Identification of a Basolateral Membrane Potassium Channel from Teleost Intestinal Epithelial Cells

1991 ◽  
Vol 159 (1) ◽  
pp. 45-64
Author(s):  
CHRISTOPHER A. LORETZ ◽  
CHARLES R. FOURTNER
Keyword(s):  
Membrane Potential ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Basolateral Membrane ◽  
K Channel ◽  
Ca Channel ◽  
Intestinal Epithelial ◽  
Channel Activity ◽  
Open Probability ◽  
Physiological Range

Using patch-clamp techniques, a Ca2+-dependent, voltage-gated K+ channel [K(Ca) channel] was isolated from the basolateral membrane of NaCl-absorbing intestinal epithelial cells of the goby Gillichthys mirabilis. This K(Ca) channel had a high conductance (approximately 150 pS) in the physiological range of membrane potential. Conclusive identification as a K+ channel is supported by dependence of the reversal potential for single-channel current on the K+ concentration gradient and the ability of Ba2+, Cs+ and other pharmacological agents to block the channel. The channel was highly selective for K+ over Na+ (PNa/PK=0.04). Channel activity, expressed as open probability (Po), was dependent on membrane potential with depolarization increasing Po over the physiological range in the presence of Ca2+. Channel activity was also dependent on cytoplasmic-side Ca2+. Po was reduced to near-zero levels following EGTA chelation of Ca2+ in the solution bathing the cytoplasmic face of excised membrane patches; channel activity was most sensitive to changes in Ca2+ concentration between 10nmoll−1 and 10μmoll−1. This K(Ca) channel may be one of several avenues for K+ exit across the basolateral cell membrane and, as such, may play roles in both transepithelial salt transport and maintenance of intracellular ionic composition.

scholarly journals Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

2014 ◽  
Vol 306 (5) ◽  
pp. C460-C470 ◽  
Author(s):  
Kiril L. Hristov ◽  
Amy C. Smith ◽  
Shankar P. Parajuli ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Regulation ◽  
Pkc Activation

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility.

Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells

1995 ◽  
Vol 108 (1) ◽  
pp. 369-377 ◽  
Author(s):  
K.L. Soole ◽  
M.A. Jepson ◽  
G.P. Hazlewood ◽  
H.J. Gilbert ◽  
B.H. Hirst
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Intestinal Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Gpi Anchor ◽  
Sorting Signal

To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated. The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and susceptibility to phosphoinositol-specific phospholipase C. In polarized MDCK cells, EGE' was localized almost exclusively to the apical cell surface, while in polarized intestinal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane. Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral. EGE' delivered to the basolateral cell surface was transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells. However, the mis-sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.

Na+ pump inhibition downregulates an ATP-sensitive K+ channel in rabbit proximal convoluted tubule

1993 ◽  
Vol 264 (4) ◽  
pp. F760-F764 ◽  
Author(s):  
A. M. Hurst ◽  
J. S. Beck ◽  
R. Laprade ◽  
J. Y. Lapointe
Keyword(s):  
Basolateral Membrane ◽  
Proximal Convoluted Tubule ◽  
K Channel ◽  
Bathing Solution ◽  
Channel Activity ◽  
Open Probability ◽  
Functional Link ◽  
Tightly Coupled ◽  
Stimulation Of

In several epithelial and nonepithelial tissues a functional link between the basolateral Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) and a basolateral K+ conductance has been established. However, the nature of this link is unclear. We have previously identified a K+ channel on the basolateral membrane of the proximal convoluted tubule perfused in vitro, the activity of which is increased by stimulation of Na+ transport [J. S. Beck, A. M. Hurst, J.-Y. Lapointe, and R. Laprade. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F496-F501, 1993]. In the present study we investigate whether basolateral membrane K+ channel activity is tightly coupled to Na(+)-K(+)-ATPase activity. In cell-attached patches (150 mM K+ pipette), following stimulation of channel activity by addition of Na(+)-cotransported solutes to the tubule lumen, mean channel open probability (NPo) was reduced from 0.35 +/- 0.09 to 0.14 +/- 0.06 (n = 7, P < 0.05) by blocking the Na(+)-K(+)-ATPase with 100 microM strophanthidin. In excised patches the channel was reversibly blocked by 2 mM ATP from the cytosolic face of the patch, such that NPo fell to 20.1 +/- 7.0% (n = 5, P < 0.001) of control and recovered to 52.2 +/- 11.2% (n = 5, P < 0.05) after washout of ATP. Diazoxide, a putative opener of ATP-sensitive K+ channels, when added to the bathing solution of an unstimulated tubule (microperfused in the absence of Na(+)-cotransported solutes), increased NPo from 0.046 +/- 0.035 to 0.44 +/- 0.2 (n = 6, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic acid inhibits K channels in basolateral membrane of the thick ascending limb

