1. Volume 134, Issue 4, Pages 1058-1069 (April 2008)
Localization, Trafficking, and Significance for Acid Secretion of Parietal Cell Kir4.1 and KCNQ1 K+ Channels 
Marc–André Kaufhold, Anja Krabbenhöft, Penghong Song, Regina Engelhardt, Brigitte Riederer, Michael Fährmann, Nikolaj Klöcker, Winfried Beil, Michael Manns, Susan J. Hagen, Ursula Seidler 
Gastroenterology 
Volume 134, Issue 4, Pages (April 2008)
DOI: /j.gastro
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2. Figure 1
Inhibitory effect of a high selectivity KCNQ1 blocker (HMR 1556) on 14C-aminopyrine accumulation into cultured rat parietal cells (A and B) and acid secretion in isolated murine gastric mucosa (C and D). The highly selective KCNQ1 inhibitor HMR 1556 resulted in approximately 70% inhibition for histamine-stimulated (100 μmol/L) 14C-AP at 1 μmol/L, which is 1–3 concentration logs above the IC50 reported for this substance37 (A) but only in approximately 40% inhibition of carbachol-stimulated (100 μmol/L) 14C-AP uptake (B). Time course of acid secretion in the absence (C) and presence (D) of 10−5 mol/L of the highly specific KCNQ1 inhibitor HMR1556. It is evident that the response to forskolin is significantly slowed in the presence of HMR 1556 in the serosal and luminal perfusate, but secretory rates 50 minutes poststimulation are not significantly different (8.04 ± 1.2 μmol · h−1 · cm−2 in the presence of 10−5 mol/L HMR1556 vs ± 1.32 μmol · h−1 · cm−2 in its absence). N = 5 different stomachs.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

3. Figure 2
Colocalization of Kir4.1 and KNCQ1 with H+/K+ ATPase in resting state tubulovesicles (TV) and secretory membranes (AS) from rat stomach. (A, upper panel) Anti-Kir4.1 antibody was able to precipitate H+/K+ ATPase from all H+/K+ ATPase-containing membrane fractions. (A, middle panel) Anti H+/K+ ATPase antibody was able to precipitate both Kir4.1 and KCNQ1 from the H+/K+ ATPase-containing fractions. (A, lower panel) Coimmunoprecipitation of H+/K+ ATPase, Kir4.1, and KCNQ1 from immunoisolated TV (IP-TV) and secretory apical membrane (AS) but not from immunoisolated basolateral (IP-BLM) or the resting state apical membrane (AR). Specificity of the staining is documented by complete removal of the band by preincubation with the peptides against which the antibodies were made (BP). (B) Anti-H+/K+ ATPase antibody coprecipitated both Kir4.1 and KCNQ1 from the TV and IP-TV, as well as the AS and IP-AS fraction (B, upper panel). Similarly, either anti-Kir 4.1 (B, middle panel) or anti-KCNQ1 antibodies (B, lower panel) coprecipitated H+/K+ ATPase. Because IP fractions are approximately 5-fold enriched in H+/K+ ATPase, but have lost approximately 80% of the membrane protein content of the original TV fraction, the similar band size for H+/K+ ATPase and Kir4.1 in the enriched and the immunoisolated fraction signifies strong colocalization of both proteins.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

4. Figure 3
Localization and trafficking of Kir4.1, KNCQ1 and H+/K+ ATPase in rabbit parietal cells. (A) In rabbit gastric membranes, Kir4.1 is exclusively localized on tubulovesicles and stimulation-associated secretory membrane, whereas both the basolateral and the resting state apical membrane are free of staining. Both KCNQ1 variants are also found in AS and TV, and a faint band for the second isoform of KCNQ1 is detected in the resting state AR. (B and C) Density gradient of the microsomal membrane pellet from resting state (B) and stimulated (C) rabbit gastric mucosa. In the resting state, when tubulovesicles are part of the microsomal membrane pellet, Kir4.1 colocalized with H+/K+-ATPase in the same density fractions, whereas KCNQ1 showed a slightly wider density distribution (B). In the microsomal pellet from stimulated gastric mucosa, H+/K+-ATPase has completely disappeared and Kir4.1 almost completely because both have translocated to the secretory membrane. On the other hand, significant quantities of KCNQ1 are found in the microsomal membrane pellet after stimulation (C).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

5. Figure 4
Lack of colocalization of Kir4.1 and KCNQ1. Immunoprecipitation (IP) of both tubulovesicles and secretory membranes was performed with antibodies against H+/K+-ATPase (A–D), against KCNQ1 (B and D), and against Kir4.1 (A and C), and the precipitates were tested for the presence of H+/K+-ATPase, as well as the respective other K+ channel. Although IP with anti-H+/K+ATPase results in coprecipitation of both Kir4.1 and KCNQ1, antibodies against either Kir4.1 or KCNQ1 resulted in coprecipitation of H+/K+-ATPase but not the other K+ channel (Figure 4A–D, third columns).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

6. Figure 5
Immunohistochemistry of rat gastric mucosal cryosections from resting state mucosa stained with antibody against Kir4.1 (green), H+/K+-ATPase (red), and KCNQ1 (blue). In resting state mucosa, diffuse cytoplasmic staining with all 3 antibodies was seen (A). (B) Double staining with antibodies against Kir4.1 and H+/K+-ATPase. (C) Double staining with antibodies against KCNQ1 and H+/K+-ATPase.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

7. Figure 6
Immunohistochemistry of rat gastric mucosal cryosections from in vivo stimulated mucosa stained with antibody against Kir4.1 (green), H+/K+-ATPase (red), and KCNQ1 (blue). After in vivo stimulation of acid secretion, virtually complete translocation of Kir4.1 and H+/K+-ATPase staining to the secretory membranes is observed, whereas diffuse blue staining for KCNQ1 is still observed within the cytoplasm (A). (B) Complete colocalization (yellow) of Kir4.1 and H+/K+-ATPase and (C) partial colocalization (white) of KCNQ1 and H+/K+-ATPase. Most KNCQ1 staining (blue) remains in the cytoplasm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

8. Figure 7
Immunocytochemistry of isolated rat parietal cell in culture. (A) Resting state. (B) After stimulation in the culture dish. Complete translocation of Kir4.1 and H+/K+-ATPase to the secretory vesicle is observed, whereas, for KCNQ1, partial translocation to the secretory vesicle membrane has occurred, but considerable diffuse staining in the cytoplasm remains.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Translocation of EGFP-Kir4.1 after stimulation of transfected parietal cells. (A) With time-lapse confocal microscopy, migration of EGFP-tagged Kir4.1 from the diffuse cytoplasmic localization to the membrane of the secretory vesicles can be observed after stimulation (B). The staining of a cultured, fixed, and air-dried parietal cell with anti-H+/K+-ATPase antibody outlines the translocation of H+/K+-ATPase into the membrane of the secretory vesicle, similar to that of Kir4.1 (B–D). The increase in EGFP fluorescence in the membrane of the enlarging secretory vesicle was measured in a time-dependent (C) and stimulation-dependent (D) manner. (E) Western blot with an anti-GFP antibody demonstrates that only intact EGFP-Kir4.1 (with the appropriate molecular weight) is expressed in cultured parietal cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions