Akiyuki Taruno

How do taste cells lacking synapses mediate neurotransmission? CALHM1, a voltage-gated ATP channel.

CALHM1 was recently demonstrated to be a voltage‐gated ATP‐permeable ion channel and to serve as a bona fide conduit for ATP release from sweet‐, umami‐, and bitter‐sensing type II taste cells. Calhm1 is expressed in taste buds exclusively in type II cells and its product has structural and functional similarities with connexins and pannexins, two families of channel protein candidates for ATP release by type II cells. Calhm1 knockout in mice leads to loss of perception of sweet, umami, and bitter compounds and to impaired gustatory nerve responses to these tastants. These new studies validate the concept of ATP as the primary neurotransmitter from type II cells to gustatory neurons. Furthermore, they identify voltage‐gated ATP release through CALHM1 as an essential molecular mechanism of ATP release in taste buds. We discuss these new findings, as well as unresolved issues in peripheral taste signaling that we hope will stimulate future research.

Regulation of Epithelial Sodium Transport via Epithelial Na+ Channel

Renal epithelial Na+ transport plays an important role in homeostasis of our body fluid content and blood pressure. Further, the Na+ transport in alveolar epithelial cells essentially controls the amount of alveolar fluid that should be kept at an appropriate level for normal gas exchange. The epithelial Na+ transport is generally mediated through two steps: (1) the entry step of Na+ via epithelial Na+ channel (ENaC) at the apical membrane and (2) the extrusion step of Na+ via the Na+, K+-ATPase at the basolateral membrane. In general, the Na+ entry via ENaC is the rate-limiting step. Therefore, the regulation of ENaC plays an essential role in control of blood pressure and normal gas exchange. In this paper, we discuss two major factors in ENaC regulation: (1) activity of individual ENaC and (2) number of ENaC located at the apical membrane.

Actions of Quercetin, a Polyphenol, on Blood Pressure

Disorder of blood pressure control causes serious diseases in the cardiovascular system. This review focuses on the anti-hypertensive action of quercetin, a flavonoid, which is one of the polyphenols characterized as the compounds containing large multiples of phenol structural units, by varying the values of various blood pressure regulatory factors, such as vascular compliance, peripheral vascular resistance, and total blood volume via anti-inflammatory and anti-oxidant actions. In addition to the anti-inflammatory and anti-oxidant actions of quercetin, we especially describe a novel mechanism of quercetin’s action on the cytosolic Cl− concentration ([Cl−]c) and novel roles of the cytosolic Cl− i.e., (1) quercetin elevates [Cl−]c by activating Na+-K+-2Cl− cotransporter 1 (NKCC1) in renal epithelial cells contributing to Na+ reabsorption via the epithelial Na+ channel (ENaC); (2) the quercetin-induced elevation of [Cl−]c in renal epithelial cells diminishes expression of ENaC leading to a decrease in renal Na+ reabsorption; and (3) this reduction of ENaC-mediated Na+ reabsorption in renal epithelial cells drops volume-dependent elevated blood pressure. In this review, we introduce novel, unique mechanisms of quercetin’s anti-hypertensive action via activation of NKCC1 in detail.

Quercetin is a useful medicinal compound showing various actions including control of blood pressure, neurite elongation and epithelial ion transport

Quercetin has multiple potential to control various cell function keeping our body condition healthy. In this review article, we describe the molecular mechanism on how quercetin exerts its action on blood pressure, neurite elongation and epithelial ion transport based from a viewpoint of cytosolic Cl- environments, which is recently recognized as an important signaling factor in various types of cells. Recent studies show various roles of cytosolic Cl- in regulation of blood pressure and neurite elongation, and prevention from bacterial and viral infection. We have found the stimulatory action of quercetin on Cl- transporter, Na+-K+-2Cl- cotransporter 1 (NKCC1; an isoform of NKCC), which has been recognized as one of the most interesting, fundamental actions of quercetin. In this review article, based on this stimulatory action of quercetin on NKCC1, we introduce the molecular mechanism of quercetin on: 1) blood pressure, 2) neurite elongation, and 3) epithelial Cl- secretion including tight junction forming in epithelial tissues. 1) Quercetin induces elevation of the cytosolic Cl- concentration via activation of NKCC1, leading to anti-hypertensive action by diminishing expression of epithelial Na+ channel (ENaC), a key ion channel involved in renal Na+ reabsorption, while quercetin has no effects on the blood pressure with normal salt intake. 2) Quercetin also has stimulatory effects on neurite elongation by elevating the cytosolic Cl- concentration via activation of NKCC1 due to tubulin polymerization facilitated through Cl--induced inhibition of GTPase. 3) Further, in lung airway epithelia quercetin stimulates Cl- secretion by increasing the driving force for Cl- secretion via elevation of the cytosolic Cl- concentration: this leads to water secretion, participating in prevention of our body from bacterial and viral infection. In addition to transcellular ion transport, quercetin regulates tight junction function via enhancement of tight junction integrity by modulating expression and assembling tight junction-forming proteins. Based on these observations, it is concluded that quercetin is a useful medicinal compound keeping our body to be in healthy condition.

