K+-Driven Cl-/HCO3- Exchange Mediated by Slc4a8 and Slc4a10.

Slc4a genes encode various types of transporters, including Na+-HCO3- cotransporters, Cl-/HCO3- exchangers, or Na+-driven Cl-/HCO3- exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl-/HCO3- exchanger, which can be driven by either Na+ or K+, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na+ or K+ drive Cl-/HCO3- exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl-/HCO3- exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl- and HCO3--dependent K+ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K+ at the Na+ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K+ in the same direction as HCO3- ions in a similar fashion to that described for Na+ transport in the rat Slc4a8 structure.

A new oxime synthesized from Senecio nutans SCh. Bip (chachacoma) reduces calcium influx in the vascular contractile response in rat aorta.

Senecio nutans Sch. Bip is an endemic plant commonly employed in the Andes culture to counteract the effects of mountain sickness, and its bioactive molecules could provide new drugs for treating hypertension. The purpose was to determine whether the vascular response of the plant bioactive molecules, such as (5-acetyl-6-hydroxy-2-isopropenyl-2,3-dihydrobenzofurane; Sn-I), could be improved by a simple structural modification to synthesize oximes (Ox-Sn-I). We characterized both compounds using IR and NMR spectroscopy and Heteronuclear Multiple Quantum Coherence (HMQC). We investigated vascular relaxation mechanisms in response to Sn-I and Ox-Sn-I using rat aorta and vascular smooth muscle cells (A7r5) as experimental models. Preincubation of aortic rings with Sn-I (10-5 M) significantly (p < 0.001) decreased the contractile effect in response to phenylephrine (PE) and potassium chloride (KCl). The sensitivity (EC50) to PE significantly (p < 0.01) decreased in the presence of Sn-I (10-5 M), but not with Ox-Sn-I. Sn-I significantly (p < 0.001) reduced the PE-induced contraction under calcium-free conditions. When A7r5 cells were preincubated with Sn-I and Ox-Sn-I (10-5 M), both compounds blunted the increase in intracellular Ca2+ induced by KCl. 2,3-Dihydrobenzofurane derived from S. nutans (Sn-I) reduces the contractile response probably by blocking Ca2+ entry through voltage-gated Ca2+ channels (VGCC) in vascular smooth cells. This effect also causes relaxation in rat aorta mediated by reduction of intracellular Ca2+ concentration, rather than an increase of NO generation in endothelial vascular cells.

CFTR expression in human salivary gland acinar cells.

The key role of CFTR in secretory epithelia has been extensively documented. Additionally, CFTR plays a significant role in ion absorption in exocrine glands, including salivary and sweat glands. Most of the knowledge about CFTR expression comes from animal models such as the mouse or the rat, but there is limited information about CFTR expression in human tissues. In the present study, we assessed the expression of CFTR in human submandibular and parotid glands. Consistent with findings in rodent salivary glands, our immunolocalization studies show that CFTR is expressed in duct cells. However, CFTR expression in human salivary glands differs from that in rodents, as immunolocalization and single-cell RNA sequencing analysis from a previous study performed in the human parotid gland revealed the presence of CFTR protein and transcripts within a distinct cell cluster. Based on cell marker expression, this cluster corresponds to acinar cells. To obtain functional evidence supporting CFTR expression, we isolated human parotid acinar cells through collagenase digestion. Acinar cells displayed an anion conductance that was activated in response to cAMP-increasing agents and was effectively blocked by CFTRInh172, a known CFTR blocker. This study provides novel evidence of CFTR expression within acinar cells of human salivary glands. This finding challenges the established model positioning CFTR exclusively in duct cells from exocrine glands.NEW & NOTEWORTHY This study addresses the uncertainty about the impact of CFTR on human salivary gland function. We found CFTR transcripts in a subset of duct cells known as ionocytes, as well as in acinar cells. Isolated human parotid acinar cells exhibited Cl- conductance consistent with CFTR activity. This marks the first documented evidence of functional CFTR expression in human salivary gland acinar cells.

Antispasmodic Effect of Valeriana pilosa Root Essential Oil and Potential Mechanisms of Action: Ex Vivo and In Silico Studies.

