Kirti P. Tewari

Sites of Protein Kinase A Activation of the Human ClC-2 Cl– Channel

Human ClC-2 Cl– (hClC-2) channels are activated by protein kinase A (PKA) and low extracellular pHo. Both of these effects are prevented by the PKA inhibitor, myristoylated PKI. The aims of the present study were to identify the PKA phosphorylation site(s) important for PKA activation of hClC-2 at neutral and low pHo and to examine the relationship between PKA and low pHo activation. Recombinant hClC-2 with point mutations of consensus phosphorylation sites was prepared and stably expressed in HEK-293 cells. The responses to forskolin plus isobutylmethylxanthine at neutral and acidic pHo were studied by whole cell patch clamp in the presence and absence of phosphatase inhibitors. The double phosphorylation site (RRAT655(A) plus RGET691(A)) mutant hClC-2 lost PKA activation and low pHo activation. Either RRAT or RGET was sufficient for PKA activation of hClC-2 at pHo 7.4, as long as phosphatase inhibitors (cyclosporin A or endothal) were present. At pHo 6 only RGET was needed for PKA activation of hClC-2. Low pHo activation of hClC-2 Cl– channel activity was PKA-dependent, retained in RGET(A) mutant hClC-2, but lost in RRAT(A) mutant hClC-2. RRAT655(D) mutant hClC-2 was constitutively active and was further activated by PKA at pHo 7.4 and 6.0, consistent with the above findings. These results show that activation of hClC-2 is differentially regulated by PKA at two sites, RRAT655 and RGET691. Either RRAT655 or RGET691 was sufficient for activation at pHo 7.4. RGET, but not RRAT, was sufficient for activation at pHo 6.0. However, in the RGET691(D) mutant, there was PKA activation at pHo 6.0.

Activation Of Human Clc-2 Cl– Channels: Implications For Cystic Fibrosis

1. The ClC‐2 Cl– channels are present in the adult human lung epithelia and, therefore, are a potential target for therapy in cystic fibrosis.

1010 Methadone, but Not Morphine, Inhibits Stimulated Chloride Currents in CLC-2 Transfected Cells in Culture

G A A b st ra ct s by aggregate CBC analysis. In addition, no change was observed in relative frequency of CD34+ cells in the peripheral circulation (baseline ~0.3%) either in the short term, at peak vedolizumab serum concentrations, or through Day 141, when drug has cleared in most subjects. Forced egress of leukocytes from bone marrow, CD34+ cells in particular, represents a potential means of JC virus reactivation. These data indicate important mechanistic distinctions between vedolizumab and natalizumab that may portend a lower risk of PML. To date, neither PML nor any other opportunistic infection has been reported for vedolizumab.

Kir2.1 K+ Channels of the Gastric Parietal Cell

The purpose of this article is to review the literature regarding the characteristics of the gastric parietal cell apical membrane K+ channel and provide new evidence that suggests that the inward rectifying K+ channel, Kir2.1, may be involved in K+ recycling at the apical membrane of the gastric parietal cell. It has been previously reported that gastric H+/K+ ATPase-containing vesicles exhibit a 10 pS K+ channel when measured in planar lipid bilayers. The native gastric K+ channel in these vesicles is insensitive to ATP, is active in the absence of Ca2+, is stable at an extracellular pH of 3.0, and is activated by PKA. A literature search suggested that Kir2.1 has many of the properties of this channel. This is the first report to show that recombinant rabbit Kir2.1 K+ channels are active in the presence of ATP, in the absence of Ca2+, and are stable at an extracellular pH of 3.0. Preliminary results demonstrate that Kir2.1 is also PKA activated. Kir2.1 also exhibits single channel currents that are of similar magnitude as the native channel under these conditions. In an accompanying paper, Grahammer et al suggest from 293 B inhibition of acid secretion that KCNQ1 is the K+ channel involved in K+ recycling at the apical membrane. Kir2.1 is not inhibited by 293B. Acid accumulation by H+/K+ ATPase-containing vesicles from stimulated rabbits is insensitive to 293B. This finding strongly suggests that KCNQ1 is not involved in K+ recycling at the apical membrane of the gastric parietal cell. Kir2.1 is a candidate for this process.

