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FEBS Letters 588 (2014) 1396–1402

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Review and pannexin channels and their signaling ⇑ ⇑ Gerhard Dahl , Kenneth J. Muller

Department of Physiology and Biophysics, University of Miami School of Medicine, 1600 NW 10th Ave, Miami, FL 33136, USA

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Article history: are bifunctional membrane proteins in invertebrates, forming gap junctions as well as Received 12 February 2014 non-junctional membrane channels (innexons). Their vertebrate analogues, the pannexins, have Accepted 6 March 2014 not only lost the ability to form gap junctions but are also prevented from it by glycosylation. Available online 14 March 2014 Pannexins appear to form only non-junctional membrane channels (pannexons). The membrane channels formed by pannexins and innexins are similar in their biophysical and pharmacological Edited by Michael Koval, Brant E. Isakson, Robert G. Gourdie and Wilhelm Just properties. Innexons and pannexons are permeable to ATP, are present in glial cells, and are involved in activation of microglia by calcium waves in . Directional movement and accumula- tion of microglia following nerve injury, which has been studied in the leech which has unusually Keywords: Innexin large glial cells, involves at least 3 signals: ATP is the ‘‘go’’ signal, NO is the ‘‘where’’ signal and Pannexin arachidonic acid is a ‘‘stop’’ signal. Glia Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Microglia ATP

1. Pannexons and innexons jump between cells and cell layers that were not in junctional communication or even in direct contact [5]. This observation It was only recently that the pannexin/innexin membrane proved that gap junctions were not the exclusive pathway for wave channels were discovered [1–3], and yet these ‘‘new’’ channels propagation and that there had to be an extracellular signal mole- mediate several ancient forms of important intercellular communi- cule mediating wave transmission to non-contiguous cells. That cation in animals ranging from primitive invertebrates to humans. signal was eventually identified as ATP [5,6]. It also became appar- The list of the channels’ functions and properties has shifted and ent that some part of ATP release was sensitive to become refined each year to the point that a review seems timely, channel inhibitors [7], leading to a plethora of papers using such with emphasis not only on diverse functions but on common pharmacological evidence to conclude that ‘‘hemichan- features of the channels, such as serving as conduits for release nels’’ are involved. As of late 2013, a PubMed search with the terms of ATP from cells. ‘‘hemichannels’’ and ‘‘ATP’’, for example, yielded 209 publications. The channels also present a problem of evolutionary interest. The complication of the term ‘‘connexin hemichannels’’ is not only When vertebrates evolved from invertebrates, a major step was in its misleading name, but also its physiological relevance. The the appearance of , new gap junctional molecules that term is derived from the fact that gap junction channels, which functioned much like the old innexins in invertebrates. This cre- span the two membranes of contacting cells, are composed of ated a divergence that remains puzzling and has highlighted the two parts, each one residing in a plasma membrane of apposing need to understand the various roles of innexins. The vertebrates cells (hemi-gap junction channels) that join to form the complete retained the innexins as pannexins; in evolving, they stopped gap junction channel. The physiological relevance comes into ques- forming gap-junctions between cells but continued as membrane tion when the conditions are considered under which connexin channels to the extracellular space, which we now know to be ‘‘hemichannel’’ activity is observed [8–10]. Extremely low extracel- one of the original functions of innexins, as described below. lular calcium concentrations and high positive potentials are required to see channel activity. 2. Calcium waves, ATP release, and pannexins/innexins When pannexins were discovered, on the basis of homology to innexins in vertebrates through a database search [3], they were Soon after the discovery of calcium waves and their spreading initially considered to represent a redundant system to the conn- through gap junctions [4], it was discovered that the waves could exin gap junctions. However, gap junction formation by pannexins is unlikely for a number of reasons [11–13], and to date there is no evidence that pannexin-based gap junction channels exist ⇑ Corresponding authors. naturally, despite the fact that the related innexins do make gap E-mail addresses: [email protected] (G. Dahl), [email protected] (K.J. Muller).

