Distinct TRP channels are required for warm and cool avoidance in Drosophila melanogaster

The ability to sense and respond to subtle variations in environmental temperature is critical for animal survival. Animals avoid temperatures that are too cold or too warm and seek out temperatures favorable for their survival. At the molecular level, members of the transient receptor potential (TRP) family of cation channels contribute to thermosensory behaviors in animals from flies to humans. In Drosophila melanogaster larvae, avoidance of excessively warm temperatures is known to require the TRP protein dTRPA1. Whether larval avoidance of excessively cool temperatures also requires TRP channel function, and whether warm and cool avoidance use the same or distinct TRP channels has been unknown. Here we identify two TRP channels required for cool avoidance, TRPL and TRP. Although TRPL and TRP have previously characterized roles in phototransduction, their function in cool avoidance appears to be distinct, as neither photoreceptor neurons nor the phototransduction regulators NORPA and INAF are required for cool avoidance. TRPL and TRP are required for cool avoidance; however they are dispensable for warm avoidance. Furthermore, cold-activated neurons in the larvae are required for cool but not warm avoidance. Conversely, dTRPA1 is essential for warm avoidance, but not cool avoidance. Taken together, these data demonstrate that warm and cool avoidance in the Drosophila larva involves distinct TRP channels and circuits.     

Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis

Epithelia frequently segregate transport processes to specific cell types, presumably for improved efficiency and control. The molecular players underlying this functional specialization are of particular interest. In Drosophila, the renal (Malpighian) tubule displays the highest per-cell transport rates known and has two main secretory cell types, principal and stellate. Electrogenic cation transport is known to reside in the principal cells, whereas stellate cells control the anion conductance, but by an as-yet-undefined route. Here, we resolve this issue by showing that a plasma membrane chloride channel, encoded by ClC-a, is exclusively expressed in the stellate cell and is required for Drosophila kinin-mediated induction of diuresis and chloride shunt conductance, evidenced by chloride ion movement through the stellate cells, leading to depolarization of the transepithelial potential. By contrast, ClC-a knockdown had no impact on resting secretion levels. Knockdown of a second CLC gene showing highly abundant expression in adult Malpighian tubules, ClC-c, did not impact depolarization of transepithelial potential after kinin stimulation. Therefore, the diuretic action of kinin in Drosophila can be explained by an increase in ClC-a–mediated chloride conductance, over and above a resting fluid transport level that relies on other (ClC-a–independent) mechanisms or routes. This key segregation of cation and anion transport could explain the extraordinary fluid transport rates displayed by some epithelia.Epithelia provide essential barrier and vectorial transport capabilities intrinsic to the success of higher organisms. Depending on their roles and specializations, epithelia may be termed tight or leaky, based on their electrical conductance, which in turn depends on the patency of their paracellular spaces. Tight epithelia are thought to have a highly restricted paracellular route because of prominent tight junctions (septate junctions in insects), so constraining transepithelial fluxes to a transcellular route. Leaky epithelia, traditionally associated with high flux rates, lack such prominent tight junctions.Insect renal (Malpighian) tubules move fluid at the highest rates observed in biology, and thus would seem to be ideal candidates for leaky epithelia. However, insect tubule cells are typically large and multinucleate or polyploid (large cell diameters minimize the junctional circumference per unit transporting membrane), and the cells are also surrounded by prominent septate junctions (1). Nonetheless, it has been argued that, in tubules of the dengue fever mosquito Aedes aegypti, the rapid neurohormonally controlled chloride shunt conductance is paracellular, caused by remodeling of the septate junctions with switch-like speed under the influence of diuretic peptides of the kinin family (2, 3).Drosophila melanogaster is a member of one of the largest insect orders, the Diptera, and these tubules are distinguished by a prominent secondary cell type, the stellate cell. The powerful transgenic toolbox available for Drosophila allows cell-specific contributions to tissue-level function to be probed; by expressing the luminescent calcium reporter apoaequorin transgenically in specific cell types, it was possible to show that kinin signals specifically through intracellular calcium in only the stellate cells (4)—and not the principal cells—consistent with the observed expression of the kinin receptor in just stellate cells (5). The biogenic amine tyramine also acts similarly; that is, it stimulates fluid secretion by activating a chloride shunt conductance by raising intracellular calcium levels in the stellate cells (6, 7). Thus—at least in Drosophila—the stellate cell plays a crucial role in transducing the diuretic kinin signal into a rapid increase in chloride conductance (8).In practice, both transcellular and paracellular routes of chloride conductance are likely to contribute to both resting and kinin-stimulated fluid secretion (Fig. 1), but the relative importance of the two routes is not known. Here, we use Drosophila transgenics—combined with physiology, electrophysiology, and imaging—to show that a CLC chloride channel encoded by ClC-a/CG31116 is a basolateral and apical plasma membrane chloride channel, uniquely localized to stellate cells, that is essential for neuropeptide-stimulated, but not resting, levels of secretion. Furthermore, by generating Drosophila that were transgenic for a membrane-targeted fluorescent chloride reporter, we demonstrate that tubule stellate cells displayed a characteristic intracellular chloride signature upon kinin stimulation. Therefore, we have resolved this issue in Drosophila; kinin stimulates chloride flux by a transcellular route that is confined to the stellate cells.Open in a separate windowFig. 1.Models for chloride flux. After kinin (K) or tyramine (Tyr) induction, chloride could take a paracellular route through septate junctions (SJ) or a transcellular route through yet-unidentified chloride channels in the stellate cells.     