Infusions of Valeriana pilosa are commonly used in Peruvian folk medicine for treating gastrointestinal disorders. This study aimed to investigate the spasmolytic and antispasmodic effects of Valeriana pilosa essential oil (VPEO) on rat ileum. The basal tone of ileal sections decreased in response to accumulative concentrations of VPEO. Moreover, ileal sections precontracted with acetylcholine (ACh), potassium chloride (KCl), or barium chloride (BaCl2) were relaxed in response to VPEO by a mechanism that depended on atropine, hyoscine butylbromide, solifenacin, and verapamil, but not glibenclamide. The results showed that VPEO produced a relaxant effect by inhibiting muscarinic receptors and blocking calcium channels, with no apparent effect on the opening of potassium channels. In addition, molecular docking was employed to evaluate VPEO constituents that could inhibit intestinal contractile activity. The study showed that α-cubebene, ß-patchoulene, ß-bourbonene, ß-caryophyllene, α-guaiene, γ-muurolene, valencene, eremophyllene, and δ-cadinene displayed the highest docking scores on muscarinic acetylcholine receptors and voltage-gated calcium channels, which may antagonize M2 and/or M3 muscarinic acetylcholine receptors and block voltage-gated calcium channels. In summary, VPEO has both spasmolytic and antispasmodic effects. It may block muscarinic receptors and calcium channels, thus providing a scientific basis for its traditional use for gastrointestinal disorders.

A hydroalcoholic extract of Senecio nutans SCh. Bip (Asteraceae); its effects on cardiac function and chemical characterization.

ETHNOPHARMACOLOGY RELEVANCE: The plant Senecio nutans SCh. Bip. is used by Andean communities to treat altitude sickness. Recent evidence suggests it may produce vasodilation and negative cardiac inotropy, though the cellular mechanisms have not been elucidated. PURPOSE: To determinate the mechanisms action of S. nutans on cardiovascular function in normotensive animals. METHODS: The effect of the extract on rat blood pressure was measured with a transducer in the carotid artery and intraventricular pressure by a Langendorff system. The effects on sheep ventricular intracellular calcium handling and contractility were evaluated using photometry. Ultra-high-performance liquid-chromatography with diode array detection coupled with heated electrospray-ionization quadrupole-orbitrap mass spectrometric detection (UHPLC-DAD-ESI-Q-OT-MSn) was used for extract chemical characterization. RESULTS: In normotensive rats, S. nutans (10 mg/kg) reduced mean arterial pressure (MAP) by 40% (p < 0.05), causing a dose-dependent coronary artery dilation and decreased left ventricular pressure. In isolated cells, S. nutans extract (1 µg/ml) rapidly reduced the [Ca2+]i transient amplitude and sarcomere shorting by 40 and 49% (p < 0.001), respectively. The amplitude of the caffeine evoked [Ca2+]i transient was reduced by 24% (p < 0.001), indicating reduced sarcoplasmic reticulum (SR) Ca2+ content. Sodium-calcium exchanger (NCX) activity increased by 17% (p < 0.05), while sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) activity was decreased by 21% (p < 0.05). LC-MS results showed the presence of vitamin C, malic acid, and several antioxidant phenolic acids reported for the first time. Dihydroeuparin and 4-hydroxy-3-(3-methylbut-2-enyl) acetophenone were abundant in the extract. CONCLUSION: In normotensive animals, S. nutans partially reduces MAP by decreasing heart rate and cardiac contractility. This negative inotropy is accounted for by decreased SERCA activity and increased NCX activity which reduces SR Ca2+ content. These results highlight the plant's potential as a source of novel cardio-active phytopharmaceuticals or nutraceuticals.

A Direct Interaction between Cyclodextrins and TASK Channels Decreases the Leak Current in Cerebellar Granule Neurons.