T1876 Lubiprostone Activation of Cl− Currents Does Not Involve CA2+, cAMP, or PKA

Introduction Lubiprostone stimulates electrogenic Cltransport in human intestinal T84 cells and Clcurrents in HEK293 cells stably transfected with recombinant human ClC-2 (hClC-2) with an EC50 of approximately 20 nM [Cuppoletti et al AJP 287:C1173, 2004]. The purpose of this study was to determine whether lubiprostone (over the concentration range that activates electrogenic Cltransport) causes changes in [Ca]i, cAMP or PKA mediated phosphorylation. Methods T84 cells, HEK293 cells and HEK293 cells stably expressing hClC-2 or mutant hClC-2 were used. T84 cell [Ca]i, was measured using the calcium indicator dye indo-1/AM and intracellular [cAMP] was determined using a commercial ELISA kit. A commercial FLIPER assay was used to measure [Ca]i, and [cAMP] in HEK293 cells. Cl currents were measured by whole cell patch clamp. Results Lubiprostone at 1, 10 or 100 nM did not increase [Ca]i, in T84 cells and only slightly increased [cAMP] from 0.56 ± 0.02 to 4.0 ± 0.3 pmole/105 cells (n=4, P<0.001) at 100 nM (5 times the EC50 for lubiprostone activation of Cl currents). 1 μM PGE1 significantly (P<0.0005) increased [Ca]i from 80.8 ± 2.4 nM (control) to 1175.0 ± 18.7 nM (n=3) and greatly increased [cAMP] to 37.6 ± 0.6 pmole/105 cells (n=4) (P<0.0005). In contrast, 100 nM lubiprostone had no effect on [Ca]i or cAMP in HEK293 cells. Nevertheless hClC-2 in HEK293 cells is activated by lubiprostone, thereby demonstrating that neither [Ca]i or cAMP signaling are involved in lubiprostone activation of hClC-2. To rule out involvement of PKA phosphorylation of hClC-2 in lubiprostone activation of Clcurrents, a mutant hClC-2 Clchannel lacking the two unique PKA consensus phosphorylation sites (RRAT and RGET) essential for PKA activation of ClC-2, was used [Cuppoletti, et al JBC 279:21849, 2004]. In wild-type hClC-2 expressing HEK293 cells, control Clcurrents at -140 mV (n=6) of -27.2 ± 3.5 pA/pF, significantly increased (P<0.0005) to -100.9 ± 6.5 pA/pF with 20 nM lubiprostone and subsequently were reduced with 500 μM CdCl2 to control levels, -29.9 ± 5.6 pA/pF (P<0.0005). Cl currents in mutant hClC-2 expressing HEK293 cells was also significantly (P<0.0005) increased with 20 nM lubiprostone and inhibited by CdCl2 (n=6): control, -27.1 ± 6.9 pA/pF; with lubiprostone, -81.9 ± 5.1 (P<0.0005); after CdCl2, -29.4 ± 3.7 pA/pF (P<0.0005). Conclusions Activation of Clcurrents by lubiprostone is independent of [Ca]i and cAMP signaling and PKA mediated phosphorylation of hClC-2. More direct action of lubiprostone on hClC-2 Clchannels may be involved. Supported by Sucampo Pharmaceuticals, Inc. E001288 Conclusions Activation of Clcurrents by lubiprostone is independent of [Ca]i and cAMP signaling and PKA mediated phosphorylation of hClC-2. More direct action of lubiprostone on hClC-2 Clchannels may be involved.

Characterization, Regulation and Localization of the Gastric Cl- Channel Associated with Gastric Acid Secretion

The Cl-channel of the apical membrane of the gastric parietal cell was characterized in native rabbit H/K ATPase-containing membrane vesicles in bilayer reconstitution studies. It is a linear Cl- channel which exhibits activation by reduction of extracellular pH and PKA. The ClC-2 Cl- channel cloned from rabbit gastric mucosa and expressed in Xenopus laevis oocytes, exhibited similar electrophysiological characteristics and regulation as the native gastric Cl- channel. Potential PKA phosphorylation sites in rabbit ClC-2 appear to underlie PKA activation. The pH sensor on the extracellular surface of ClC-2 was identified as E419 and amidation resulted in activation of the channel. Recombinant ClC-2 stably expressed in HEK-293 cells showed Cl- currents that were activated by forskolin and IBMX, low extracellular pH, amidation, arachidonic acid and acid-activated omeprazole. Using immunofluorescent confocal microscopy, ClC-2 was localized to the secretory canalicular membrane of stimulated parietal cells and to intracellular structures (tubulovesicles) in resting parietal cells, which was confirmed by immunogold electron microscopy. Distribution of the gastric H/K ATPase and ClC-2 were similar. These findings strongly support the view that ClC-2 is the Cl- channel in the apical secretory membrane of the gastric parietal cell which, together and in concert with the H/K ATPase and a K+ channel, result in HCl secretion.