Wasted TMEM16A channels are rescued by phosphatidylinositol 4,5-bisphosphate

Recently there has been a flurry of interest in the regulation of the homo-dimeric calcium-activated chloride channel ANO1 (also known as TMEM16A) by phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂). These recent studies show that upon Ca²⁺ binding, PI(4,5)P₂ cooperates to maintain the conductive state of ANO1. PI(4,5)P₂ does so by binding to sites or modules on the protein’s cytosolic side. These findings add a new function to the PI(4,5)P₂ repertoire and a new dimension to ANO1 gating.     

Phosphate stimulates CFTR Cl- channels.

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.     

On the mechanism of MgATP-dependent gating of CFTR Cl- channels

CFTR, the product of the gene mutated in cystic fibrosis, is an ATPase that functions as a Cl(-) channel in which bursts of openings separate relatively long interburst closed times (tauib). Channel gating is controlled by phosphorylation and MgATP, but the underlying molecular mechanisms remain controversial. To investigate them, we expressed CFTR channels in Xenopus oocytes and examined, in excised patches, how gating kinetics of phosphorylated channels were affected by changes in [MgATP], by alterations in the chemical structure of the activating nucleotide, and by mutations expected to impair nucleotide hydrolysis and/or diminish nucleotide binding affinity. The rate of opening to a burst (1/tauib) was a saturable function of [MgATP], but apparent affinity was reduced by mutations in either of CFTR's nucleotide binding domains (NBDs): K464A in NBD1, and K1250A or D1370N in NBD2. Burst duration of neither wild-type nor mutant channels was much influenced by [MgATP]. Poorly hydrolyzable nucleotide analogs, MgAMPPNP, MgAMPPCP, and MgATPgammaS, could open CFTR channels, but only to a maximal rate of opening approximately 20-fold lower than attained by MgATP acting on the same channels. NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Corresponding NBD1 mutations did not affect timing of burst termination in normal, hydrolytic conditions. However, when hydrolysis at NBD2 was impaired, the NBD1 mutation K464A shortened the prolonged open bursts. In light of recent biochemical and structural data, the results suggest that: nucleotide binding to both NBDs precedes channel opening; at saturating nucleotide concentrations the rate of opening to a burst is influenced by the structure of the phosphate chain of the activating nucleotide; normal, rapid exit from bursts occurs after hydrolysis of the nucleotide at NBD2, without requiring a further nucleotide binding step; if hydrolysis at NBD2 is prevented, exit from bursts occurs through a slower pathway, the rate of which is modulated by the structure of the NBD1 catalytic site and its bound nucleotide. Based on these and other results, we propose a mechanism linking hydrolytic and gating cycles via ATP-driven dimerization of CFTR's NBDs.     

Calmodulin regulation of TMEM16A and 16B Ca2+-activated chloride channels

Ca²⁺-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle contraction, olfactory transduction, and cell proliferation. Whether and how the ubiquitous Ca²⁺ sensor calmodulin (CaM) regulates the activity of TMEM16A and 16B channels has been controversial and the subject of an ongoing debate. Recently, using a bioengineering approach termed ChIMP (Channel Inactivation induced by Membrane-tethering of an associated Protein) we argued that Ca²⁺-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI) of some TMEM16A splice variants. In this review, we discuss these findings in the context of previous and recent results relating to Ca²⁺-dependent regulation of TMEM16A/16B channels and the putative role of CaM. We further discuss potential future directions for these nascent ideas on apoCaM regulation of TMEM16A/16B channels, noting that such future efforts will benefit greatly from the pioneering work of Dr. David T. Yue and colleagues on CaM regulation of voltage-dependent calcium channels.     

Functional Swapping between Transmembrane Proteins TMEM16A and TMEM16F

The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca²⁺-dependent Cl⁻ ion transport, and TMEM16F is responsible for Ca²⁺-dependent phospholipid scrambling. Here we established assay systems for the Ca²⁺-dependent Cl⁻ channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F^−/− immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl⁻ channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl⁻ channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.     

Developmental expression of the calcium‐activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium

Calcium‐activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium‐activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 657–675, 2014     

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na⁺ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na⁺. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.     

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na⁺ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na⁺. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4''-Diisothiocyano-2,2''-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.     