Two pore domain potassium channels (K2P) are strongly expressed in the nervous system (CNS), where they play a central role in excitability. These channels give rise to background K+ currents, also known as IKSO (standing-outward potassium current). We detected the expression in primary cultured cerebellar granule neurons (CGNs) of TWIK-1 (K2P1), TASK-1 (K2P3), TASK-3 (K2P9), and TRESK (K2P18) channels by immunocytochemistry and their association with lipid rafts using the specific lipids raft markers flotillin-2 and caveolin-1. At the functional level, methyl-ß-cyclodextrin (MßCD, 5 mM) reduced IKSO currents by ~40% in CGN cells. To dissect out this effect, we heterologously expressed the human TWIK-1, TASK-1, TASK-3, and TRESK channels in HEK-293 cells. MßCD directly blocked TASK-1 and TASK-3 channels and the covalently concatenated heterodimer TASK-1/TASK-3 currents. Conversely, MßCD did not affect TWIK-1- and TRESK-mediated K+ currents. On the other hand, the cholesterol-depleting agent filipin III did not affect TASK-1/TASK-3 channels. Together, the results suggest that neuronal background K+ channels are associated to lipid raft environments whilst the functional activity is independent of the cholesterol membrane organization.

Novel Oxime Synthesized from a Natural Product of Senecio nutans SCh. Bip. (Asteraceae) Enhances Vascular Relaxation in Rats by an Endothelium-Independent Mechanism.

Senecio nutans Sch. Bip. and its constituents are reported to have antihypertensive effects. We isolated metabolite−1, a natural compound from S. nutans (4-hydroxy-3-(isopenten-2-yl)-acetophenone), and synthesized novel oxime − 1 (4-hydroxy-3-(isopenten-2-yl)-acetophenoxime) to evaluate their effect on vascular reactivity. Compounds were purified (metabolite−1) or synthetized (oxime−1) and characterized using IR and NMR spectroscopy and Heteronuclear Multiple Quantum Coherence (HMQC). Using pharmacological agents such as phenylephrine (PE) and KCl (enhancing contraction), acetylcholine (ACh), L-NAME (nitric oxide (NO) and endothelial function), Bay K8644-induced CaV1.2 channel (calcium channel modulator), and isolated aortic rings in an organ bath setup, the possible mechanisms of vascular action were determined. Pre-incubation of aortic rings with 10−5 M oxime−1 significantly (p < 0.001) decreased the contractile response to 30 mM KCl. EC50 to KCl significantly (p < 0.01) increased in the presence of oxime−1 (37.72 ± 2.10 mM) compared to that obtained under control conditions (22.37 ± 1.40 mM). Oxime−1 significantly reduced (p < 0.001) the contractile response to different concentrations of PE (10−7 to 10−5 M) by a mechanism that decreases Cav1.2-mediated Ca2+ influx from the extracellular space and reduces Ca2+ release from intracellular stores. At a submaximal concentration (10−5 M), oxime−1 caused a significant relaxation in rat aorta even without vascular endothelium or after pre-incubate the tissue with L-NAME. Oxime−1 decreases the contractile response to PE by blunting the release of Ca2+ from intracellular stores and blocking of Ca2+ influx by channels. Metabolite−1 reduces the contractile response to KCl, apparently by reducing the plasma membrane depolarization and Ca2+ influx from the extracellular space. These acetophenone derivates from S. nutans (metabolite−1 and oxime−1) cause vasorelaxation through pathways involving an increase of the endothelial NO generation or a higher bioavailability, further highlighting that structural modification of naturally occurring metabolites can enhance their intended pharmacological functions.

Nocturnal Light Pollution Induces Weight Gain in Mice and Reshapes the Structure, Functions, and Interactions of Their Colonic Microbiota.

In mammals, the daily variation in the ecology of the intestinal microbiota is tightly coupled to the circadian rhythm of the host. On the other hand, a close correlation between increased body weight and light pollution at night has been reported in humans and animal models. However, the mechanisms underlying such weight gain in response to light contamination at night remain elusive. In the present study, we tested the hypothesis that dim light pollution at night alters the colonic microbiota of mice, which could correlate with weight gain in the animals. By developing an experimental protocol using a mouse model that mimics light contamination at night in urban residences (dLAN, dim light at night), we found that mice exposed to dLAN showed a significant weight gain compared with mice exposed to control standard light/dark (LD) photoperiod. To identify possible changes in the microbiota, we sampled two stages from the resting period of the circadian cycle of mice (ZT0 and ZT10) and evaluated them by high-throughput sequencing technology. Our results indicated that microbial diversity significantly differed between ZT0 and ZT10 in both LD and dLAN samples and that dLAN treatment impacted the taxonomic composition, functions, and interactions of mouse colonic microbiota. Together, these results show that bacterial taxa and microbial metabolic pathways might be involved with the mechanisms underlying weight gain in mice subjected to light contamination at night.