Serotonergic neurons are targets for leptin in the monkey

Leptin is a secretory product of adipocytes that has been shown to affect food intake, metabolism, and reproduction. One site of leptin's action is the central nervous system, where the leptin receptor (Ob-R) messenger ribonucleic acid (mRNA) and protein are expressed in discrete areas. In both the rat and monkey, Ob-R mRNA has been localized in the Raphe nuclei of the brainstem. Neurons in the Raphe nuclei are the primary source of serotonin in the brain. Serotonergic pathways influence both feeding and reproduction, and these cells are plausible direct targets for leptin's action. We used double label in situ hybridization and computerized image analysis to determine whether serotonergic neurons in the brainstem of the female pigtailed macaque (Macaca nemestrina) express Ob-R mRNA. We observed that many cells in the Raphe nuclei express serotonin transporter mRNA, a marker of serotonergic cells, and Ob-R mRNA. Based on quantitative analysis, the highest number of cells that express both serotonin transporter and Ob-R mRNAs were found in the caudal dorsal Raphe and median Raphe nuclei; fewer double labeled cells were situated in the caudal linear nucleus and rostral median Raphe, whereas double labeled cells occurred infrequently in the rostral dorsal Raphe. These observations suggest that leptin may act on serotonergic cells to mediate some of its effects on ingestive behavior, metabolism, and reproduction.     

Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop

Ca(2+)-activated Cl(-) channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca(2+) has recently become possible with the discovery that Ano1 (TMEM16a) is an essential subunit of CaCCs. Ano1 can be gated by Ca(2+) or by voltage in the absence of Ca(2+), but Ca(2+)- and voltage-dependent gating are very closely coupled. Here we identify a region in the first intracellular loop that is crucial for both Ca(2+) and voltage sensing. Deleting (448)EAVK in the first intracellular loop dramatically decreases apparent Ca(2+) affinity. In contrast, mutating the adjacent amino acids (444)EEEE abolishes intrinsic voltage dependence without altering the apparent Ca(2+)affinity. Voltage-dependent gating of Ano1 measured in the presence of intracellular Ca(2+) was facilitated by anions with high permeability or by an increase in [Cl(-)](e). Our data show that the transition between closed and open states is governed by Ca(2+) in a voltage-dependent manner and suggest that anions allosterically modulate Ca(2+)-binding affinity. This mechanism provides a unified explanation of CaCC channel gating by voltage and ligand that has long been enigmatic.     

