F508del-CFTR increases intracellular Ca(2+) signaling that causes enhanced calcium-dependent Cl(-) conductance in cystic fibrosis.

In many cells, increase in intracellular calcium ([Ca(2+)](i)) activates a Ca(2+)-dependent chloride (Cl(-)) conductance (CaCC). CaCC is enhanced in cystic fibrosis (CF) epithelial cells lacking Cl(-) transport by the CF transmembrane conductance regulator (CFTR). Here, we show that in freshly isolated nasal epithelial cells of F508del-homozygous CF patients, expression of TMEM16A and bestrophin 1 was unchanged. However, calcium signaling was strongly enhanced after induction of expression of F508del-CFTR, which is unable to exit the endoplasmic reticulum (ER). Since receptor-mediated [Ca(2+)](i) increase is Cl(-) dependent, we suggested that F508del-CFTR may function as an ER chloride counter-ion channel for Ca(2+). This was confirmed by expression of the double mutant F508del/G551D-CFTR, which remained in the ER but had no effects on [Ca(2+)](i). Moreover, F508del-CFTR could serve as a scavenger for inositol-1,4,5-trisphosphate [IP3] receptor binding protein released with IP(3) (IRBIT). Our data may explain how ER-localized F508del-CFTR controls intracellular Ca(2+) signaling.

Stimulation of renin secretion by catecholamines is dependent on adenylyl cyclases 5 and 6.

The sympathetic nervous system stimulates renin release from juxtaglomerular cells via the ß-adrenoreceptor-cAMP pathway. Recent in vitro studies have suggested that the calcium-inhibited adenylyl cyclases (ACs) 5 and 6 possess key roles in the control of renin exocytosis. To investigate the relative contribution of AC5 and AC6 to the regulation of renin release in vivo we performed experiments using AC5 and AC6 knockout mice. Male AC5(-/-) mice exhibited normal plasma renin concentrations, renal renin synthesis (mRNA and renin content), urinary volume, and systolic blood pressure. In male AC6(-/-) mice, plasma renin concentration (AC6(-/-): 732 ± 119; AC6 (+/+): 436 ± 78 ng of angiotensin I per hour*mL(-1); P<0.05), and renin synthesis were stimulated associated with an increased excretion of dilute urine (1.55-fold; P<0.05) and reduced blood pressure (-10.6 mm Hg; P<0.001). Stimulation of plasma renin concentration by a single injection of the ß-adrenoreceptor agonist isoproterenol (10 mg/kg IP) was significantly attenuated in AC5(-/-) (male: -20%; female: -33%) compared with wild-type mice in vivo. The mitigation of the plasma renin concentration response to isoproterenol was even more pronounced in AC6(-/-) (male: -63%; female: -50% versus AC6(+/+)). Similarly, the effects of isoproterenol, prostaglandin E2, and pituitary adenylyl cyclase-activating polypeptide on renin release from isolated perfused kidneys were attenuated to a higher extent in AC6(-/-) (-51% to -98% versus AC6(+/+)) than in AC5(-/-) (-31% to 46% versus AC5(+/+)). In conclusion, both AC5 and AC6 are involved in the stimulation of renin secretion in vivo, and AC6 is the dominant isoforms in this process.

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

Bestrophins form Ca(2+)-activated Cl(-) channels and regulate intracellular Ca(2+) signaling. We demonstrate that bestrophin 1 is localized in the endoplasmic reticulum (ER), where it interacts with stromal interacting molecule 1, the ER-Ca(2+) sensor. Intracellular Ca(2+) transients elicited by stimulation of purinergic P2Y(2) receptors in HEK293 cells were augmented by hBest1. The p21-activated protein kinase Pak2 was found to phosphorylate hBest1, thereby enhancing Ca(2+) signaling and activation of Ca(2+)-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 Ca(2+) deposits. hBest1 is, therefore, important for Ca(2+) handling of the ER store and may resemble the long-suspected counterion channel to balance transient membrane potentials occurring through inositol triphosphate (IP(3))-induced Ca(2+) release and store refill. Thus, bestrophin 1 regulates compartmentalized Ca(2+) signaling that plays an essential role in Best macular dystrophy, inflammatory diseases such as cystic fibrosis, as well as proliferation.

Bestrophin and TMEM16-Ca(2+) activated Cl(-) channels with different functions.