Activation of the Ae4 (Slc4a9) cation-driven Cl-/HCO3- exchanger by the cAMP-dependent protein kinase in salivary gland acinar cells.

Ae4 transporters are critical for Cl- uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl-/HCO3- exchanger activity in SMG acinar cells from Ae2-/- mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to ß-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH2-terminal domain according to a homology model of Ae4. NH2-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl-/HCO3- exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173.NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon ß-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.

Short Chain Fatty Acids Effect on Chloride Channel ClC-2 as a Possible Mechanism for Lubiprostone Intestinal Action.

Lubiprostone, a 20-carbon synthetic fatty acid used for the treatment of constipation, is thought to act through an action on Cl- channel ClC-2. Short chain fatty acids (SCFAs) are produced and absorbed in the distal intestine. We explore whether SCFAs affect ClC-2, re-examine a possible direct effect of lubiprostone on ClC-2, and use mice deficient in ClC-2 to stringently address the hypothesis that the epithelial effect of lubiprostone targets this anion channel. Patch-clamp whole cell recordings of ClC-2 expressed in mammalian cells are used to assay SCFA and lubiprostone effects. Using chamber measurements of ion current in mice deficient in ClC-2 or CFTR channels served to analyze the target of lubiprostone in the distal intestinal epithelium. Intracellular SCFAs had a dual action on ClC-2, partially inhibiting conduction but, importantly, facilitating the voltage activation of ClC-2. Intra- or extracellular lubiprostone had no effect on ClC-2 currents. Lubiprostone elicited a secretory current across colonic epithelia that was increased in mice deficient in ClC-2, consistent with the channel's proposed proabsorptive function, but absent from those deficient in CFTR. Whilst SCFAs might exert a physiological effect on ClC-2 as part of their known proabsorptive effect, ClC-2 plays no part in the lubiprostone intestinal effect that appears mediated by CFTR activation.

Withaferin A suppresses breast cancer cell proliferation by inhibition of the two-pore domain potassium (K2P9) channel TASK-3.

Withaferin A (WFA), a C5,C6-epoxy steroidal lactone isolated from the medicinal plant Withania somnifera (L.) Dunal, inhibits growth of tumor cells in different cancer types. However, the mechanisms underlying the effect of WFA on tumor cells are not fully understood. In the present study, we evaluated the blockade of TASK-3 channels by WFA in TASK-3-expressing HEK-293 cells. Explore if the WFA-mediated TASK-3 blockade can be used as a pharmacological tool to decrease the cell viability in cancer cells. A combination of functional experiments (patch-clamp, gene downregulation, overexpression and pharmacological inhibition) and molecular docking analysis were used to get insights into the mechanism by which the inhibition of TASK-3 by WFA affects the growth and viability of cancer cells. Withaferin A was found to inhibit the activity of TASK-3 channels. The inhibitory effect of Withaferin A on TASK-3 potassium currents was dose-dependent and independent of voltage. Molecular modeling studies identified putative WFA-binding sites in TASK-3 channel involved the channel blockade. In agreements with the molecular modeling predictions, mutation of residues F125 to A (F125A), L197 to V (L197 V) and the double mutant F125A-L197 V markedly decreased the WFA-induced inhibition of TASK-3. Finally, the cytotoxic effect of WFA was tested in MDA-MB-231 human breast cancer cells transfected with TASK-3 or shRNA that decreases TASK-3 expression. Together, our results show that the cytotoxic effect of WFA on fully transformed MDA-MB-231 cells depends on the expression of TASK-3. Herein, we also provide insights into the mechanism of TASK-3 inhibition by WFA.

Correction: Concha, G., et al. The Insensitivity of TASK-3 K2P Channels to External Tetraethylammonium (TEA) Partially Depends on the Cap Structure. Int. J. Mol. Sci. 2018, 19, 2437.

We would like to submit the following correction to our published paper [...].

Physiological cAMP-elevating secretagogues differentially regulate fluid and protein secretions in mouse submandibular and sublingual glands.

The mechanisms underlying the functional differences in sympathetic and parasympathetic regulation of the major salivary glands have received little attention. The acute effects of parasympathetic muscarinic (carbachol)-dependent and combined parasympathetic-dependent plus cAMP-dependent pathways on fluid secretion rates, ion composition, and protein content were assessed using a newly developed ex vivo preparation that allows the simultaneous perfusion of the mouse submandibular (SMGs) and sublingual glands (SLGs). Our results confirm that the muscarinic-dependent pathway accounts for the bulk of salivation in SMGs and SLGs, whereas costimulation with a cAMP-increasing agent (forskolin, isoproterenol, or vasoactive intestinal peptide) did not increase the flow rate. Costimulation with carbachol plus the ß-adrenergic agonist isoproterenol decreased the concentration of NaCl and produced a substantial increase in the protein and Ca2+ content of SMG but not SLG saliva, consistent with a sparse sympathetic innervation of the SLGs. On the other hand, forskolin, which bypasses receptors to increase intracellular cAMP by directly activating the enzyme adenylate cyclase, enhanced the secretion of protein and Ca2+ by both the SMGs and SLGs. In contrast, isoproterenol and vasoactive intestinal peptide specifically stimulated protein secretion in SMG and SLG salivas, respectively. In summary, cAMP-dependent signaling does not play a major role in the stimulation of fluid secretion in SMGs and SLGs, whereas each cAMP-increasing agonist behaves differently in a gland-specific manner suggesting differential expression of G protein-coupled receptors in the epithelial cells of SMGs and SLGs.

The Insensitivity of TASK-3 K2P Channels to External Tetraethylammonium (TEA) Partially Depends on the Cap Structure.

Two-pore domain K⁺ channels (K2P) display a characteristic extracellular cap structure formed by two M1-P1 linkers, the functional role of which is poorly understood. It has been proposed that the presence of the cap explains the insensitivity of K2P channels to several K⁺ channel blockers including tetraethylammonium (TEA). We have explored this hypothesis using mutagenesis and functional analysis, followed by molecular simulations. Our results show that the deletion of the cap structure of TASK-3 (TWIK-related acid-sensitive K⁺ channel) generates a TEA-sensitive channel with an IC50 of 11.8 ± 0.4 mM. The enhanced sensitivity to TEA displayed by the cap-less channel is also explained by the presence of an extra tyrosine residue at position 99. These results were corroborated by molecular simulation analysis, which shows an increased stability in the binding of TEA to the cap-less channel when a ring of four tyrosine is present at the external entrance of the permeation pathway. Consistently, Y99A or Y205A single-residue mutants generated in a cap-less channel backbone resulted in TASK-3 channels with low affinity to external TEA.

A Mathematical Model Supports a Key Role for Ae4 (Slc4a9) in Salivary Gland Secretion.

We develop a mathematical model of a salivary gland acinar cell with the objective of investigating the role of two [Formula: see text] exchangers from the solute carrier family 4 (Slc4), Ae2 (Slc4a2) and Ae4 (Slc4a9), in fluid secretion. Water transport in this type of cell is predominantly driven by [Formula: see text] movement. Here, a basolateral [Formula: see text] adenosine triphosphatase pump (NaK-ATPase) and a [Formula: see text]-[Formula: see text]-[Formula: see text] cotransporter (Nkcc1) are primarily responsible for concentrating the intracellular space with [Formula: see text] well above its equilibrium potential. Gustatory and olfactory stimuli induce the release of [Formula: see text] ions from the internal stores of acinar cells, which triggers saliva secretion. [Formula: see text]-dependent [Formula: see text] and [Formula: see text] channels promote ion secretion into the luminal space, thus creating an osmotic gradient that promotes water movement in the secretory direction. The current model for saliva secretion proposes that [Formula: see text] anion exchangers (Ae), coupled with a basolateral [Formula: see text] ([Formula: see text]) (Nhe1) antiporter, regulate intracellular pH and act as a secondary [Formula: see text] uptake mechanism (Nauntofte in Am J Physiol Gastrointest Liver Physiol 263(6):G823-G837, 1992; Melvin et al. in Annu Rev Physiol 67:445-469, 2005. https://doi.org/10.1146/annurev.physiol.67.041703.084745 ). Recent studies demonstrated that Ae4 deficient mice exhibit an approximate [Formula: see text] decrease in gland salivation (Peña-Münzenmayer et al. in J Biol Chem 290(17):10677-10688, 2015). Surprisingly, the same study revealed that absence of Ae2 does not impair salivation, as previously suggested. These results seem to indicate that the Ae4 may be responsible for the majority of the secondary [Formula: see text] uptake and thus a key mechanism for saliva secretion. Here, by using 'in-silico' Ae2 and Ae4 knockout simulations, we produced mathematical support for such controversial findings. Our results suggest that the exchanger's cotransport of monovalent cations is likely to be important in establishing the osmotic gradient necessary for optimal transepithelial fluid movement.

