Maxi-anion Channel Research Articles

Maxi-anion channel as a candidate pathway for osmosensitive ATP release from mouse astrocytes in primary culture

In the present study, we aimed to evaluate the pathways contributing to ATP release from mouse astrocytes during hypoosmotic stress. We first examined the expression of mRNAs for proteins constituting possible ATP-releasing pathways that have been suggested over the past several years. In RT-PCR analysis using both control and osmotically swollen astrocytes, amplification of cDNA fragments of expected size was seen for connexins (Cx32, Cx37, Cx43), pannexin 1 (Px1), the P2X7 receptor, MRP1 and MDR1, but not CFTR. Inhibitors of exocytotic vesicular release, gap junction hemi-channels, CFTR, MRP1, MDR1, the P2X7 receptor, and volume-sensitive outwardly rectifying chloride channels had no significant effects on the massive ATP release from astrocytes. In contrast, the hypotonicity-induced ATP release from astrocytes was most effectively inhibited by gadolinium (50 muM), an inhibitor of the maxi-anion channel, which has recently been shown to serve as a pathway for ATP release from several other cell types. Thus, we propose that the maxi-anion channel constitutes a major pathway for swelling-induced ATP release from cultured mouse astrocytes as well.

Spatial Distribution of Maxi-Anion Channel on Cardiomyocytes Detected by Smart-Patch Technique

Spatial distribution of maxi-anion channels in rat cardiomyocytes were studied by applying the recently developed patch clamp technique under scanning ion conductance microscopy, called the “smart-patch” technique. In primary-cultured neonatal cells, the channel was found to be unevenly distributed over the cell surface with significantly lower channel activity in cellular extensions compared with the other parts. Local ATP release, detected using a PC12 cell-based biosensor technique, also exhibited a similar pattern. The maxi-anion channel activity could not be detected in freshly isolated adult cardiomyocytes by the conventional patch-clamp with 2-MΩ pipettes. However, when fine-tipped 15–20 MΩ pipettes were targeted to only Z-line areas, we observed, for the first time, the maxi-anion events. Smart-patching different regions of the cell surface, we found that the channel activity was maximal at the openings of T-tubules and along Z-lines, but was significantly decreased in the scallop crest area. Thus, it is concluded that maxi-anion channels are concentrated at the openings of T-tubules and along Z-lines in adult cardiomyocytes. This study showed that the smart-patch technique provides a powerful method to detect a unitary event of channels that are localized at some specific site in the narrow region.

Oxygen-glucose deprivation induces ATP release via maxi-anion channels in astrocytes

ATP represents a major gliotransmitter that serves as a signaling molecule for the cross talk between glial and neuronal cells. ATP has been shown to be released by astrocytes in response to a number of stimuli under nonischemic conditions. In this study, using a luciferin-luciferase assay, we found that mouse astrocytes in primary culture also exhibit massive release of ATP in response to ischemic stress mimicked by oxygen-glucose deprivation (OGD). Using a biosensor technique, the local ATP concentration at the surface of single astrocytes was found to increase to around 4 μM. The OGD-induced ATP release was inhibited by Gd3+ and arachidonic acid but not by blockers of volume-sensitive outwardly rectifying Cl− channels, cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance-related protein (MRP), connexin or pannexin hemichannels, P2X7 receptors, and exocytotic vesicular transport. In cell-attached patches on single astrocytes, OGD caused activation of maxi-anion channels that were sensitive to Gd3+ and arachidonic acid. The channel was found to be permeable to ATP4− with a permeability ratio of PATP/PCl = 0.11. Thus, it is concluded that ischemic stress induces ATP release from astrocytes and that the maxi-anion channel may serve as a major ATP-releasing pathway under ischemic conditions.