http://dx.doi.org/10.1016/j.febslet.2014.03.007 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. G. Dahl, K.J. Muller / FEBS Letters 588 (2014) 1396–1402 1397 junctions. In hindsight, the junctional coupling reported initially The biophysical properties of innexin membrane channels [1] was an artifact of the high levels of expression in the oocyte, (innexons) are very similar to those of Panx channels, with single where some pannexons were consequently without their usual channel properties of innexons as complex as those observed for glycosylation and were thus able to dock with pannexons overex- pannexons [17]. The maximal single channel conductance is pressed in an adjacent, contacting oocyte. Thus, pannexin was a 500 pS and, like pannexons, innexons exhibit multiple subcon- protein in search of a function. Because of the similar pharmacol- ductance states (Fig. 1). Furthermore, innexons open in response + ogy of innexins and connexins, it was intuited that pannexins to mechanical stress and are activated by increased [K ]o at the would behave similarly. This assumption was proven to be correct; resting like pannexons. all gap junction inhibitors also inhibit pannexin channels, as Concordant with the high single channel conductance, both summarized in a recent review [14]. innexin and Panx1 channels exhibit permeability to larger Given the overlapping pharmacologies of connexins and pan- molecules than the small ions typically permeating ion channels nexins, it was reasonable to consider that pannexins might be [17–21]. Channels formed by both proteins allow the passage of responsible for ATP release, as originally attributed to connexin both cationic and anionic dyes, which also are known to cross from ‘‘hemichannels’’. Systematic testing of this hypothesis in various cell to cell through gap junctions. Physiologically more relevant is cell types yielded evidence for a major role of Panx1 channels in the permeation of the signal molecule ATP from the cytoplasm to ATP release under physiological and pathological conditions [15]. the extracellular space through innexin and Panx1 channels. Invertebrates that are not chordates do not have connexins, but The similarity between pannexons and innexons also extends to their cells can generate calcium waves. Because of the sequence the pharmacological properties of the channels. Both types of similarity between pannexins and innexins, it was obvious to test channels are inhibited by cytoplasmic acidification, carbenoxolone, the ability of innexins to form patent unpaired channels in addition arachidonic acid, and Brilliant Blue G [17,22,23]. Thus, the overall to their well-documented formation of gap junction channels. In- similarity between innexins and pannexins extends well beyond deed, several innexins were found to be bifunctional, forming the limited similarity between the amino acid sequences. gap junctions and unpaired membrane channels, called ‘‘innex- ons’’, allowing the flux of small molecules including ATP across the plasma membrane [16,17]. 4. Activation mechanisms for innexin and pannexin channels

Both innexin and pannexin channels can be activated by mem- 3. Biophysical properties of Panx1 and innexin channels brane potential changes [18,24]. While activation by voltage is a convenient method to study basic properties of pannexin channels, Single channel records among the pannexins presently are only this activation method is far from physiological, since positive available for Panx1. This channel, called a ‘‘pannexon’’, is unusual holding potentials in excess of +20 mV are required for Panx1 in its high conductance and the presence of multiple subconduc- channel opening. Panx2 channels open at even more extreme posi- tance states [18]. The maximal conductance of the Panx1 channel tive membrane potentials [25]. Innexin channels are somewhat is 500 pS. However, even with maximal stimulation, this high more sensitive to voltage changes and can be activated by depolar- conductance typically is not sustained, but the channel dwells for ization to 20 mV. The positive potentials required to open variable durations in one of the multiple subconductance states pannexin channels are unlikely to occur in vivo. Indeed, membrane (Fig. 1). potential-induced ATP release from nerve terminals is mainly, if not exclusively, vesicular [26–29]. Positive potentials are typically only associated with action potentials, which are generally very a brief and thus would not support efficient ATP release from cells. c More significantly, the electrochemical gradient would not be favorable for such release either. While the concentration gradient represents a driving force for ATP efflux, the electrical gradient Panx1 would oppose such flux. Indeed, a recent study demonstrated that ATP flux through connexin ‘‘hemichannels’’, which only open at extremely positive potentials, did not occur while the membrane potential was stepped to +80 mV [30]. Instead, a brief ATP release was associated with the tail currents after stepping the membrane potential back to 40 mV. While tail currents are very useful anal- o ysis tools, they are an experimental artifact. In vivo, membrane channels do not work under voltage clamp conditions. It would 20 pA need a combination of slowly inactivating depolarizing channels combined with fast opening hyperpolarizing channels to approxi- mate the situation encountered experimentally in tail currents. 