Analysis of Blocker-labeled Channels Reveals the Dependence of Recycling Rates of ENaC on the Total Amount of Recycled Channels

Trafficking is one of the primary mechanisms of epithelial Na+ channel (ENaC) regulation. Although it is known that ENaCs are recycled between the apical membrane and the intracellular channel pool, it has been difficult to investigate the recycling of ENaCs; especially endogenously expressed ENaCs. The aim of the present study is to investigate if the recycling rates of ENaCs depend on the total amount of recycled ENaCs. To accomplish this point, we established a novel method to estimate the total amount of recycled ENaCs and the ENaC recycling rates by using a specific blocker (benzamil) of ENaC with a high-affinity for functional label of the channels in recycling. Applying this method, we studied if a decrease in the total amount of ENaCs caused by brefeldin A (5 µg/mL, 1 h) affects respectively the rates of insertion and endocytosis of ENaCs to and from the apical membrane in monolayers of renal epithelial A6 cells. Our observations indicate that: 1) both insertion and endocytosis rates of ENaC increase when the total amount of ENaCs decreases, 2) the increase in the insertion rate is larger than that in the endocytosis rate, and 3) this larger increase in the insertion rate than the endocytosis rate caused by the decrease in the total amount of ENaCs plays an important role in preventing Na+ transport from drastically diminishing due to a decrease in the total amount of ENaCs. The newly established analysis of blocker-labeled ENaCs in the present study provides a useful tool to investigate the recycling of endogenously expressed ENaCs.

Post-translational palmitoylation controls the voltage gating and lipid raft association of the CALHM1 channel

Calcium homeostasis modulator 1 (CALHM1), a new voltage‐gated ATP‐ and Ca2+‐permeable channel, plays important physiological roles in taste perception and memory formation. Regulatory mechanisms of CALHM1 remain unexplored, although the biophysical disparity between CALHM1 gating in vivo and in vitro suggests that there are undiscovered regulatory mechanisms. Here we report that CALHM1 gating and association with lipid microdomains are post‐translationally regulated through the process of protein S‐palmitoylation, a reversible attachment of palmitate to cysteine residues. Our data also establish cysteine residues and enzymes responsible for CALHM1 palmitoylation. CALHM1 regulation by palmitoylation provides new mechanistic insights into fine‐tuning of CALHM1 gating in vivo and suggests a potential layer of regulation in taste and memory.

Simulation of Cl− Secretion in Epithelial Tissues: New Methodology Estimating Activity of Electro-Neutral Cl− Transporter

Transcellular Cl− secretion is, in general, mediated by two steps; (1) the entry step of Cl− into the cytosolic space from the basolateral space across the basolateral membrane by Cl− transporters, such as Na+-K+-2Cl− cotransporter (NKCC1, an isoform of NKCC), and (2) the releasing step of Cl− from the cytosolic space into the luminal (air) space across the apical membrane via Cl− channels, such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Transcellular Cl− secretion has been characterized by using various experimental techniques. For example, measurements of short-circuit currents in the Ussing chamber and patch clamp techniques provide us information on transepithelial ion movements via transcellular pathway, transepithelial conductance, activity (open probability) of single channel, and whole cell currents. Although many investigators have tried to clarify roles of Cl− channels and transporters located at the apical and basolateral membranes in transcellular Cl− secretion, it is still unclear how Cl− channels/transporters contribute to transcellular Cl− secretion and are regulated by various stimuli such as Ca2+ and cAMP. In the present study, we simulate transcellular Cl− secretion using mathematical models combined with electrophysiological measurements, providing information on contribution of Cl− channels/transporters to transcellular Cl− secretion, activity of electro-neutral ion transporters and how Cl− channels/transporters are regulated.