Genetic Demonstration That the Plasma Membrane Maxianion Channel and Voltage-dependent Anion Channels Are Unrelated Proteins

The maxianion channel is widely expressed in many cell types, where it fulfills a general physiological function as an ATP-conductive gate for cell-to-cell purinergic signaling. Establishing the molecular identity of this channel is crucial to understanding the mechanisms of regulated ATP release. A mitochondrial porin (voltage-dependent anion channel (VDAC)) located in the plasma membrane has long been considered as the molecule underlying the maxianion channel activity, based upon similarities in the biophysical properties of these two channels and the purported presence of VDAC protein in the plasma membrane. We have deleted each of the three genes encoding the VDAC isoforms individually and collectively and demonstrate that maxianion channel (approximately 400 picosiemens) activity in VDAC-deficient mouse fibroblasts is unaltered. The channel activity is similar in VDAC1/VDAC3-double-deficient cells and in double-deficient cells with the VDAC2 protein depleted by RNA interference. VDAC deletion slightly down-regulated, but never abolished, the swelling-induced ATP release. The lack of correlation between VDAC protein expression and maxianion channel activity strongly argues against the long held hypothesis of plasmalemmal VDAC being the maxianion channel.

Gene deletion and silencing refutes the long held hypothesis that maxi-anion channel is a plasmalemmal VDAC

Gene deletion and silencing refutes the long held hypothesis that maxi-anion channel is a plasmalemmal VDAC

ATP release via anion channels.

ATP serves not only as an energy source for all cell types but as an ‘extracellular messenger-for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg2+ and/or H+ salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP4- in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed.

Renal Cell-to-Cell Communication via Extracellular ATP

In the kidney, macula densa cells communicate with the mesangial cell-afferent arteriolar smooth muscle cell complex through ATP signaling. This signaling process involves release of ATP across the macula densa basolateral membrane through a maxi anion channel and the interaction of ATP with purinergic P2 receptors.

FUNCTION OF CHLORIDE CHANNELS IN THE KIDNEY

Numerous Cl- channels have been identified in the kidney using physiological approaches and thus are thought to be involved in a range of physiological processes, including vectorial transepithelial Cl- transport, cell volume regulation, and vesicular acidification. In addition, expression of genes from several Cl- channel gene families has also been observed. However, the molecular characteristics of a number of Cl- channels within the kidney are still unknown, and the physiological roles of Cl- channels identified by molecular means remain to be determined. A gene knockout approach using mice might shed further light on the characteristics of these various Cl- channels. In addition, study of diseases involving Cl- channels (channelopathies) might clarify the physiological role of specific Cl- channels. To date, more is known about CLC Cl- channels than any other Cl- channels within the kidney. This review focuses on the physiological roles of CLC Cl- channels within the kidney, particularly kidney-specific ClC-K Cl- channels, as well as the recently identified maxi anion channel in macula densa, which is involved in tubulo-glomerular feedback.

Wide Nanoscopic Pore of Maxi-Anion Channel Suits its Function as an ATP-Conductive Pathway

The newly proposed function of the maxi-anion channel as a conductive pathway for ATP release requires that its pore is sufficiently large to permit passage of a bulky ATP 4− anion. We found a linear relationship between relative permeability of organic anions of different size and their relative ionic mobility (measured as the ratio of ionic conductance) with a slope close to 1, suggesting that organic anions tested with radii up to 0.49 nm (lactobionate) move inside the channel by free diffusion. In the second approach, we, for the first time, succeeded in pore sizing by the nonelectrolyte exclusion method in single-channel patch-clamp experiments. The cutoff radii of PEG molecules that could access the channel from intracellular (1.16 nm) and extracellular (1.42 nm) sides indicated an asymmetry of the two entrances to the channel pore. Measurements by symmetrical two-sided application of PEG molecules yielded an average functional pore radius of ∼1.3 nm. These three estimates are considerably larger than the radius of ATP 4− (0.57–0.65 nm) and MgATP 2− (∼0.60 nm). We therefore conclude that the nanoscopic maxi-anion channel pore provides sufficient room to accommodate ATP and is well suited to its function as a conductive pathway for ATP release in cell-to-cell communication.

Macula densa basolateral ATP release is regulated by luminal [NaCl] and dietary salt intake.

