KCNN4 Expression Research Articles

Ursodeoxycholic acid attenuates colonic epithelial secretory function

Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl(-) secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl(-) secretory responses to the Ca(2+) and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n = 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na(+)/K(+)-ATPase activity and basolateral K(+) channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg(-1)) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50-200 μm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea.

Abstract 3037: Role of KCNN4 in ovarian cancer and its pharmacologic activation increases sensitivity to cisplatin

Abstract Ovarian cancer is the second most common and most lethal gynecologic cancer in the United States with an estimated 15520 deaths expected from 21,650 cases. Patients with stage I disease have a relatively favorable survival approaching 80%, however most patients are first diagnosed with stage III and IV metastatic disease and have very poor prognosis. Expression of potassium channel genes has been shown to affect chemosensitivity and cell proliferation in several tumor types. We examined the expression of the known potassium channel genes in ovarian cancer and the presumptive cells of ovarian cancer origin. Affymetrix U133 Plus2.0 array data identified that the KCNN4 gene was up-regulated in 15 of 20 stage IIIC or IV serous ovarian cancers compared to eight cases of normal ovarian surface epithelium. This result was confirmed by quantitative real-time polymerase chain reactions (qRT-PCR) using an independent set of 25 ovarian cancers compared to 10 normals. Also, we assessed expression of KCNN4 in a large set (101 ovarian cases) of publically available microarray data (Berchuck, et al. Clin Cancer Res, 2009) and normal fallopian tube. These data confirmed that KCNN4 was highly-expressed in ovarian cancer and also showed expression in normal fallopian tube epithelial cells -the other suspected candidate for the cell of origin of ovarian cancers. KCNN4 forms the intermediate conductance Ca2+ – activated K+ channel (IKCa channel), which serves as a highly sensitive environmental sensor of intracellular calcium. Studies in other tumor types suggest that potassium channels are integral components of cellular proliferation. siRNA and shRNA mediated knockdown of KCNN4 resulted in decreased cell proliferation in cell culture models of ovarian cancer suggesting an essential role for KCNN4 in ovarian cancer cell growth and viability. Furthermore we hypothesized that by pharmacologically activating these channels we could further increase the killing of ovarian cancer cells by cis-platinum. We found that 9 of 11 ovarian cancer cell lines demonstrated functionally active IKCa channels and we were able to increase the sensitivity of these cells to cis-platinum following IKCa channel activation with 1-EBIO -a specific activator of IKCa channels. The chemo-sensitizing effect of treating these cells with the activator was reversed by adding clotrimazole -a specific inhibitor of the IKCa channels, further suggesting this effect was mediated through KCNN4. Furthermore, co-treatment of 1-EBIO and a non-lethal dose of cis-platinum to OVCAR5 cells resulted in apoptotic cell death suggesting a chemo-sensitizing role for IKCa channels. Preliminary data in vivo mimic some of the in vitro results. Our data shows that KCNN4 is integral to ovarian cancer cell growth and that sensitization with pharmacologic activators such as 1-EBIO may have future therapeutic benefit for increasing cancer cell sensitivity to chemotherapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3037. doi:10.1158/1538-7445.AM2011-3037

T1845 Enhanced Mucosal Expression of KCNN4 and Bk Channels Contribute to Aldosterone Induced K+ Secretion in Distal Colon

Background: Mammalian colon plays important roles in body K+ homeostasis, as it both actively absorbs and secretes K+. Aldosterone (aldo) stimulates both active electroneutral K+ absorption and active electrogenic K+ secretion in rat distal colon. Increased H,K-ATPase expression has been shown as a mechanism for enhanced K+ absorption. However, although both intermediate conductance (KCNN4) and large conductance (BK) K+ channels are present on the mucosal membranes, a mechanism responsible for aldo increased K+ secretion is not known. Aim: Studies were initiated to identify whether KCNN4 and/or BK channels mediate the aldo enhanced K+ secretion in rat distal colon. Methods: Aldo animals were produced by feeding Na-free diet for 6-7 days. Mucosal to serosal (m-s) and serosal to mucosal (s-m) 86Rb (K+ surrogate) fluxes were measured in stripped colonic mucosa mounted under voltage clamp condition in presence of mucosal benzamil (50 μM; ENaC blocker). Western blot analyses were performed using anti-KCNN4-abc (developed in our laboratory) and BKα (Santa Cruz) antibodies. RTQ-PCR analyses were performed using standard technique. Results: Under basal condition, mucosal addition of VO4 (H,K-ATPase inhibitor) inhibited the net K+ absorption (0.42 ± 0.06 vs -0.04 ± 0.03 μEq/h.cm2; p < 0.001) in normal rat distal colon. Serosal addition of carbachol (CCH) induced net K secretion (-0.04 ± 0.03 vs 0.48 ± 0.06 μEq/h.cm2; p < 0.001). The CCH-induced net K+ secretion was inhibited by 60% and 40% by mucosal TRAM-34 (KCNN4 channel blocker) and iberiotoxin (IbTX; BK channel blocker), respectively. Either simultaneous presence of TRAM-34 and IbTX or Ba2+ (nonspecific K+ channel blocker) completely inhibited the CCH-induced K secretion. Aldo reversed the net K absorption in normal colon to net K+ secretion (0.42 ± 0.06 vs -1.34 ± 0.22 μEq/h.cm2; p < 0.001). Mucosal addition of TRAM34 and IbTX inhibited the aldo-induced net K+ secretion by 33% and 55%, respectively. Simultaneous presence of TRMA-34 and IbTX, and Ba2+ inhibited the aldo induced net K secretion by 84% and 80%, respectively. Western blot analyses indicated that aldo enhanced mucosal KCNN4 and BKα specific protein expression by 2.2 and 3.1 fold, respectively. RTQ-PCR analyses indicated that mucosal KCNN4 and BKα channel specific mRNA abundances were increased by 3.0 and 3.5 fold in aldo animals, respectively. Conclusions: 1) Both KCNN4 and BK channels mediate CCHand aldo-induced K+ secretion; 2) Enhanced expression of both mucosal KCNN4 and BK channels mediate the aldo induced K+ secretion; and 3) Aldo regulates both KCNN4 and BK channel expression at transcriptional level.