2002 ◽  
Vol 283 (3) ◽  
pp. F407-F414 ◽  
Author(s):  
Rui-Min Gu ◽  
Wen-Hui Wang
Keyword(s):  
Arachidonic Acid ◽  
Basolateral Membrane ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Cytochrome P 450

We have used the patch-clamp technique to study the effect of arachidonic acid (AA) on the basolateral K channels in the medullary thick ascending limb (mTAL) of rat kidney. An inwardly rectifying 50-pS K channel was identified in cell-attached and inside-out patches in the basolateral membrane of the mTAL. The channel open probability ( P o) was 0.51 at the spontaneous cell membrane potential and decreased to 0.25 by 30 mV hyperpolarization. The addition of 5 μM AA decreased channel activity, identified as NP o, from 0.58 to 0.08 in cell-attached patches. The effect of AA on the 50-pS K channel was specific because 10 μM cis-11,14,17-eicosatrienoic acid had no significant effect on channel activity. To determine whether the effect of AA was mediated by AA per se or by its metabolites, we examined the effect of AA on channel activity in the presence of indomethacin, an inhibitor of cyclooxygenase, or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), an inhibitor of cytochrome P-450 monooxygenase. Inhibition of cyclooxygenase increased channel activity from 0.54 to 0.9. However, indomethacin did not abolish the inhibitory effect of AA on the 50-pS K channel. In contrast, inhibition of cytochrome P-450 metabolism not only increased channel activity from 0.49 to 0.83 but also completely abolished the effect of AA. Moreover, addition of DDMS can reverse the inhibitory effect of AA on channel activity. The notion that the effect of AA was mediated by cytochrome P-450-dependent metabolites of AA is also supported by the observation that addition of 100 nM of 20-hydroxyeicosatetraenoic acid, a main metabolite of AA in the mTAL, can mimic the effect of AA. We conclude that AA inhibits the 50-pS K channel in the basolateral membrane of the mTAL and that the effect of AA is mainly mediated by cytochrome P-450-dependent metabolites of AA.

Subcloning, localization, and expression of the rat intestinal sodium-hydrogen exchanger isoform 8

2005 ◽  
Vol 289 (1) ◽  
pp. G36-G41 ◽  
Author(s):  
Hua Xu ◽  
Rongji Chen ◽  
Fayez K. Ghishan
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Early Life ◽  
Intestinal Epithelial Cells ◽  
Protein Localization ◽  
Basolateral Membrane ◽  
Gene Expression Pattern ◽  
Intestinal Epithelial ◽  
Sodium Hydrogen ◽  
Apical Localization

Apically expressed intestinal and renal sodium-hydrogen exchangers (NHEs) play a major role in Na+ absorption. Our previous studies on NHE ontogeny have shown that NHE-2 and NHE-3 are expressed at very low levels in young animals. Furthermore, single and/or double NHE-2 and NHE-3 knockout mice display no obvious abnormalities before weaning. These observations suggest that other transporter(s) may be involved in intestinal Na+ absorption during early life. The present studies were designed to clone the novel rat intestinal NHE-8 cDNA and to decipher the NHE-8 protein localization and gene expression pattern during different developmental stages. The rat NHE-8 cDNA has 2,160 bp and encodes a 575-amino acid protein. An antibody against NHE-8 protein was developed. Immunohistochemistry staining indicated apical localization of NHE-8 protein in rat intestinal epithelial cells. The apical localization of NHE-8 was also confirmed by its presence in brush-border membrane and its absence in basolateral membrane preparations. Northern blotting utilizing a NHE-8-specific probe demonstrated higher NHE-8 mRNA expression in young animals compared with adult animals. Western blot analysis revealed a similar pattern. Tissue distribution with multiple human tissue RNA blot showed that NHE-8 was expressed in multiple tissues including the gastrointestinal tract. In conclusion, we have cloned the full-length NHE-8 cDNA from rat intestine and further showed its apical localization in intestinal epithelial cells. We have also shown that NHE-8 gene expression and protein expression were regulated during ontogeny. Our data suggests that NHE-8 may play an important role in intestinal Na+ absorption during early life.