In the past, a number of candidates have been proposed to form Ca(2+) activated Cl(-) currents, but it is only recently that two families of proteins, the bestrophins and the TMEM16-proteins, recapitulate reliably the properties of Ca(2+) activated Cl(-) currents. Bestrophin 1 is strongly expressed in the retinal pigment epithelium, but also at lower levels in other cell types. Bestrophin 1 may form Ca(2+) activated chloride channels and, at the same time, affect intracellular Ca(2+) signaling. In epithelial cells, bestrophin 1 probably controls receptor mediated Ca(2+) signaling. It may do so by facilitating Ca(2+) release from the endoplasmic reticulum, thereby indirectly activating membrane localized Ca(2+)-dependent Cl(-) channels. In contrast to bestrophin 1, the Ca(2+) activated Cl(-) channel TMEM16A (anoctamin 1, ANO1) shows most of the biophysical and pharmacological properties that have been attributed to Ca(2+)-dependent Cl(-) channels in various tissues. TMEM16A is broadly expressed in both mouse and human tissues and is of particular importance in epithelial cells. Thus exocrine gland secretion as well as electrolyte transport by both respiratory and intestinal epithelia requires TMEM16A. Because of its role for Ca(2+)-dependent Cl(-) secretion in human airways, it is likely to become a prime target for the therapy of cystic fibrosis lung disease, caused by defective cAMP-dependent Cl(-) secretion. It will be very exciting to learn, how TMEM16A and other TMEM16-proteins are activated upon increase in intracellular Ca(2+), and whether the other nine members of the TMEM16 family also form Cl(-) channels with properties similar to TMEM16A.

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 Ca(2+)-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 P2Y(2) receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca(2+) and a Ca(2+)-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 I(Cl,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.

Bestrophin 1 promotes epithelial-to-mesenchymal transition of renal collecting duct cells.

Bestrophin 1 (Best1) controls intracellular Ca(2+) concentration, induces Ca(2+)-activated Cl(-) conductance, and increases proliferation of colon carcinoma cells. Here, we show that expression of Best1 in mouse renal collecting duct (CD) cells causes i) an increase in cell proliferation, ii) a loss of amiloride-sensitive Na(+) absorption, iii) induction of Ca(2+)-dependent Cl(-) conductance (CaCC), and iv) epithelial-to-mesenchymal transition. During conditions of high proliferation or when we exposed CD cells to serum or TGF-beta1, we observed upregulation of Best1, increased CaCC, redistribution of the epithelial-to-mesenchymal transition marker beta-catenin, and upregulation of vimentin. In contrast, suppression of Best1 by RNAi inhibited proliferation, reduced CaCC, and downregulated markers of EMT. CaCC and expression of Best1 were independent of the cell cycle but clearly correlated to cell proliferation and cell density. During renal inflammation in LPS-treated mice or after unilateral ureteral obstruction, we observed transient upregulation of Best1. These data indicate that repression of cell proliferation, CaCC, and expression of Best1 occurs during mesenchymal-to-epithelial transition once CD cells polarize and terminally differentiate. These results may suggest a role for Best1 in renal fibrosis and tissue repair.

Functional assembly and purinergic activation of bestrophins.

Proteins of the bestrophin family produce Ca(2+)-activated Cl(-) currents and regulate voltage-gated Ca(2+) channels. Bestrophin 1 was first identified in the retinal pigment epithelium. Four human paralogs (hBest1-hBest4) exist, and for some bestrophins, dimeric and heterotetrameric structures have been proposed. Here, we demonstrate that hBest1-hBest4 induce Cl(-) conductances of different amplitudes when expressed in HEK293 cells and when activated through purinergic stimulation. hBest1 mutants that are known to cause autosomal dominant macular dystrophy (Best disease) did not produce a Cl(-) current. Bestrophins were colocalized and showed molecular and functional interaction in HEK293 cells, overexpressing hBest1 and hBest2 or hBest4. Interaction was confirmed in airway epithelial cells coexpressing endogenous bestrophins. A fraction of hBest2 and hBest4 was expressed in the membrane, while most of hBest1 was found in the endoplasmic reticulum. Nevertheless, hBest1 has a clear role for the adenosine triphosphate (ATP; or uridine triphosphate)-induced Cl(-) current in both HEK293 and Calu-3 cells. Since native epithelial tissues typically express several bestrophin paralogs, these proteins may exist as heterooligomeric structures.

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 beta- and gamma-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 (alphabeta(S631A)gamma(T599A)) 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 alphabeta(S631A)gamma(T599A)-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of alphabetagamma-ENaC. Channels lacking the Nedd4-2 binding motif in beta-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 beta-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes.