CLCA proteins were discovered in bovine trachea and named for a calcium-dependent chloride conductance found in trachea and in other secretory epithelial tissues. At least four closely located gene loci in the mouse and the human code for independent isoforms of CLCA proteins. Full-length CLCA proteins have an unprocessed mass ratio of ∼100 kDa. Three of the four human loci code for the synthesis of membrane-associated proteins. CLCA proteins affect chloride conductance, epithelial secretion, cell-cell adhesion, apoptosis, cell cycle control, mucus production in asthma, and blood pressure. There is a structural and probable functional divergence between CLCA isoforms containing or not containing β4-integrin binding domains. Cell cycle control and tumor metastasis are affected by isoforms with the binding domains. These isoforms are expressed prominently in smooth muscle, in some endothelial cells, in the central nervous system, and also in secretory epithelial cells. The isoform with disrupted β4-integrin binding (hCLCA1, pCLCA1, mCLCA3) alters epithelial mucus secretion and ion transport processes. It is preferentially expressed in secretory epithelial tissues including trachea and small intestine. Chloride conductance is affected by the expression of several CLCA proteins. However, the dependence of the resulting electrical signature on the expression system rather than the CLCA protein suggests that these proteins are not independent Ca2+-dependent chloride channels, but may contribute to the activity of chloride channels formed by, or in conjunction with, other proteins.

Two Cl− conductances have been described in the apical membrane of both human and murine proximal airway epithelia that are thought to play predominant roles in airway hydration: (1) CFTR, which is cAMP regulated and (2) the Ca2+-activated Cl− conductance (CaCC) whose molecular identity is uncertain. In addition to second messenger regulation, cross talk between these two channels may also exist and, whereas CFTR is absent or defective in cystic fibrosis (CF) airways, CaCC is preserved, and may even be up-regulated. Increased CaCC activity in CF airways is controversial. Hence, we have investigated the effects of CFTR on CaCC activity and have also assessed the relative contributions of these two conductances to airway surface liquid (ASL) height (volume) in murine tracheal epithelia. We find that CaCC is up-regulated in intact murine CF tracheal epithelia, which leads to an increase in UTP-mediated Cl−/volume secretion. This up-regulation is dependent on cell polarity and is lost in nonpolarized epithelia. We find no role for an increased electrical driving force in CaCC up-regulation but do find an increased Ca2+ signal in response to mucosal nucleotides that may contribute to the increased Cl−/volume secretion seen in intact epithelia. CFTR plays a critical role in maintaining ASL height under basal conditions and accordingly, ASL height is reduced in CF epithelia. In contrast, CaCC does not appear to significantly affect basal ASL height, but does appear to be important in regulating ASL height in response to released agonists (e.g., mucosal nucleotides). We conclude that both CaCC and the Ca2+ signal are increased in CF airway epithelia, and that they contribute to acute but not basal regulation of ASL height.

Little is known about the postinternalization trafficking of surface-expressed voltage-gated potassium channels. Here, for the first time, we investigate into which of four major trafficking pathways a voltage-gated potassium channel is targeted after internalization. In both a cardiac myoblast cell line and in HEK293 cells, channels were found to internalize and to recycle quickly. Upon internalization, Kv1.5 rapidly associated with Rab5-and Rab4-positive endosomes, suggesting that the channel is internalized via a Rab5-dependent pathway and rapidly targeted for recycling to the plasma membrane. Nevertheless, as indicated by colocalization with Rab7, a fraction of the channels are targeted for degradation. Recycling through perinuclear endosomes is limited; colocalization with Rab11 was evident only after 24 h postsurface labelling. Expression of dominant negative (DN) Rab constructs significantly increased Kv1.5 functional expression. In the myoblast line, Rab5DN increased Kv1.5 current densities to 1305 ± 213 pA pF −1 from control 675 ± 81.6 pA pF −1 . Rab4DN similarly increased Kv1.5 currents to 1382 ± 155 pA pF −1 from the control 522 ± 82.7 pA pF −1 at +80 mV. Expression of the Rab7DN increased Kv1.5 currents 2.5-fold in HEK293 cells but had no significant effect in H9c2 myoblasts, and, unlike the other Rab GTPases tested, over-expression of wild-type Rab7 decreased Kv1.5 currents in the myoblast line. Densities fell to 573 ± 96.3 pA pF −1 from the control 869 ± 135.5 pA pF −1 . The Rab11DN was slow to affect Kv1.5 currents but had comparable effects to other dominant negative constructs after 48 h. With the exception of Rab11DN and nocodazole, the effects of interference with microtubule-dependent trafficking by nocodazole or p50 overexpression were not additive with the Rab dominant negatives. The Rab GTPases thus constitute dynamic targets by which cells may modulate Kv1.5 functional expression.

The objectives of this study were to determine the effect of an increase in diet fermentability on 1) the rate and extent to which short-chain fatty acid (SCFA) absorption pathways adapt relative to changes in Na(+) transport, 2) the epithelial surface area (SA), and 3) the barrier function of the bovine ruminal epithelium. Twenty-five Holstein steer calves were assigned to either the control diet (CON; 91.5% hay and 8.5% supplement) or a moderately fermentable diet (50% hay; 41.5% barley grain (G), and 8.5% supplement) fed for 3 (G3), 7 (G7), 14 (G14), or 21 days (G21). All calves were fed at 2.25% body weight at 0800. Calves were killed (at 1000), and ruminal tissue was collected to determine the rate and pathway of SCFA transport, Na(+) transport and barrier function in Ussing chambers. Tissue was also collected for SA measurement and gene expression. Mean reticular pH decreased from 6.90 for CON to 6.59 for G7 and then increased (quadratic P < 0.001). While effective SA of the ruminal epithelium was not affected (P > 0.10) by dietary treatment, the net Na(+) flux increased by 125% within 7 days (quadratic P = 0.016). Total acetate and butyrate flux increased from CON to G21, where passive diffusion was the primary SCFA absorption pathway affected. Increased mannitol flux, tissue conductance, and tendencies for increased expression of IL-1β and TLR2 indicated reduced rumen epithelium barrier function. This study indicates that an increase in diet fermentability acutely increases Na(+) and SCFA absorption in the absence of increased SA, but reduces barrier function.

The CLCA gene family produces both secreted and membrane-associated proteins that modulate ion-channel function, drive mucus production and have a poorly understood pleiotropic effect on airway inflammation. The primary up-regulated human CLCA ortholog in airway inflammation is hCLCA1. Here we show that this protein can activate airway macrophages, inducing them to express cytokines and to undertake a pivotal role in airway inflammation. In a U-937 airway macrophage–monocyte cell line, conditioned media from HEK 293 cells heterologously expressing hCLCA1 (with or without fetal bovine serum) increased the levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and IL-8). This effect was independent of the metalloprotease domain of hCLCA1. Primary porcine alveolar macrophages were similarly activated, demonstrating the effect was not cell line dependent. Similarly, immuno-purified hCLCA1 at physiologically relevant concentration of ~100 pg/mL was able to activate macrophages and induce pro-inflammatory response. This cytokine response increased with higher concentration of immuno-purified hCLCA1. These findings demonstrate the ability of hCLCA1 to function as a signaling molecule and activate macrophages, central regulators of airway inflammation.