2 sec However, most ATP release in vivo occurs at or near the resting b membrane potential, where connexin hemichannels are closed c and Panx1 channels are activated by other means than membrane voltage changes. Inx3 We have pointed out early on that the activation of Panx1 chan- nels by physiological stimuli can occur at the resting membrane potential [11]. A series of stimuli have been identified to open o pannexin and innexin channels by voltage independent means. These physiological stimuli include low oxygen environment, Fig. 1. Single channel activity in excised membrane patches containing Panx1 (a) or mechanical stress, increased cytoplasmic calcium ion concentra- Hminx3 (b). The membrane potential was clamped at 100 mV. Both the bath and tion, and more indirect activation of the channels by ligands to the pipette solutions contained 150 mM potassium gluconate. Both channels have sub-conductance states, as previously reported for Hminx2. membrane receptors. Under pathological conditions, the channel 1398 G. Dahl, K.J. Muller / FEBS Letters 588 (2014) 1396–1402 can also be activated by increased extracellular potassium ion control of oxygen supply to tissues. This process most likely occurs concentration. multiple times during a day, and thus within a small fraction of the One of the physiological stimuli for pannexin channels found lifetime of the cell. soon after discovery of these proteins is a low oxygen environment surrounding erythrocytes or neurons (ischemia) [19,31,32]. Re- 5. Inactivation of pannexin and innexin channels duced oxygen causes ATP release from erythrocytes, which triggers a propagated calcium wave in the endothelial cell layer of blood Cytoplasmic acidification results in closure of all connexin and vessels, thereby producing NO to relax vascular smooth muscle. innexin gap junction channels [17,18]. Similarly, low cytoplasmic In this way, oxygen content of blood regulates oxygen supply to pH closes innexin and Panx1 membrane channels. The sensitivity tissues, such as skeletal muscle [33]. ATP release from erythrocytes of some of the various channels to pH changes is less than others, is particularly interesting, since these cells do not contain vesicles with some closed only at pH lower than the physiological range. and the release therefore must be non-vesicular and, therefore, Extracellular ATP is an important regulator of Panx1 and prob- probably channel mediated. Panx1 has been identified as a major ably also of innexin membrane channels [47]. Since these can be ATP release mediator [19,34] and Panx1 opens in physiologically ATP release channels, it may seem odd that the permeant inhibits relevant oxygen concentrations [31]. Oxygen sensing in the carotid its own permeation pathway. But the positive feedback resulting body also appears to involve Panx1 [35], where Panx1 in glia-like from purinergic receptors opening Panx1 channels needs a safe- cells is activated by ATP through P2Y receptors and acts as a signal guard against hyperactivation of the receptor/channel complex, amplifier, causing release of more ATP to activate neurons. and the negative feedback loop provided by the autoinhibition of Shear stress is a widespread physiological stimulus. For exam- the Panx1 channel by ATP provides it. The safeguard is needed be- ple, mechanical stimuli release ATP from erythrocytes and airway cause, for example, P2X7R and Panx1 signal to the inflammasome, epithelial cells [36,37]. It appears that mechanosensitivity is an and excessive signaling through this pathway can induce apopto- inherent basic feature of ATP release channels, since in other cell tic/pyroptotic cell death. Under physiological conditions, such types, such as astrocytes, mechanical stress can replace the ‘‘phys- excessive signaling probably is curbed by the autoinhibition of iological’’ stimulus, even when the stimulus is yet unknown. Both Panx1 by ATP. The inhibitory binding site for ATP on Panx1 is lo- innexin and pannexin channels contained in excised membrane cated within the extracellular loops of the protein [48] and shares patches can be activated by mechanical stress, indicating a direct features with that of the P2X7R. With the exception of oxidized action or a short signaling chain affecting channel open probability ATP [49], all ligands for the P2X7R tested so far also inhibit the [17,18]. Panx1 channel. In this context it does not matter whether the li- Both innexin and pannexin channels contained in excised mem- gands act as agonists or