Involvement of K+ channel expression and Ca2+ in migration of differentiated intestinal epithelial cells after wounding

2001 ◽  
Vol 120 (5) ◽  
pp. A146
Author(s):  
Rao N. Jaladanki ◽  
Li Li ◽  
Oleksandr Platoshyn ◽  
Vera A. Golovina ◽  
Jason X-J Yuan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
K Channel ◽  
Intestinal Epithelial ◽  
Channel Expression

Tu1846 Flagellin Response via TLR5 on Basolateral Membrane of Primary Intestinal Epithelial Cells is Regulated by Notch Signaling

2012 ◽  
Vol 142 (5) ◽  
pp. S-859-S-860
Author(s):  
Kiichiro Tsuchiya ◽  
Xiu Zheng ◽  
Yoshihito Kano ◽  
Nobukatsu Horita ◽  
Ryuichi Okamoto ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Notch Signaling ◽  
Intestinal Epithelial Cells ◽  
Basolateral Membrane ◽  
Intestinal Epithelial

Involvement of K+ channel expression and Ca2+ in migration of differentiated intestinal epithelial cells after wounding

2001 ◽  
Vol 120 (5) ◽  
pp. A146-A146
Author(s):  
R JALADANKI ◽  
L LI ◽  
O PLATOSHYN ◽  
V GOLOVINA ◽  
J YUAN ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
K Channel ◽  
Intestinal Epithelial ◽  
Channel Expression

DUSP16 IS A NOVEL IBD GENE IMPLICATED IN THE REGULATION OF DIFFERENTIATION AND HOMEOSTASIS OF INTESTINAL EPITHELIAL CELLS

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S29-S30
Author(s):  
Jessy Ntunzwenimana ◽  
Azadeh Alikashani ◽  
Claudine Beauchamp ◽  
Jean Paquette ◽  
Gabrielle Boucher ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Map Kinases ◽  
Intestinal Epithelial Cells ◽  
Cell Model ◽  
Basolateral Membrane ◽  
Intestinal Lumen ◽  
Interleukin 17 ◽  
Intestinal Epithelial ◽  
Intestinal Pathogens ◽  
Ht 29

Abstract Inflammatory bowel disease (IBD) are chronic inflammatory diseases including Crohn’s disease (CD) and ulcerative colitis (UC). More than 200 genomic regions have been identified and validated (association values〈 5x10-8) to be associated with CD, UC or IBD. These regions may contain multiple genes and the current challenge lies in identifying the causal gene in each of these. To address this problem, we performed a functional genomic screen of 145 genes from validated IBD loci, in a relevant intestinal epithelial cell model (HT-29). The results of this transcriptome-based screening revealed that the candidate IBD gene DUSP16 (a dual specificity phosphatase targeting MAP kinases (MAPKs) phosphorylation) as well as the known IBD gene KSR1 (a scaffold protein regulating the spatiotemporal activation of the ERK) regulate the expression of genes involved in intestinal differentiation and homeostasis. They induce, among others, the expression of the PIGR gene that encodes the polymeric immunoglobulin receptor. PIGR plays a role in transporting dimeric IgA molecules from the basolateral membrane of epithelial cells to the intestinal lumen, via transcytosis, where they play an essential role in protecting the epithelium against intestinal pathogens. Our hypothesis is that DUSP16 and KSR1 modulate the activity of MAPKs in intestinal epithelial cells to induce PIGR expression, thus participating in the maintenance of homeostasis of the intestinal barrier. To better understand how DUSP16 modulates the expression of PIGR, we used an approach of over- expression (cDNA) and knockdown (shRNA) of DUSP16 in HT-29 cells. Our results confirmed that DUSP16 induction increases the expression of PIGR, whereas a knockdown of DUSP16 reduces the basal level of PIGR. Next we confirmed by Western Blot that the induction of DUSP16 was accompanied by a decrease in MAPK phosphorylation. The involvement of MAPKs was also confirmed through the use of chemical inhibitors specific for each MAPK, with inhibition of ERK and p38 showing the strongest induction of PIGR expression. We are currently analyzing known functional mutants of DUSP16 and KSR1 to determine their impact on MAPK activity and on PIGR expression. This work supports a role for PIGR in disease pathogenesis, adding to two recent studies that documented that patients suffering from UC accumulated somatic mutations in a group of genes regulating the expression of PIGR by Interleukin 17. The mutated genes, including PIGR, were positively selected in inflamed tissues, indicating the importance of the biological function occupied by this gene in the maintenance of homeostasis. In conclusion, our study successfully identified functional links between two genes from independent IBD loci, and suggests that these DUSP16 and KSR1 play a role in the process of epithelial transcytosis and the development of IBD.

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