Dynamic and differential regulation of NKCC1 by calcium and cAMP in the native human colonic epithelium

Abstract

The capacity of the intestine to secrete fluid is dependent on the basolateral Na + -K + -2Cl − co-transporter (NKCC1). Given that cAMP and Ca2+ signals promote sustained and transient episodes of fluid secretion, respectively, this study investigated the differential regulation of functional NKCC1 membrane expression in the native human colonic epithelium. Tissue sections and colonic crypts were obtained from sigmoid rectal biopsy tissue samples. Cellular location of NKCC1, Na + -K + -ATPase, M3 muscarinic acetylcholine receptor (M 3 AChR) and lysosomes was examined by immunolabelling techniques. NKCC1 activity (i.e. bumetanide-sensitive NH 4 + uptake), intracellular Ca 2+ and cell volume were assessed by 2 ,7 -bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), Fura-2 and differential interference contrast/calcein imaging. Unstimulated NKCC1 was expressed on basolateral membranes and exhibited a topological expression gradient, predominant at the crypt base. Cholinergic Ca2+ signals initiated at the crypt base and spread along the crypt axis. In response, NKCC1 underwent a Ca 2+ -dependent 4 h cycle of recruitment to basolateral membranes, activation, internalization, degradation and re-expression. Internalization was prevented by the epidermal growth factor receptor kinase inhibitor tyrphostin-AG1478, and re-expression was prohibited by the protein synthesis inhibitor cylcoheximide; the lysosome inhibitor chloroquine promoted accumulation of NKCC1 vesicles. NKCC1 internalization and re-expression were accompanied by secretory volume decrease and bumetanide-sensitive regulatory volume increase, respectively. In contrast, forskolin (i.e. cAMP elevation)-stimulated NKCC1 activity was sustained, and membrane expression and cell volume remained constant. Co-stimulation with forskolin and acetylcholine promoted dramatic recruitment of NKCC1 to basolateral membranes and prolonged the cycle of co-transporter activation, internalization and re-expression. In conclusion, persistent NKCC1 activation by cAMP is constrained by a Ca 2+ -dependent cycle of co-transporter internalization, degradation and re-expression; this is a novel mechanism to limit intestinal fluid loss.

Disruption of the K+ Channel β-Subunit KCNE3 Reveals an Important Role in Intestinal and Tracheal Cl− Transport

“…This model (Fig. 6A) agrees with the co-expression of CFTR (57), NKCC1 (58,59), and KCNQ1/KCNE3 in colonic crypts, the site of fluid and electrolyte secretion (60). In a minimal model, KCNQ1/KCNE3 would provide the only basolateral K ϩ conductance, and CFTR would be the sole apical Cl ever, this picture is not complete, because carbachol increased I sc in the presence of forskolin when KCNQ1/KCNE3 was either inhibited by C293B or absent in kcne3 Ϫ/Ϫ colon (Fig.…”

Sorbitol Dehydrogenase Overexpression and Other Aspects of Dysregulated Protein Expression in Human Precancerous Colorectal Neoplasms: A Quantitative Proteomics Study

“…The sodium-and potassium-coupled chloride cotransporter SLC12A2 is expressed on the basolateral membrane of the normal colon epithelium, where its recruitment and activation are regulated by calcium and cAMP. Loss of SLC12A2 leads to impaired chloride secretion in the intestine (48,49), but to our knowledge, there are no published data linking this protein to colon cancer. The fifth markedly overexpressed protein was SET, one of the five proteins that make up the inhibitor of acetyltranferases (INHAT) complex.…”

Dissociated Gender-Specific Effects of Recurrent Seizures on GABA Signaling in CA1 Pyramidal Neurons: Role of GABAAReceptors

“…Low bumetanide concentrations preferentially inhibit NKCC1, decreasing E GABA (Russell, 2000;Dzhala et al, 2005;Banke and McBain, 2006;Reynolds et al, 2007). NKCC1-ir was similar among the groups, suggesting that bumetanide either inhibited other chloride transporters or led to posttranslational inactivation of NKCC1.…”