antagonists at the receptor. This feature brane patches have been shown to exhibit increased open proba- makes it difficult to distinguish between contributions of P2X7R bility when exposed to increased cytoplasmic calcium and Panx1 to various physiological events by pharmacological concentrations [17,38]. This is consistent with the role of the chan- means. The affinity of ATP to Panx1 is lower than to P2X7R nels in regeneratively propagated calcium waves. It should be [47,48], permitting sequential activation of positive and negative mentioned that, on the basis of indirect evidence, one study has re- feedback control mechanisms. Thus, extracellular ATP can bind to ported finding no effect of elevated calcium on the channels [39]. P2X7R, activate Panx1 and induce ATP release, which gets attenu- Panx1 channels can be activated indirectly by ligands binding to ated once the extracellular ATP concentration reaches a sufficiently their appropriate membrane receptors. This includes ATP binding high concentration to affect Panx1. to P2Y [38] or P2X7 receptors [20,40]. Similarly, glutamate binding Currents through both Panx1 and leech innexin (Hminx2) chan- to NMDA receptors has been shown to open Panx1 channels in nels are attenuated by arachidonic acid [50]. This effect appears to neurons [41]. be exerted by the compound itself, because a non-metabolizable Increased extracellular potassium ion concentration opens inn- form of arachidonic acid (eicosatetraynoic acid) exerts the same ef- exin and Panx1 channels independently of the effect of this ion on fect. Both compounds also inhibit the release of ATP from Panx1 the membrane potential. Under voltage clamp conditions the effect expressing oocytes [50]. of potassium ions was observed over a wide range of voltages [42]. Besides these potentially physiological regulators of Panx1 and The threshold concentration for the potassium ions is circa 10 mM innexin channels, diverse pharmacological inhibitors of these + [43] and the K -induced Panx1 currents increase with higher K+ channels have been identified [14]. In addition to the aforemen- + concentrations. Such K concentrations do not occur under physio- tioned gap junction blockers are substances not associated with logical conditions, since control mechanisms keep variations of gap junctions such as transport blockers (probenecid), potassium + extracellular K concentration to a minimum. However, under channel inhibitors (), mitochondrial inhibitors + pathological conditions extracellular K can reach values as high (bongkrekic acid), anti-malaria drugs (mefloquine, artemesinin), as 60 mM as observed in models of ischemic stroke [44,45]. Thus, chloride channel inhibitors (4,40-Diisothiocyano-2,20-stilbenedi- + K could well be a Panx1 activator in vivo and as such may be in- sulfonic acid), and P2X7R ligands (Brilliant Blue G). volved in secondary cell death [15]. An alternate activation mechanism for Panx1 channels has been reported to be cleavage of carboxy terminal amino acids by Cas- 6. Glia, microglia, and the role of pannexins/innexins pase 3 at a consensus site for this enzyme [46]. This mechanism in signaling injury of Panx1 activation rapidly gained support and is now widely ac- cepted, although it is clear that this activation mechanism cannot Glial cells, or glia, are the principal non-neuronal cells in the be broadly used by cells. Such cleavage is irreversible, causing cell nervous system. They acquired their name, which means ‘‘glue’’ death. Thus, while Panx1 activation by Caspase 3 cleavage is an in Greek, because of the way they closely surround nerve cells, important process late in the apoptotic signaling chain (after the including their dendrites and axons. It is this close association that cell is committed to apoptosis), this activation mechanism is unli- helps glia to act both as chemical and electrical insulators between kely to operate in most other cell types where Panx1 activation is a nerve cells and to supply important metabolites and signaling mol- rapidly and totally reversible process. The reversible activation of ecules. When Kuffler and Potter sought to make the first electrical Panx1 applies to all the other mechanisms described above, includ- measurements of glial cell properties, they turned to leech glia be- ing erythrocytes that exhibit Panx1 mediated ATP release for cause the glia in leech are large – up to 5 mm long and 100 lmin G. Dahl, K.J. Muller / FEBS Letters 588 (2014) 1396–1402 1399 diameter [51]. Kuffler and others soon found that the physiological injury to be important in microglial cell responses, the separate properties of mammalian glia are similar to leech glia, including function of NO in directing ATP-activated microglia to lesions has their gap junctional coupling to each other but not to neurons yet only been identified in the leech. [52]. We now know that a general property of glia is their genera- The leech nerve cord lies within a blood vessel so that the