Regulation of paracellular Na+ and Cl− conductances by hydrostatic pressure

The effect of hydrostatic pressure on the paracellular ion conductance (Gp) composed of the Na+ conductance (GNa) and the Cl− conductance (GCl) has been Investigated. Gp, GNa and GCl were time‐dependently increased after applying an osmotic gradient generated by NaCl with basolateral hypotonicity. Hydrostatic pressure (1–4 cm H2O) applied from the basolateral side enhanced the osmotic gradient‐induced increase in Gp, GNa and GCl in a magnitude‐dependent manner, while the hydrostatic pressure applied from the apical side diminished the osmotic gradient‐induced increase in Gp, GNa and GCl. How the hydrostatic pressure influences Gp, GNa and GCl under an isosmotic condition was also investigated. Gp, GNa and GCl were stably constant under a condition with basolateral application of sucrose canceling the NaCl‐generated osmotic gradient (an isotonic condition). Even under this stable condition, the basolaterally applied hydrostatic pressure drastically elevated Gp, GNa and GCl, while apically applied hydrostatic pressure had little effect on Gp, GNa or GCl. Taken together, these observations suggest that certain factors controlled by the basolateral osmolality and the basolaterally applied hydrostatic pressure mainly regulate the Gp, GNa and GCl.

Current-direction/amplitude-dependent single channel gating kinetics of mouse pannexin 1 channel: a new concept for gating kinetics

The detailed single-channel gating kinetics of mouse pannexin 1 (mPanx1) remains unknown, although mPanx1 is reported to be a voltage-activated anion-selective channel. We investigated characteristics of single-channel conductances and opening and closing rates of mPanx1 using patch-clamp techniques. The unitary current of mPanx1 shows outward rectification with single-channel conductances of ~20 pS for inward currents and ~80 pS for outward currents. The channel open time for outward currents (Cl− influx) increases linearly as the amplitude of single channel currents increases, while the open time for inward currents (Cl− efflux) is constant irrespective of changes in the current amplitude, as if the direction and amplitude of the unitary current regulates the open time. This is supported by further observations that replacement of extracellular Cl− with gluconate− diminishes the inward tail current (Cl− efflux) at a membrane potential of −100 mV due to the lowered outward current (gluconate− influx) at membrane potential of 100 mV. These results suggest that the direction and rate of charge-carrier movement regulate the open time of mPanx1, and that the previously reported voltage-dependence of Panx1 channel gating is not directly mediated by the membrane potential but rather by the direction and amplitude of currents through the channel.

Analysis of Aprotinin, a Protease Inhibitor, Action on the Trafficking of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells Using a Mathematical Model

Background/Aim: Epithelial Na+ channels (ENaC) play a crucial role in control of blood pressure by regulating renal Na+ reabsorption. Intracellular trafficking of ENaC is one of the key regulators of ENaC function, but a quantitative description of intracellular recycling of endogenously expressed ENaC is unavailable. We attempt here to provide a model for intracellular recycling after applying a protease inhibitor under hypotonic conditions. Methods: We simulated the ENaC-mediated Na+ transport in renal epithelial A6 cells measured as short-circuit currents using a four-state mathematical ENaC trafficking model. Results: We developed a four-state mathematical model of ENaC trafficking in the cytosol of renal epithelial cells that consists of: an insertion state of ENaC that can be trafficked to the apical membrane state (insertion rate); an apical membrane state of ENaC conducting Na+ across the apical membrane; a recycling state containing ENaC that are retrieved from the apical membrane state (endocytotic rate) and then to the insertion state (recycling rate) communicating with the apical membrane state or to a degradation state (degradation rate). We studied the effect of aprotinin (a protease inhibitor) blocking protease-induced cleavage of the extracellular loop of γ ENaC subunit on the rates of intracellular ENaC trafficking using the above-defined four-state mathematical model of ENaC trafficking and the recycling number relative to ENaC staying in the apical membrane. We found that aprotinin significantly reduced the insertion rate of ENaC to the apical membrane by 40%, the recycling rate of ENaC by 81%, the cumulative time of an individual ENaC staying in the apical membrane by 32%, the cumulative life-time after the first endocytosis of ENaC by 25%, and the cumulative Na+ absorption by 31%. The most interesting result of the present study is that cleavage of ENaC affects the intracellular ENaC trafficking rate and determines the residency time of ENaC, indicating that more active cleaved ENaCs stay longer at the apical membrane contributing to transcellular Na+ transport via an increase in recycling of ENaC to the apical membrane. Conclusion: The extracellular protease-induced cleavage of the extracellular loop of γ ENaC subunit increases transcellular epithelial Na+ transport by elevating the recycling rate of ENaC due to an increase in the recycling rate of ENaCs associated with increases in the insertion rate of ENaC.