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.
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.
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 AC5 and AC6 possess key roles in the control of renin exocytosis. In order 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 (PRC), renal renin synthesis (mRNA and renin content), urinary volume and systolic blood pressure. In male AC6−/− mice, PRC (AC6−/−: 732 ± 119, AC6 +/+: 436 ± 78 ng angI/h*ml, 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 mmHg, p < 0.001). Stimulation of PRC by a single injection of the ß-adrenoreceptor agonist isoproterenol (10 mg/kg, i.p.) was significantly attenuated in AC5−/− (male −20%, female −33%) compared to wildtype mice in vivo. The mitigation of the PRC response to isoproterenol was even more pronounced in AC6−/− (male −63%, female −50% vs. AC6+/+). Similarly, the effects of isoproterenol, prostaglandin E2 and pituitary adenylyl cyclase-activating polypeptide (PACAP) on renin release from isolated perfused kidneys were attenuated to a higher extent in AC6−/− (−51 to −98% vs. AC6+/+) than in AC5−/− (−31 to 46% vs. 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.
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