liv- tion of calcium waves [53–56], which may be generated physiolog- ing cord can be removed to a nutrient saline free of blood cells; ically and cause release of ATP, thereby signaling to neurons under these conditions its large glia, neurons and the microglia [57,58]. Less is known about the effects of glial calcium waves are clearly visible, the microglia respond to nerve lesions, and and the associated release of ATP on other cells, such as microglia, axons regenerate synaptic connections [74,75]. One can inject particularly under pathological conditions. the single giant glial cell that wraps thousands of axons and Microglial cells were first described by del Rio Hortega and oth- microglia in the nerve between two ganglia with siRNA to knock ers who used silver carbonate to stain both leech and mammalian down innexin expression and with other substances such as cal- microglia and saw that they were similar in diverse species [59]. cium-sensitive dyes [73]. These and related experiments to mea- Although the name microglia associates them with glia, microglial sure ATP and NO release and to administer pharmacological cells are the immune cells of the central nervous system (CNS), agents while watching microglial cells move have shown that a responding rapidly to CNS injury by moving to lesions, where they glial-specific innexin is required to release ATP, which triggers phagocytize cellular debris and microorganisms. Just as in mam- microglia to move to lesions, beginning hundreds of micrometers mals, leech microglia to not arise from ectodermal progenitors as away immediately after injury, when the calcium wave sweeps glial cells do; microglia enter the nervous system during develop- down the glial cell [63,73]. With the various cells maintaining ment and, in their resting state, are evenly distributed in the brain their same relationships as in vivo, it has been possible to show and rest of the central nervous system. In the course of evolution, that the direction that distant microglia travel depends on NO; adaptive immunity has increased the range of immune responses moreover, when glial innexins are blocked or knocked down, of vertebrate microglia, which nonetheless have retained the basic ATP added exogenously restores movement of microglia to morphology, physiology and behavior of their invertebrate coun- lesions and their accumulation there over the course of a few terparts, including leech microglia [60–65]. hours. Fig. 2 diagrams and summarizes the sequence of events Nerve injury is the classic means by which microglia become controlling migration of leech microglia to lesions, beginning in activated from their usual resting state. At rest, microglia are dis- their resting state (top panel), their activation and movement tributed throughout the nervous system as highly branched cells, toward a lesion (middle panel), and their accumulation there their branches extending into regions each occupying hundreds (bottom panel). The higher concentration of NO generated at the of lm3 that tile the brain. Even for resting microglia the branches lesion contributes to microglia stopping there [76,77], as does appear to be in continual movement, however [66]. Within min- arachidonic acid generated at lesions, which has been shown to utes of nerve or brain trauma, such as a crush or penetration inhibit the glial cell’s Hminx2 as it does Panx1 [22]. wound, microglia near the lesion change their shape, becoming In the mammalian brain, pannexons are expressed principally amoeboid, and move toward the lesion. This change is mimicked in glia [1]. Likewise, in the leech, where there is rapid loss of dye by microglia cultured in vitro, which have purinergic receptors from leech glia through innexons that are inhibited by carbenox-

[67], in response to extracellular ATP [68], which was known to olone or CO2 [17] but limited dye loss from neurons, it appears be released by nerve damage. The same study used a Boyden that innexins that form ATP-releasing channels operate primarily chamber, in which a filter with pores large enough for microglia in glia. Following the discovery of innexins less than 2 decades to cross separated the cells from a similar saline on the other side, ago in fruit flies and nematodes, they have been primarily consid- but containing ATP. The movement of microglia to the side of the ered as the molecular basis of gap junctions. The variety of cell filter in contact with the ATP solution was interpreted as indicating specific innexins suggested that, as for connexins in vertebrates, that ATP is a chemo-attractant for microglia; an alternative inter- innexins with different sequences and biophysical properties pretation, that ATP diffusing across the membrane simply triggered could underlie distinct functions of the gap junctions. For exam- microglia to move, some crossing the membrane in the presence of ple, in nematodes there have been 25 [78], fruit flies 8 (with mul- ATP, was unlikely because