Function of the CLC chloride channels and their implication in human pathology

To date, nine chloride channels belonging to the family of CLC chloride channels have been identified. They are localized either in plasma membranes or in intracellular vesicles (endosomes or lysosomes) and can have an ubiquitus or a more restrained tissue distribution. Recent studies on ClC-K1, ClC-2, ClC-3, ClC-5 and ClC-7 knockout mice and the identification of human inherited diseases caused by mutations of some of these chloride channels (myotonia congenita for ClC-1, Bartter disease for ClC-Kb, Dent's disease for ClC-5 and osteopetrose for ClC-7) have provided lines of direct evidence of the physiological relevance and importance of these chloride channels in the transport of chloride and in the endocytosis and transcytosis of proteins in specialized cells from the kidney and other tissues.     

From the Cover: Supercell tornadoes are much stronger and wider than damage-based ratings indicate

Tornadoes cause damage, injury, and death when intense winds impact structures. Quantifying the strength and extent of such winds is critical to characterizing tornado hazards. Ratings of intensity and size are based nearly entirely on postevent damage surveys [R. Edwards et al., Bull. Am. Meteorol. Soc. 94, 641–653 (2013)]. It has long been suspected that these suffer low bias [C. A. Doswell, D. W. Burgess, Mon. Weather Rev. 116, 495–501 (1988)]. Here, using mapping of low-level tornado winds in 120 tornadoes, we prove that supercell tornadoes are typically much stronger and wider than damage surveys indicate. Our results permit an accurate assessment of the distribution of tornado intensities and sizes and tornado wind hazards, based on actual wind-speed observations, and meaningful comparisons of the distribution of tornado intensities and sizes with theoretical predictions. We analyze data from Doppler On Wheels (DOW) radar measurements of 120 tornadoes at the time of peak measured intensity. In striking contrast to conventional damage-based climatologies, median tornado peak wind speeds are ∼60 m⋅s⁻¹, capable of causing significant, Enhanced Fujita Scale (EF)-2 to -3, damage, and 20% are capable of the most intense EF-4/EF-5 damage. National Weather Service (NWS) EF/wind speed ratings are 1.2 to 1.5 categories (∼20 m⋅s⁻¹) lower than DOW observations for tornadoes documented by both the NWS and DOWs. Median tornado diameter is 250 to 500 m, with 10 to 15% >1 km. Wind engineering tornado-hazard-model predictions and building wind resistance standards may require upward adjustment due to the increased wind-damage risk documented here.

Tornadoes cause direct harm to people, infrastructure, and communities (1). Quantifying tornado risk requires accurate knowledge of their wind speeds and the size of the areas at risk from these intense winds. However, since direct measurements of tornado winds are rare, tornado intensity and size are nearly always inferred indirectly from postevent damage surveys applying the Fujita (F) or Enhanced Fujita (EF) scales (2–5) to infer maximum wind speeds. Statistics concerning tornado frequency, intensity, and size are derived from these surveys. However, because most tornadoes do not damage well-engineered structures, from which the most intense wind speeds can be inferred, and many occur in primarily rural areas, damage-based tornado wind speed and size estimations are likely severely low biased (6–11). A limited climatology (12), using Doppler On Wheels (DOW) radar data (13–15), suggested that tornadoes may be larger and more intense than indicated by these surveys. In-situ observations of wind speeds reliably demonstrable to be inside the radius of maximum winds of tornadoes are very rare (16, 17) and inadequate for deriving a statistically meaningful climatology. It is no exaggeration to state that, until now, statistics concerning even the most basic characteristics of tornadoes, including intensity and size, could not be quantified with confidence.     