The Role of Na:K:2Cl Cotransporter 1 (NKCC1/SLC12A2) in Dental Epithelium during Enamel Formation in Mice.

Na+:K+:2Cl- cotransporters (NKCCs) belong to the SLC12A family of cation-coupled Cl- transporters. We investigated whether enamel-producing mouse ameloblasts express NKCCs. Transcripts for Nkcc1 were identified in the mouse dental epithelium by RT-qPCR and NKCC1 protein was immunolocalized in outer enamel epithelium and in the papillary layer but not the ameloblast layer. In incisors of Nkcc1-null mice late maturation ameloblasts were disorganized, shorter and the mineral density of the enamel was reduced by 10% compared to wild-type controls. Protein levels of gap junction protein connexin 43, Na+-dependent bicarbonate cotransporter e1 (NBCe1), and the Cl--dependent bicarbonate exchangers SLC26A3 and SLC26A6 were upregulated in Nkcc1-null enamel organs while the level of NCKX4/SLC24A4, the major K+, Na+ dependent Ca2+ transporter in maturation ameloblasts, was slightly downregulated. Whole-cell voltage clamp studies on rat ameloblast-like HAT-7 cells indicated that bumetanide increased ion-channel activity conducting outward currents. Bumetanide also reduced cell volume of HAT-7 cells. We concluded that non-ameloblast dental epithelium expresses NKCC1 to regulate cell volume in enamel organ and provide ameloblasts with Na+, K+ and Cl- ions required for the transport of mineral- and bicarbonate-ions into enamel. Absence of functional Nkcc1 likely is compensated by other types of ion channels and ion transporters. The increased amount of Cx43 in enamel organ cells in Nkcc1-null mice suggests that these cells display a higher number of gap junctions to increase intercellular communication.

Ae4 (Slc4a9) is an electroneutral monovalent cation-dependent Cl-/HCO3- exchanger.

Ae4 (Slc4a9) belongs to the Slc4a family of Cl(-)/HCO3 (-) exchangers and Na(+)-HCO3 (-) cotransporters, but its ion transport cycle is poorly understood. In this study, we find that native Ae4 activity in mouse salivary gland acinar cells supports Na(+)-dependent Cl(-)/HCO3 (-) exchange that is comparable with that obtained upon heterologous expression of mouse Ae4 and human AE4 in CHO-K1 cells. Additionally, whole cell recordings and ion concentration measurements demonstrate that Na(+) is transported by Ae4 in the same direction as HCO3 (-) (and opposite to that of Cl(-)) and that ion transport is not associated with changes in membrane potential. We also find that Ae4 can mediate Na(+)-HCO3 (-) cotransport-like activity under Cl(-)-free conditions. However, whole cell recordings show that this apparent Na(+)-HCO3 (-) cotransport activity is in fact electroneutral HCO3 (-)/Na(+)-HCO3 (-) exchange. Although the Ae4 anion exchanger is thought to regulate intracellular Cl(-) concentration in exocrine gland acinar cells, our thermodynamic calculations predict that the intracellular Na(+), Cl(-), and HCO3 (-) concentrations required for Ae4-mediated Cl(-) influx differ markedly from those reported for acinar secretory cells at rest or under sustained stimulation. Given that K(+) ions share many properties with Na(+) ions and reach intracellular concentrations of 140-150 mM (essentially the same as extracellular [Na(+)]), we hypothesize that Ae4 could mediate K(+)-dependent Cl(-)/HCO3 (-) exchange. Indeed, we find that Ae4 mediates Cl(-)/HCO3 (-) exchange activity in the presence of K(+) as well as Cs(+), Li(+), and Rb(+) In summary, our results strongly suggest that Ae4 is an electroneutral Cl(-)/nonselective cation-HCO3 (-) exchanger. We postulate that the physiological role of Ae4 in secretory cells is to promote Cl(-) influx in exchange for K(+)(Na(+)) and HCO3 (-) ions.

