose that the C terminus of β-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes.

Regulation of Cl− secretion by AMPK in vivo

Previous in vitro studies suggested that Cl− currents produced by the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) are inhibited by the α1 isoform of the adenosine monophosphate (AMP)-stimulated kinase (AMPK). AMPK is a serine/threonine kinase that is activated during metabolic stress. It has been proposed as a potential mediator for transport–metabolism coupling in epithelial tissues. All previous studies have been performed in vitro and thus little is known about the regulation of Cl− secretion by AMPK in vivo. Using AMPKα1−/− mice and wild-type littermates, we demonstrate that phenformin, an activator of AMPK, strongly inhibits cAMP-activated Cl− secretion in mouse airways and colon, when examined in ex vivo in Ussing chamber recordings. However, phenformin was equally effective in AMPKα1−/− and wild-type animals, suggesting additional AMPK-independent action of phenformin. Phenformin inhibited CFTR Cl− conductance in basolaterally permeabilized colonic epithelium from AMPKα1+/+ but not AMPKα1−/− mice. The inhibitor of AMPK compound C enhanced CFTR-mediated Cl− secretion in epithelial tissues of AMPKα1−/− mice, but not in wild-type littermates. There was no effect on Ca2+-mediated Cl− secretion, activated by adenosine triphosphate or carbachol. Moreover CFTR-dependent Cl− secretion was enhanced in the colon of AMPKα1−/− mice, as indicated in Ussing chamber ex vivo and rectal PD measurements in vivo. Taken together, these data suggest that epithelial Cl− secretion mediated by CFTR is controlled by AMPK in vivo.

Regulation of ENaC biogenesis by the stress response protein SERP1

Cystic fibrosis lung disease is caused by reduced Cl− secretion along with enhanced Na+ absorption, leading to reduced airway surface liquid and compromised mucociliary clearance. Therapeutic strategies have been developed to activate cystic fibrosis transmembrane conductance regulator (CFTR) or to overcome enhanced Na+ absorption by the epithelial Na+ channel (ENaC). In a split-ubiquitin-based two-hybrid screening, we identified stress-associated ER protein 1 (SERP1)/ribosome-associated membrane protein 4 as a novel interacting partner for the ENaC β-subunit. SERP1 is induced during cell stress and interacts with the molecular chaperone calnexin, thus controlling early biogenesis of membrane proteins. ENaC activity was measured in the human airway epithelial cell lines H441 and A549 and in voltage clamp experiments with ENaC-overexpressing Xenopus oocytes. We found that expression of SERP1 strongly inhibits amiloride-sensitive Na+ transport. SERP1 coimmunoprecipitated and colocalized with βENaC in the endoplasmic reticulum, together with the chaperone calnexin. In contrast to the inhibitory effects on ENaC, SERP1 appears to promote expression of CFTR. Taken together, SERP1 is a novel cochaperone and regulator of ENaC expression.

Functional Genomics Assays to Study CFTR Traffic and ENaC Function

As several genomes have been sequenced, post-genomic approaches like transcriptomics and proteomics, identifying gene products differentially expressed in association with a given pathology, have held promise both of understanding the pathways associated with the respective disease and as a fast track to therapy. Notwithstanding, these approaches cannot distinguish genes and proteins with mere secondary pathological association from those primarily involved in the basic defect(s). New global strategies and tools identifying gene products responsible for the basic cellular defect(s) in CF pathophysiology currently being performed are presented here. These include high-content screens to determine proteins affecting function and trafficking of CFTR and ENaC.