Ca2+ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and abscisic acid

In stomatal guard cells of higher-plant leaves, abscisic acid (ABA) evokes increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) by means of Ca(2+) entry from outside and release from intracellular stores. The mechanism(s) for Ca(2+) flux across the plasma membrane is poorly understood. Because [Ca(2+)](i) increases are voltage-sensitive, we suspected a Ca(2+) channel at the guard cell plasma membrane that activates on hyperpolarization and is regulated by ABA. We recorded single-channel currents across the Vicia guard cell plasma membrane using Ba(2+) as a charge-carrying ion. Both cell-attached and excised-patch measurements uncovered single-channel events with a maximum conductance of 12.8 +/- 0.4 pS and a high selectivity for Ba(2+) (and Ca(2+)) over K(+) and Cl(-). Unlike other Ca(2+) channels characterized to date, these channels rectified strongly toward negative voltages with an open probability (P(o)) that increased with [Ba(2+)] outside and decreased roughly 10-fold when [Ca(2+)](i) was raised from 200 nM to 2 microM. Adding 20 microM ABA increased P(o), initially by 63- to 260-fold; in both cell-attached and excised patches, it shifted the voltage sensitivity for channel activation, and evoked damped oscillations in P(o) with periods near 50 s. A similar, but delayed response was observed in 0.1 microM ABA. These results identify a Ca(2+)-selective channel that can account for Ca(2+) influx and increases in [Ca(2+)](i) triggered by voltage and ABA, and they imply a close physical coupling at the plasma membrane between ABA perception and Ca(2+) channel control.     

Tissue-specific regulatory differences for the alcohol dehydrogenase genes of Hawaiian Drosophila are conserved in Drosophila melanogaster transformants.

Naturally occurring regulatory variation is a source of genetic variability that is well documented but poorly understood. Two members of the Hawaiian picture-winged Drosophila, D. affinidisjuncta and D. hawaiiensis, display markedly different levels of alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) in the larval midgut and Malpighian tubules. To analyze the regulation of the alcohol dehydrogenase genes from these two species, their homologous alcohol dehydrogenase genes were cloned and introduced, via P element-mediated transformation, into the germ line of Drosophila melanogaster. Histochemical and electrophoretic analyses of larval transformants demonstrate that major differences in the tissue-specific levels of alcohol dehydrogenase production are characteristic of the alcohol dehydrogenase genes themselves. While these results do not directly address possible species-specific differences in the tissue distribution of trans-acting regulatory components, they indicate that demonstrable differences in cis-dominant regulatory information are sufficient to account for the observed regulatory variation.     

Insights into the structure, distribution and function of the cardiac chloride channels.

This review describes the properties and distribution of the three major types of chloride currents that have been studied in cardiac tissue. These include a cAMP- and protein kinase A-dependent current, a calcium-activated current and a swelling-induced current. The study of cardiac anion currents is a less mature field than the study of cardiac cation currents. Consequently, less is known regarding the structure, molecular identity and physiological role of anion currents in comparison to cardiac cation currents. Where known, the available molecular and structural information is also discussed. Although there is no proven physiological role for cardiac chloride currents, the possible clinical electrophysiological roles of cardiac chloride currents are discussed.     