Ae4 (Slc4a9) Anion Exchanger Drives Cl- Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells.

Transcellular Cl(-) movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na(+)-K(+)-2Cl(-) cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl(-) above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl(-) uptake pathway concentrates Cl(-) ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl(-)/HCO3 (-) exchangers. We found that salivation stimulated by muscarinic and ß-adrenergic receptor agonists was normal in the submandibular glands of Ae2(-/-) mice. In contrast, saliva secretion was reduced by 35% in Ae4(-/-) mice. The decrease in salivation was not related to loss of Na(+)-K(+)-2Cl(-) cotransporter or Na(+)/H(+) exchanger activity in Ae4(-/-) mice but correlated with reduced Cl(-) uptake during ß-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl(-)/HCO3 (-) exchanger activity revealed that HCO3 (-)-dependent Cl(-) uptake was reduced in the acinar cells of Ae2(-/-) and Ae4(-/-) mice. Moreover, Cl(-)/HCO3 (-) exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl(-)/HCO3 (-) exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.

A fluid secretion pathway unmasked by acinar-specific Tmem16A gene ablation in the adult mouse salivary gland.

Activation of an apical Ca(2+)-activated Cl(-) channel (CaCC) triggers the secretion of saliva. It was previously demonstrated that CaCC-mediated Cl(-) current and Cl(-) efflux are absent in the acinar cells of systemic Tmem16A (Tmem16A Cl(-) channel) null mice, but salivation was not assessed in fully developed glands because Tmem16A null mice die within a few days after birth. To test the role of Tmem16A in adult salivary glands, we generated conditional knockout mice lacking Tmem16A in acinar cells (Tmem16A(-/-)). Ca(2+)-dependent salivation was abolished in Tmem16A(-/-) mice, demonstrating that Tmem16A is obligatory for Ca(2+)-mediated fluid secretion. However, the amount of saliva secreted by Tmem16A(-/-) mice in response to the ß-adrenergic receptor agonist isoproterenol (IPR) was comparable to that seen in controls, indicating that Tmem16A does not significantly contribute to cAMP-induced secretion. Furthermore, IPR-stimulated secretion was unaffected in mice lacking Cftr (Cftr(∆F508/∆F508)) or ClC-2 (Clcn2(-/-)) Cl(-) channels. The time course for activation of IPR-stimulated fluid secretion closely correlated with that of the IPR-induced cell volume increase, suggesting that acinar swelling may activate a volume-sensitive Cl(-) channel. Indeed, Cl(-) channel blockers abolished fluid secretion, indicating that Cl(-) channel activity is critical for IPR-stimulated secretion. These data suggest that ß-adrenergic-induced, cAMP-dependent fluid secretion involves a volume-regulated anion channel. In summary, our results using acinar-specific Tmem16A(-/-) mice identify Tmem16A as the Cl(-) channel essential for muscarinic, Ca(2+)-dependent fluid secretion in adult mouse salivary glands.

Ca²âº-dependent K⁺ channels in exocrine salivary glands.

In the last 15 years, remarkable progress has been realized in identifying the genes that encode the ion-transporting proteins involved in exocrine gland function, including salivary glands. Among these proteins, Ca(2+)-dependent K(+) channels take part in key functions including membrane potential regulation, fluid movement and K(+) secretion in exocrine glands. Two K(+) channels have been identified in exocrine salivary glands: (1) a Ca(2+)-activated K(+) channel of intermediate single channel conductance encoded by the KCNN4 gene, and (2) a voltage- and Ca(2+)-dependent K(+) channel of large single channel conductance encoded by the KCNMA1 gene. This review focuses on the physiological roles of Ca(2+)-dependent K(+) channels in exocrine salivary glands. We also discuss interesting recent findings on the regulation of Ca(2+)-dependent K(+) channels by protein-protein interactions that may significantly impact exocrine gland physiology.