One component of the macula densa (MD) tubuloglomerular feedback (TGF) signaling pathway may involve basolateral release of ATP through a maxi-anion channel. Release of ATP has previously been studied during a maximal luminal NaCl concentration ([NaCl](L)) stimulus (20-150 mmol/l). Whether MD ATP release occurs during changes in [NaCl](L) within the physiological range (20-60 mmol/l) has not been examined. Also, because TGF is known to be enhanced by low dietary salt intake, we examined the pattern of MD ATP release from salt-restricted rabbits. Fluorescence microscopy, with fura 2-loaded cultured mouse mesangial cells as biosensors, was used to assess ATP release from the isolated, perfused thick ascending limb containing the MD segment. The mesangial biosensor cells, which contain purinergic receptors and elevate intracellular Ca(2+) concentration ([Ca(2+)](i)) on ATP binding, were placed adjacent to the MD basolateral membrane. Elevations in [NaCl](L) between 0 and 80 mmol/l, in 20-mmol/l increments, caused stepwise increases in [Ca(2+)](i), with the highest increase at [NaCl](L) of approximately 60 mmol/l. Luminal furosemide at 10(-4) mol/l blocked ATP release, which suggests that the efflux of ATP required MD Na-2Cl-K cotransport. A low-salt diet for 1 wk increased the magnitude of [NaCl](L)-dependent elevations in biosensor [Ca(2+)](i) by twofold, whereas high-salt intake had no effect. In summary, ATP release occurs over the same range of [NaCl](L) (20-60 mmol/l) previously reported for TGF responses, and, similar to TGF, ATP release was enhanced by dietary salt restriction. Thus these two findings are consistent with the role of MD ATP release as a signaling component of the TGF pathway.

ATP-conducting maxi-anion channel: a new player in stress-sensory transduction.

The regulated release of ATP is a fundamental process in cell-to-cell signaling. The electrogenic translocation of ATP via an anion channel has been suggested as one possible mechanism of the release. In this review, we survey possible candidate channels for this pathway. The maxi-anion channel characterized by an exceedingly large unitary conductance has been a stray channel with regard to its function. A newly discovered property, its ATP conductivity and its activation in response to stress signals, indicates that this channel has a central role in stress-sensory transduction for cell volume regulation and tubuloglomerular feedback.

Macula densa cell signaling involves ATP release through a maxi anion channel.

Macula densa cells are unique renal biosensor cells that detect changes in luminal NaCl concentration ([NaCl](L)) and transmit signals to the mesangial cellafferent arteriolar complex. They are the critical link between renal salt and water excretion and glomerular hemodynamics, thus playing a key role in regulation of body fluid volume. Since identification of these cells in the early 1900s, the nature of the signaling process from macula densa cells to the glomerular contractile elements has remained unknown. In patch-clamp studies of macula densa cells, we identified an [NaCl](L)-sensitive ATP-permeable large-conductance (380 pS) anion channel. Also, we directly demonstrated the release of ATP (up to 10 microM) at the basolateral membrane of macula densa cells, in a manner dependent on [NaCl](L), by using an ATP bioassay technique. Furthermore, we found that glomerular mesangial cells respond with elevations in cytosolic Ca(2+) concentration to extracellular application of ATP (EC(50) 0.8 microM). Importantly, we also found increases in cytosolic Ca(2+) concentration with elevations in [NaCl](L), when fura-2-loaded mesangial cells were placed close to the basolateral membrane of macula densa cells. Thus, cell-to-cell communication between macula densa cells and mesangial cells, which express P2Y(2) receptors, involves the release of ATP from macula densa cells via maxi anion channels at the basolateral membrane. This mechanism may represent a new paradigm in cell-to-cell signal transduction mediated by ATP.

Macula densa cell signaling.

Macula densa cells are renal sensor elements that detect changes in distal tubular fluid composition and transmit signals to the glomerular vascular elements. This tubuloglomerular feedback mechanism plays an important role in regulating glomerular filtration rate and blood flow. Macula densa cells detect changes in luminal sodium chloride concentration through a complex series of ion transport-related intracellular events. NaCl entry via a Na:K:2Cl cotransporter and Cl exit through a basolateral channel lead to cell depolarization and increases in cytosolic calcium. Na/H exchange (NHE2) results in cell alkalization, whereas intracellular [Na] is regulated by an apically located H(Na)-K ATPase and not by the traditional basolateral Na:K ATPase. Communication from macula densa cells to the glomerular vascular elements involves ATP release across the macula densa basolateral membrane through a maxi-anion channel. The adaptation of multi-photon microscopy is providing new insights into macula densa-glomerular signaling.