G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block

Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies “sick sinus” syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Ca_v1.3 Ca²⁺ channels (Ca_v1.3^−/−) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K⁺ channel (I_KACh) could rescue SSS and heart block in Ca_v1.3^−/− mice. We found that genetic inactivation of I_KACh abolished SSS symptoms in Ca_v1.3^−/− mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of I_KACh either in Ca_v1.3^−/− mice or following selective inhibition of Ca_v1.3-mediated L-type Ca²⁺ (I_Ca,L) current in vivo. Ablation of I_KACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from I_KACh suppression achieved by gene therapy or selective pharmacological inhibition.Pacemaker activity of the sinoatrial node (SAN) controls heart rate under physiological conditions. Abnormal generation of SAN automaticity underlies “sick sinus” syndrome (SSS), a pathological condition manifested when heart rate is not sufficient to meet the physiological requirements of the organism (1). Typical hallmarks of SSS include SAN bradycardia, chronotropic incompetence, SAN arrest, and/or exit block (1–3). SSS carries incapacitating symptoms, such as fatigue and syncope (1–3). A significant percentage of patients with SSS present also with tachycardia-bradycardia syndrome (3). SSS can also be associated with atrioventricular (AV) conduction block (heart block) (1–3). Although aging is a known intrinsic cause of SSS (4), this disease appears also in the absence of any associated cardiac pathology and displays a genetic legacy (1, 2). Heart disease or drug intake can induce acquired SSS (2). Symptomatic SSS requires the implantation of an electronic pacemaker. SSS accounts for about half of all pacemaker implantations in the United States (5, 6). The incidence of SSS has been forecasted to increase during the next 50 y, particularly in the elder population (7). Furthermore, it has been estimated that at least half of SSS patients will need to be electronically paced (7). Although pacemakers are continuously ameliorated, they remain costly and require lifelong follow-up. Moreover, the implantation of an electronic pacemaker remains difficult in pediatric patients (8). Development of alternative and complementary pharmacological or molecular therapies for SSS management could improve quality of life and limit the need for implantation of electronic pacemakers.Recently, the genetic bases of some inherited forms of SSS have been elucidated (recently reviewed in 1, 9) with the discovery of mutations in genes encoding for ion channels involved in cardiac automaticity (4, 9, 10). Notably, loss of function of L-type Ca_v1.3 Ca²⁺ channels is central in some inherited forms of SSS. For instance, loss of function in Ca_v1.3-mediated L-type Ca²⁺ (I_Ca,L) current causes the sinoatrial node dysfunction and deafness syndrome (SANDD) (10). Affected individuals with SANDD present with profound deafness, bradycardia, and dysfunction of AV conduction (10). Mutation in ankyrin-B causes SSS by reduced membrane targeting of Ca_v1.3 channels (11). The relevance of Ca_v1.3 channels to SSS is demonstrated also by work on the pathophysiology of congenital heart block, where down-regulation of Ca_v1.3 channels by maternal Abs causes heart block in infants (12). Additionally, recent data show that chronic iron overload induces acquired SSS via a reduction in Ca_v1.3-mediated I_Ca,L (13).In mice and humans, Ca_v1.3 channels are expressed in the SAN, atria, and the AV node but are absent in adult ventricular tissue (14, 15). Ca_v1.3-mediated I_Ca,L plays a major role in the generation of the diastolic depolarization in SAN and AV myocytes, thereby constituting important determinants of heart rate and AV conduction velocity (14, 16). The heart rate of mice lacking Ca_v1.3 channels (Ca_v1.3^−/− mice) fairly recapitulates the hallmarks of SSS and associated symptoms, including bradycardia and tachycardia-bradycardia syndrome (17, 18). In addition, severe AV dysfunction is recorded in Ca_v1.3^−/− mice to variable degrees. Typically, these mice show first- and second-degree AV block (16, 17, 19). Complete AV block with dissociated atrial and ventricular rhythms can also be observed in these animals. The phenotype of Ca_v1.3^−/− mice thus constitutes a unique model for developing new therapeutic strategies against SSS (10).The muscarinic-gated K⁺ channel (I_KACh) is involved in the negative chronotropic effect of the parasympathetic nervous system on heart rate (20, 21). Two subunits of the G-protein activated inwardly rectifying K⁺ channels (GIRK1 and GIRK4) of the GIRK/Kir3 subfamily assemble as heterotetramers to form cardiac I_KACh channels (22). Indeed, both Girk1^−/− and Girk4^−/− mice lack cardiac I_KACh (20, 21, 23). We recently showed that silencing of the hyperpolarization-activated current “funny” (I_f) channel in mice induces a complex arrhythmic profile that can be rescued by concurrent genetic ablation of Girk4 (24). In this study, we tested the effects of genetic ablation and pharmacological inhibition of I_KACh on the Ca_v1.3^−/− mouse model of SSS. We found that Girk4 ablation or pharmacological inhibition of I_KACh rescues SSS and AV dysfunction in Ca_v1.3^−/−. Thus, our study shows that I_KACh targeting may be pursued as a therapeutic strategy for treatment of SSS and heart block.     

Pyruvate dehydrogenase complex and lactate dehydrogenase are targets for therapy of acute liver failure

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Mitochondria are selective targets for the protective effects of heat shock against oxidative injury.

Heat shock (HS) proteins (HSPs) induce protection against a number of stresses distinct from HS, including reactive oxygen species. In the human premonocytic line U937, we investigated in whole cells the effects of preexposure to HS and exposure to hydrogen peroxide (H2O2) on mitochondrial membrane potential, mass, and ultrastructure. HS prevented H2O2-induced alterations in mitochondrial membrane potential and cristae formation while increasing expression of HSPs and the protein product of bcl-2. Protection correlated best with the expression of the 70-kDa HSP, hsp70. We propose that mitochondria represent a selective target for HS-mediated protection against oxidative injury.     

Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases

The minichromosome maintenance (MCM) 2-7 helicase complex functions to initiate and elongate replication forks. Cell cycle checkpoint signaling pathways regulate DNA replication to maintain genomic stability. We describe four lines of evidence that ATM/ATR-dependent (ataxia-telangiectasia-mutated/ATM- and Rad3-related) checkpoint pathways are directly linked to three members of the MCM complex. First, ATM phosphorylates MCM3 on S535 in response to ionizing radiation. Second, ATR phosphorylates MCM2 on S108 in response to multiple forms of DNA damage and stalling of replication forks. Third, ATR-interacting protein (ATRIP)-ATR interacts with MCM7. Fourth, reducing the amount of MCM7 in cells disrupts checkpoint signaling and causes an intra-S-phase checkpoint defect. Thus, the MCM complex is a platform for multiple DNA damage-dependent regulatory signals that control DNA replication.     

Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative pore-lining basic residues significantly altered the IC₅₀ of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore.The TMEM16 (transmembrane protein 16) family consists of transmembrane proteins, of which at least two members, TMEM16A and TMEM16B, are pore-forming subunits of calcium-activated chloride channels (CaCCs) (1–4). TMEM16A and TMEM16B channels serve a variety of functions in many cell types, including secretory epithelia (5–8), gastrointestinal pacemakers (9), sensory neurons and hippocampal neurons (10–13), urethral and vascular smooth muscle (14, 15), and tumor cells (16). Hence, it is important to understand how these channels work.TMEM16A channels are activated by direct binding of intracellular calcium ions (17–19) and open to conduct currents with anion selectivity that follows an Eisenman type I sequence (1, 3); however, the mechanism of permeation in TMEM16A channels is not well-understood. Although binding of intracellular calcium at physiological concentrations is known to catalyze the passage of anions through the channel, the properties of the resulting conductance are complex. A hallmark feature of CaCC in multiple cell types, including Xenopus oocytes, is the presence of two obvious conduction modes: a voltage-dependent or outwardly rectifying mode at lower concentrations of calcium and a leak mode with Ohmic character at higher concentrations (20, 21). Whereas this phenomenon may reflect voltage-dependent calcium sensitivity of CaCC, it may also indicate the existence of multiple open states (20, 22–24). Indeed, Xenopus TMEM16A (3) and mouse TMEM16B (25) appeared to adopt multiple distinguishable conductive conformations as intracellular calcium levels increased after photolysis of caged calcium. Furthermore, the identity of the permeating anions also seems to influence gating behavior, because anions for which the channel shows preferred selectivity also seem to facilitate CaCC activation in both Xenopus oocytes (23) and cells heterologously expressing mouse TMEM16B (26). Taken together, these findings suggest that the anion pore and the calcium-dependent gating machinery may be tightly coupled to one another, whereby increases in intracellular calcium may modify chloride-binding properties in the pore. These observations further raise the question of whether the molecular determinants of anion selectivity may vary in different distinct open states that may be occupied preferentially at various intracellular calcium levels. In light of these considerations, it is intriguing that a recently published crystal structure of a TMEM16 family protein from the Nectria hematococcus fungus, although not showing obvious channel behavior itself, has a calcium-binding pocket in close physical proximity to the fifth transmembrane helix, which harbors a residue corresponding to K584 of TMEM16A and Q559 of TMEM16F (27). This residue has been implicated to underlie the differences in ion selectivity between the TMEM16A and TMEM16F channels, because the anion selectivity of TMEM16A is reduced by the K584Q mutation and the cation selectivity of TMEM16F is reduced by the Q559K mutation (28).It is important to delineate the location and properties of the pore of TMEM16A channels. Whereas the TMEM16A pore has been proposed to be between the fifth and sixth of either 8 (17, 19, 29) or 10 transmembrane segments (27) and several positively charged residues were proposed to contribute to pore properties (1, 2), whether and how these residues influence permeation remain unclear as the channel’s topological arrangement has come into clearer focus using biochemical, biophysical, and structural biological methods (17, 19, 27, 29). It is also important to identify and characterize pharmacological modulators of the TMEM16A channel pore. Of the channel blockers that have been identified, some are of low potency [DIDS and tannic acid (30)] and/or nonspecific [niflumic acid, anthracene-9-carboxylic acid, NPPB, CaCC-inh001, and benzbromarone (6, 31–33)], whereas others with potency in the low micromolar range (30, 34) seem to be only partially effective (T16Ainh-001) in blocking the current (31), and none have been conclusively shown to interact with the pore.To address these questions, we used two approaches to examine TMEM16A permeation behavior. First, reasoning that positively charged residues are likely involved in conferring anion selectivity to TMEM16A, we mutated all of the basic residues within the putative pore region as well as several others previously queried by Martinez–Torres and coworkers (35) for contribution to apparent anomalous mole fraction effects and in helices shown to reside nearby in the fungal TMEM16 crystal structure (27), and we screened for those that prevented shifts in anion selectivity with higher intracellular calcium. Second, we tested compounds isolated from a high-throughput small molecule screen based on iodide quenching of YFP fluorescence (2, 34, 36) to search for pore blockers. From these compounds, we identified two with voltage- and anion-dependent blocking properties that appeared to display some preference for specific open states, because they blocked the channel with greater potency at elevated intracellular calcium. Here, we report that alanine substitution for four basic residues caused not only alterations of anion selectivity but also, shifted the concentration dependence of these pore blockers, suggesting that the affinity between the pore and the blockers was being affected by those mutations of putative pore-lining residues.     