Differences in electrogenic secretion of electrolytes in colonic epithelium of inbred mouse strains.

The mouse is the animal model of choice in studies of the physiology and pathophysiology of intestinal fluid and electrolyte transport. In the course of our research in colon physiology we have observed differences in electrical tissue properties and responses to agonists that induce electrogenic secretion of electrolytes among mice strains. Such differences are important enough to preclude comparisons between strains and might be crucial when choosing the genetic background for knockout of genes of interest. We have now investigated some of these differences by electrophysiological methods. Colonic tissue from Black Swiss, 129Sv and B6 mice were placed in Ussing chambers and their responses to a series of agonists and blockers recorded. We observe that while the 3 strains have similar anion secretory responses to increasing cAMP (around ‐100 μA cm−2), when the muscarinic agonist carbachol was used the largest response was seen with B6 tissue, compared to that of Black Swiss and 129Sv colon (‐200, ‐163 and ‐133 μA cm−2 respectively). The importance of the genetic background on the severity of cystic fibrosis in mice has long been known (Rozmahel et al., Nat Genet 12: 280‐7, 1996) and has been related to differences in calcium‐dependent anion secretion. We reported previously that basolateral KCNN4 calcium‐activated potassium channels are the rate‐limiting step in calcium‐agonist‐dependent anion secretion, rather than apical CFTR chloride channels (Flores et al., J Physiol 583: 705‐17, 2007). We are studying KCNN4 expression as a possible explanation to these differences.Supported by FONDECYT 3085023

Expression of intermediate-conductance, Ca2+-activated K+channel (KCNN4) in H441 human distal airway epithelial cells

Electrophysiological studies of H441 human distal airway epithelial cells showed that thapsigargin caused a Ca(2+)-dependent increase in membrane conductance (G(Tot)) and hyperpolarization of membrane potential (V(m)). These effects reflected a rapid rise in cellular K(+) conductance (G(K)) and a slow fall in amiloride-sensitive Na(+) conductance (G(Na)). The increase in G(Tot) was antagonized by Ba(2+), a nonselective K(+) channel blocker, and abolished by clotrimazole, a KCNN4 inhibitor, but unaffected by other selective K(+) channel blockers. Moreover, 1-ethyl-2-benzimidazolinone (1-EBIO), which is known to activate KCNN4, increased G(K) with no effect on G(Na). RT-PCR-based analyses confirmed expression of mRNA encoding KCNN4 and suggested that two related K(+) channels (KCNN1 and KCNMA1) were absent. Subsequent studies showed that 1-EBIO stimulates Na(+) transport in polarized monolayers without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)), suggesting that the activity of KCNN4 might influence the rate of Na(+) absorption by contributing to G(K). Transient expression of KCNN4 cloned from H441 cells conferred a Ca(2+)- and 1-EBIO-sensitive K(+) conductance on Chinese hamster ovary cells, but this channel was inactive when [Ca(2+)](i) was <0.2 microM. Subsequent studies of amiloride-treated H441 cells showed that clotrimazole had no effect on V(m) despite clear depolarizations in response to increased extracellular K(+) concentration ([K(+)](o)). These findings thus indicate that KCNN4 does not contribute to V(m) in unstimulated cells. The present data thus establish that H441 cells express KCNN4 and highlight the importance of G(K) to the control of Na(+) absorption, but, because KCNN4 is quiescent in resting cells, this channel cannot contribute to resting G(K) or influence basal Na(+) absorption.