individual microglia moved toward a tiple splice isoforms) and in leeches 21 different innexin genes source of ATP and ADP in a Dunn chamber [68]. Whether microglial identified [79]. The numbering system for each species has gener- cell movement toward a lesion uses an additional signal that pre- ally been in the order that the genes have been discovered, so sumably forms a gradient, and whether ATP alone is important that the numbers across species typically do not correspond. Con- in vivo remains to be determined. Experiments on microglia in sistent with a variety of innexins, it has been shown that the the leech show that while extracellular ATP is required for microg- specificity of electrical connections between certain neurons in lial movement, directed movement depends upon nitric oxide the leech and presumably other organisms is regulated by cell- (NO), acting through a soluble guanylate cyclase, which is likely re- specific innexins [80,81]. Adding to the complexity is the discov- leased by a gradient of intracellular calcium (‘‘calcium wave’’) ery that some insect viruses contain vinnexins, which can form which contributes to ATP release. functional gap junctions with innexins of their insect host [82]. When glial cells such as astrocytes are mechanically perturbed Far less continues to be known about the function of unpaired or injured, they generate calcium waves spreading hundreds of innexons in invertebrates. micrometers [69] that cause microglia to respond [70]. Since cal- Close to the glia, the epithelial cells of the choroid plexus cium waves release ATP [71], which activates resting microglia release ATP through pannexons to cause epiplexus cells, which [68], and evidently ATP released from glia acts on the microglia are the plexus immune cells, to become motile [83]. The relation- [72], it is reasonable to conclude that glia carry some or all of the ship and action are similar to that between glia and microglia. signal sensed by distant microglia to indicate that there has been a nervous system injury. But for technical reasons it has been 7. Physiological release of ATP by Panx1 and innexins shown primarily in the leech that ATP released from glia activates microglial cells and signals them to move to lesions [63,73], and The foregoing sections on activation and inhibition of pannex- that the release pathway is through innexins associated with a cal- ons and innexons cite several examples of cells that release ATP cium wave that spreads hundreds of micrometers from the lesion through these channels under physiological conditions, including site [73]. As an aside, although NO has been shown in spinal cord erythrocytes, ciliated cells of the lung, and glial-like cells in the 1400 G. Dahl, K.J. Muller / FEBS Letters 588 (2014) 1396–1402

Fig. 2. Proposed schematic representation of the post-injury signals that regulate the microglial response in the leech nerve cord (after Samuels, 2010). Before Injury, in the leech a single giant glial cell ensheathes the axons and microglia of the nerve (connective). The glial innexons, formed by Hminx2, are closed at rest. The microglia (MG) display P2Y receptors (P2YR). Immediately after Injury, the lesion initiates a calcium wave which maintains a calcium gradient. The calcium wave opens innexons, permitting exit of ATP molecules, which bind to P2YR and activate the microglia, causing them to move. The increased glial calcium also generates NO, which provides direction to the microglia, which move toward the lesion, where NO is also released. Minutes to Hours after Injury, microglia accumulate at the lesion and stop there. This is because generation of arachidonic acid (ArA) causes pannexons nearest the lesion to close, reducing microglial cell activation. Elevated NO at the crush also slows microglia, while microglia distant from the lesion continue to migrate to the lesion. Modified from: Samuels, Stuart E., ‘‘Innexons, Membrane Channels for ATP, Control Microglia Migration to Nerve Lesions’’ (2009). University of Miami Open Access Dissertation. Paper 660.

carotid involved in amplifying receptor signals. Some of these evolved, for the unrelated connexins rather than pannexins examples and others are described in pannexin-focused reviews perform gap junction functions of innexins. But like pannexins, [15,84]. innexins can form channels for ATP release from cells, and both vertebrate and invertebrate glia use the pathway to signal to microglia assaults to the nervous system. The range of such ATP- 8. Summary release functions has just begun to be explored, and recent work showing several conductance states of both pannexins and innex- In one sense pannexins represent an evolutionary narrowing of ins suggests that there are many more possibly shared functions to the range of functions of the innexin proteins from which they be identified. G. Dahl, K.J. Muller / FEBS Letters 588 (2014) 1396–1402 1401

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