Sialic acid and the surface charge of delayed rectifier potassium channels

We used the whole-cell configuration of the patch-clamp technique and cultured ventricular myocytes from 7-day embryonic chicks to test the hypothesis that sialic acid residues (NANA) constitute the negative surface charge associated with delayed rectifier potassium channels. Delayed rectifier current (iK) was elicited at potentials between -40 and +60 mV. The existence of negative fixed charges close to the "gating sensor" was confirmed by a 6.8-mV negative shift of the half-activation potential (V1/2) following a 10-fold reduction of divalent cations and a 22.6-mV position shift following the addition of 10 mM NiCl2. An 8.4-mV increase in the Boltzmann equation slope factor (k) in the former experiment and a 5.5-mV decline in the latter suggested that the surface charge is not uniformly distributed. We used a high performance liquid chromatography procedure to detect freed sarcolemmal NANA and found that 71-88% was released by neuraminidase (0.2-2.0 U/ml) during 1-h treatments. Such treatments had no significant effect upon the amplitudes of iK or V1/2. On the other hand, k was increased significantly by the enzyme (2.0 U/ml), but only when Ca2+ was present. Finally, 1-h pre-treatments with neuraminidase (2.0 U/ml) had no effect on the positive shift of V1/2 induced by Ni2+. We conclude that although sarcolemmal NANA may bind Ca2+, it does not constitute the surface charge of delayed rectifier potassium channels.     

Minireview: ribonucleic acid interference for the identification of new targets for the treatment of metabolic diseases

Over the past years, RNA interference (RNAi) has exploded as a new approach to manipulate gene expression in mammalian systems. More recently, RNAi has acquired interest as a tool to identify new targets for therapeutic intervention. This review focuses on the current understanding of RNAi biology, how RNAi has been used to study the role of different genes in the pathogenesis of diabetes and obesity, and the use of RNAi screens for the identification of new targets for metabolic diseases. Also reviewed are the in vivo proof of principle experiments that provide the validation of these new targets for the development of RNAi-based therapeutics for diabetes.