Compartmentalized crosstalk of CFTR and TMEM16A (ANO1) through EPAC1 and ADCY1

Airway epithelial cells express both Ca²⁺ activated TMEM16A/ANO1 and cAMP activated CFTR anion channels. Previous work suggested a significant crosstalk of intracellular Ca²⁺ and cAMP signaling pathways, leading to activation of both chloride channels. We demonstrate that in airway epithelial cells, stimulation of purinergic or muscarinic G-protein coupled receptors (GPCRs) activates TMEM16A and CFTR. Additional expression of G_q/11 and phospholipase C coupled GPCRs strongly enhanced the crosstalk between Ca²⁺- and cAMP-dependent signaling. Knockdown of endogenous GRCRs attenuated crosstalk and functional coupling between TMEM16A and CFTR. The number of receptors did not affect expression or membrane localization of TMEM16A or CFTR, but controlled assembly of the local signalosome. GPCRs translocate Ca²⁺-sensitive adenylate cyclase type 1 (ADCY1) and exchange protein directly activated by cAMP (EPAC1) to particular plasma membrane domains containing GPCRs, CFTR and TMEM16A, thereby producing compartmentalized Ca²⁺ and cAMP signals and significant crosstalk. While biosynthesis and membrane trafficking of CFTR requires a functional Golgi apparatus, maturation and membrane trafficking of TMEM16A may occur independent of the Golgi. Because Ca²⁺ activated TMEM16A currents are only transient, continuous Cl⁻ secretion by airway epithelial cells requires CFTR. The present data also explain why receptor-dependent activation of TMEM16A is more efficient than direct stimulation by Ca²⁺.     

Sinupret Activates CFTR and TMEM16A-Dependent Transepithelial Chloride Transport and Improves Indicators of Mucociliary Clearance

Introduction

We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl⁻) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.

Methods

Primary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR^−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl⁻ transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca²⁺]_i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.

Results

Sinupret-mediated Cl⁻ secretion [ΔI_SC(µA/cm²)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR^−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca²⁺ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl⁻ secretion. Sinupret also enhanced CBF and ASL depth.

Conclusion

Sinupret stimulates CBF, promotes transepithelial Cl⁻ secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca²⁺-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl⁻ secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.     

Role of separate K+ and Cl- channels and of Na+/Cl- cotransport in volume regulation in Ehrlich cells

Cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but later recover their volume with an associated KCl loss. This regulatory volume decrease (RVD) is unaffected when nitrate is substituted for Cl- or if bumetanide or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) is added. It is inhibited by quinine, Ba2+, low pH, anticalmodulin drugs, and depletion of intracellular Ca2+. It is accelerated by the Ca2+ ionophore A23187, or by a sudden increase in external Ca2+ and at high pH. A net KCl loss is also seen after addition of ionophore A23187 in isotonic medium. Similarities are demonstrated between the KCl loss seen after addition of A23187 and the KCl loss seen during RVD. It is proposed that separate conductive K+ and Cl- channels are activated during RVD by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin. After restoration of tonicity the cells shrink initially, but recover their volume with an associated KCl uptake. This regulatory volume increase (RVI) is inhibited when NO3- is substituted for Cl-, and is also inhibited by furosemide or bumetanide, but it is unaffected by DIDS. The unidirectional Cl-flux ratio is compatible with either a coupled uptake of Na+ and Cl-, or an uptake via a K+/Na+/2Cl- cotransport system. No K+ uptake was found, however, in ouabain-poisoned cells where a bumetanide-sensitive uptake of Na+ and Cl- in nearly equimolar amounts was demonstrated. Therefore, it is proposed that the primary process during RVI is an activation of an otherwise quiescent Na+/Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump. There is a marked increase in the rate of pump activity in the absence of a detectable increase in intracellular Na+ concentration.     

单羧酸类Cl-通道阻断剂对心室肌CFTR Cl-通道的影响

本文采用全细胞膜片箝与细胞内灌注技术,观察了单羧酸类Ｃｌ＾－通道阻断剂对豚鼠心室肌囊性纤维变性膜透性调节蛋白（ＣＦＴＲ）Ｃｌ＾－电流的影响,细胞包９－ＡＣ以可逆方式增强异丙肾上腺素（ＩＳＯ）激发的ＣＦＴＲＣｌ＾－的外向电流成分,５－ｎｉｔｒｏ－２－（３－ｐｈｅｎｙｌｐｒｏｐｙｌａｍｉｎｏ）－ｂｅｎｚｏａｔｅ（ＮＰＰＢ）和二苯胺羧酸（ＤＰＣ）对ＩＳＯ发的ＣＦＴＲＣｌ＾－电流的作用呈现先增强后抑制的双     

Interdomain but not intermolecular interactions observed in CFTR channels.

Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) channels requires interdomain and/or intermolecular interactions involving different parts of the protein, yet the exact nature of those interactions remains unclear. In this study we report that treating wild type CFTR-expressing cells with oxidizing agents results in a significant reduction in the gel mobility of the protein indicative of the formation of disulfide bonds. In contrast, mutant CFTR channels in which cysteine residues in both nucleotide binding domains (NBDs) were mutated to serine, showed little change in gel mobility in oxidizing conditions. Mutation of the two cysteine residues in either the first or the second NBD alone also eliminates the change in gel mobility in oxidizing conditions. Wild type channels treated with oxidizing agents did not appear to form disulfide bonds with other proteins, suggesting that the close association that allows the formation of disulfide bonds occurs only within single proteins and not between separate channels interacting in a multimer.     

TMEM16 Proteins Produce Volume-regulated Chloride Currents That Are Reduced in Mice Lacking TMEM16A

All vertebrate cells regulate their cell volume by activating chloride channels of unknown molecular identity, thereby activating regulatory volume decrease. We show that the Ca²⁺-activated Cl⁻ channel TMEM16A together with other TMEM16 proteins are activated by cell swelling through an autocrine mechanism that involves ATP release and binding to purinergic P2Y₂ receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca²⁺ and a Ca²⁺-independent mechanism engaging extracellular-regulated protein kinases (ERK1/2). The ability of epithelial cells to activate a Cl⁻ conductance upon cell swelling, and to decrease their cell volume (regulatory volume decrease) was dependent on TMEM16 proteins. Activation of I_Cl,swell was reduced in the colonic epithelium and in salivary acinar cells from mice lacking expression of TMEM16A. Thus TMEM16 proteins appear to be a crucial component of epithelial volume-regulated Cl⁻ channels and may also have a function during proliferation and apoptotic cell death.Regulation of cell volume is fundamental to all cells, particularly during cell growth and division. External hypotonicity leads to cell swelling and subsequent activation of volume-regulated chloride and potassium channels, to release intracellular ions and to re-shrink the cells, a process termed regulatory volume decrease (RVD)³ (1). Volume-regulated chloride currents (I_Cl,swell) have dual functions during cell proliferation as well as apoptotic volume decrease (AVD), preceding apoptotic cell death (2). Although I_Cl,swell is activated in swollen cells to induce RVD, AVD takes place under normotonic conditions to shrink cells (3, 4). Early work suggested intracellular Ca²⁺ as an important mediator for activation of I_Cl,swell and volume-regulated K⁺ channels (5), whereas subsequent studies only found a permissive role of Ca²⁺ for activation of I_Cl,swell (6), reviewed in Ref. 1. In addition, a plethora of factors and signaling pathways have been implicated in activation of I_Cl,swell, making cell volume regulation an extremely complex process (reviewed in Refs. 1, 3, and 7). These factors include intracellular ATP, the cytoskeleton, phospholipase A2-dependent pathways, and protein kinases such as extracellular-regulated kinase ERK1/2 (reviewed in Refs. 1 and 7). Previous approaches in identifying swelling-activated Cl⁻ channels have been unsuccessful or have produced controversial data. Thus none of the previous candidates such as pI_Cln, the multidrug resistance protein, or ClC-3 are generally accepted to operate as volume-regulated Cl⁻ channels (reviewed in Refs. 8 and 9). Notably, the cystic fibrosis transmembrane conductance regulator (CFTR) had been shown in earlier studies to influence I_Cl,swell and volume regulation (10–12). The variable properties of I_Cl,swell suggest that several gene products may affect I_Cl,swell in different cell types.The TMEM16 transmembrane protein family consists of 10 different proteins with numerous splice variants that contain 8–9 transmembrane domains and have predicted intracellular N- and C-terminal tails (13, 16–18). TMEM16A (also called ANO1) is required for normal development of the murine trachea (14) and is associated with different types of tumors, dysplasia, and nonsyndromic hearing impairment (13, 15). TMEM16A has been identified as a subunit of Ca²⁺-activated Cl⁻ channels that are expressed in epithelial and non-epithelial tissues (16–18). Interestingly, members of the TMEM16 family have been suggested to play a role in osmotolerance in Saccharomyces cerevisiae (19). Here we show that TMEM16 proteins also contribute to I_Cl,swell and regulatory volume decrease.     

Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia

Cystic fibrosis (CF) is caused by the functional expression defect of the CF transmembrane conductance regulator (CFTR) chloride channel at the apical plasma membrane. Impaired bacterial clearance and hyperactive innate immune response are hallmarks of the CF lung disease, yet the existence of and mechanism accounting for the innate immune defect that occurs before infection remain controversial. Inducible expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuated the proinflammatory cytokines interleukin-6 (IL-6), IL-8, and CXCL1/2 in two human respiratory epithelial models under air–liquid but not liquid–liquid interface culture. Expression of wild-type but not the inactive G551D-CFTR indicates that secretion of the chemoattractant IL-8 is inversely proportional to CFTR channel activity in cftr^{∆F508/∆F508} immortalized and primary human bronchial epithelia. Similarly, direct but not P2Y receptor–mediated activation of TMEM16A attenuates IL-8 secretion in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport at the apical membrane may contribute to the excessive and persistent lung inflammation in CF and perhaps in other respiratory diseases associated with documented down-regulation of CFTR (e.g., chronic obstructive pulmonary disease). Direct pharmacological activation of TMEM16A offers a potential therapeutic strategy to reduce the inflammation of CF airway epithelia.     

Pharmacological characterization of TMEM16A currents

Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACC_inhA01 and T16A_inhA01) on TMEM16A currents. The whole cell patch clamp technique was used to record TMEM16A currents from HEK 293 cells that stably expressed human TMEM16A. Niflumic acid, A-9-C, CACC_inhA01 and T16A_inhA01 inhibited TMEM16A currents with IC₅₀ values of 12, 58, 1.7 and 1.5 µM, respectively, however, A-9-C and niflumic acid were less efficacious at negative membrane potentials. A-9-C and niflumic acid reduced the rate of TMEM16A tail current deactivation at negative membrane potentials and A-9-C (1 mM) enhanced peak TMEM16A tail current amplitude. In contrast, the inhibitory effects of CACC_inhA01 and T16A_inhA01 were independent of voltage and they did not prolong the rate of TMEM16A tail current deactivation. The effects of niflumic acid and A-9-C on TMEM16A currents were similar to previous observations on CACCs in vascular smooth muscle, strengthening the hypothesis that they are encoded by TMEM16A. However, CACC_inhA01 and T16A_inhA01 were more potent inhibitors of TMEM16A channels and their effects were not diminished at negative membrane potentials making them attractive candidates to interrogate the functional role of TMEM16A channels in future studies.     

Bestrophin Cl- channels are highly permeable to HCO3-

Bestrophin-1 (Best1) is a Cl(-) channel that is linked to various retinopathies in both humans and dogs. Dysfunction of the Best1 Cl(-) channel has been proposed to cause retinopathy because of altered Cl(-) transport across the retinal pigment epithelium (RPE). In addition to Cl(-), many Cl(-) channels also transport HCO3(-). Because HCO3(-) is physiologically important in pH regulation and in fluid and ion transport across the RPE, we measured the permeability and conductance of bestrophins to HCO3(-) relative to Cl(-). Four human bestrophin homologs (hBest1, hBest2, hBest3, and hBest4) and mouse Best2 (mBest2) were expressed in HEK cells, and the relative HCO3(-) permeability (P HCO3/PCl) and conductance (G HCO3/GCl) were determined. P HCO3/PCl was calculated from the change in reversal potential (Erev) produced by replacing extracellular Cl(-) with HCO3(-). hBest1 was highly permeable to HCO3(-) (P HCO3)/PCl = approximately 0.44). hBest2, hBest4, and mBest2 had an even higher relative HCO3(-) permeability (P HCO3/PCl = 0.6-0.7). All four bestrophins had HCO3(-) conductances that were nearly the same as Cl(-) (G HCO3/GCl = 0.9-1.1). Extracellular Na+ did not affect the permeation of hBest1 to HCO3(-). At physiological HCO3(-) concentration, HCO3(-) was also highly conductive. The hBest1 disease-causing mutations Y85H, R92C, and W93C abolished both Cl(-) and HCO3(-) currents equally. The V78C mutation changed P HCO3/PCl and G HCO3/GCl of mBest2 channels. These results raise the possibility that disease-causing mutations in hBest1 produce disease by altering HCO3(-) homeostasis as well as Cl(-) transport in the retina.     

Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity.

beta-Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamyl-beta-alanine as the sole nitrogen source and exhibits diminished beta-alanine synthase activity was used to clone analogous genes from different eukaryotes. Putative PYD3 sequences from the yeast S. kluyveri, the slime mold Dictyostelium discoideum, and the fruit fly Drosophila melanogaster complemented the pyd3 defect. When the S. kluyveri PYD3 gene was expressed in S. cerevisiae, which has no pyrimidine catabolic pathway, it enabled growth on N-carbamyl-beta-alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta-alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial N-carbamyl amidohydrolases. All three beta-alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N-carbamyl-beta-alanine, but not by uracil. This work establishes S. kluyveri as a model organism for studying pyrimidine degradation and beta-alanine production in eukaryotes.