Structural order in Pannexin 1 cytoplasmic domains

Pannexin 1 forms ion and metabolite permeable hexameric channels with abundant expression in the central nervous system and elsewhere. Although pannexin 1 does not form intercellular channels, a common channel topology and oligomerization state, as well as involvement of the intracellular carboxyl terminal (CT) domain in channel gating, is shared with connexins. In this study, we characterized the secondary structure of the mouse pannexin 1 cytoplasmic domains to complement structural studies of the transmembrane segments and compare with similar domains from connexins. A combination of structural prediction tools and circular dichroism revealed that, unlike connexins (predominately intrinsically disordered), cytosolic regions of pannexin 1 contain approximately 50% secondary structure, a majority being α-helical. Moreover, prediction of transmembrane domains uncovered a potential membrane interacting region (I360-G370) located upstream of the caspase cleavage site (D375-D378) within the pannexin 1 CT domain. The α-helical content of a peptide containing these domains (G357-S384) increased in the presence of detergent micelles providing evidence of membrane association. We also purified a pannexin 1 CT construct containing the caspase cleavage site (M374-C426), assigned the resonances by NMR, and confirmed cleavage by Caspase-3 in vitro. On the basis of these structural studies of the cytoplasmic domains of pannexin 1, we propose a mechanism for the opening of pannexin 1 channels upon apoptosis, involving structural changes within the CT domain.     

Pannexin 1 and Pannexin 3 Channels Regulate Skeletal Muscle Myoblast Proliferation and Differentiation

Pannexins constitute a family of three glycoproteins (Panx1, -2, and -3) forming single membrane channels. Recent work demonstrated that Panx1 is expressed in skeletal muscle and involved in the potentiation of contraction. However, Panxs functions in skeletal muscle cell differentiation, and proliferation had yet to be assessed. We show here that Panx1 and Panx3, but not Panx2, are present in human and rodent skeletal muscle, and their various species are differentially expressed in fetal versus adult human skeletal muscle tissue. Panx1 levels were very low in undifferentiated human primary skeletal muscle cells and myoblasts (HSMM) but increased drastically during differentiation and became the main Panx expressed in differentiated cells. Using HSMM, we found that Panx1 expression promotes this process, whereas it was impaired in the presence of probenecid or carbenoxolone. As for Panx3, its lower molecular weight species were prominent in adult skeletal muscle but very low in the fetal tissue and in undifferentiated skeletal muscle cells and myoblasts. Its overexpression (∼43-kDa species) induced HSMM differentiation and also inhibited their proliferation. On the other hand, a ∼70-kDa immunoreactive species of Panx3, likely glycosylated, sialylated, and phosphorylated, was highly expressed in proliferative myoblasts but strikingly down-regulated during their differentiation. Reduction of its endogenous expression using two Panx3 shRNAs significantly inhibited HSMM proliferation without triggering their differentiation. In summary, our results demonstrate that Panx1 and Panx3 are co-expressed in human skeletal muscle myoblasts and play a pivotal role in dictating the proliferation and differentiation status of these cells.     

Structural order in Pannexin 1 cytoplasmic domains

Pannexin 1 forms ion and metabolite permeable hexameric channels with abundant expression in the central nervous system and elsewhere. Although pannexin 1 does not form intercellular channels, a common channel topology and oligomerization state, as well as involvement of the intracellular carboxyl terminal (CT) domain in channel gating, is shared with connexins. In this study, we characterized the secondary structure of the mouse pannexin 1 cytoplasmic domains to complement structural studies of the transmembrane segments and compare with similar domains from connexins. A combination of structural prediction tools and circular dichroism revealed that, unlike connexins (predominately intrinsically disordered), cytosolic regions of pannexin 1 contain approximately 50% secondary structure, a majority being α-helical. Moreover, prediction of transmembrane domains uncovered a potential membrane interacting region (I360-G370) located upstream of the caspase cleavage site (D375-D378) within the pannexin 1 CT domain. The α-helical content of a peptide containing these domains (G357-S384) increased in the presence of detergent micelles providing evidence of membrane association. We also purified a pannexin 1 CT construct containing the caspase cleavage site (M374-C426), assigned the resonances by NMR, and confirmed cleavage by Caspase-3 in vitro. On the basis of these structural studies of the cytoplasmic domains of pannexin 1, we propose a mechanism for the opening of pannexin 1 channels upon apoptosis, involving structural changes within the CT domain.     

Pannexin1 and Pannexin3 Delivery, Cell Surface Dynamics, and Cytoskeletal Interactions

Pannexins (Panx) are a class of integral membrane proteins that have been proposed to exhibit characteristics similar to those of connexin family members. In this study, we utilized Cx43-positive BICR-M1R_k cells to stably express Panx1, Panx3, or Panx1-green fluorescent protein (GFP) to assess their trafficking, cell surface dynamics, and interplay with the cytoskeletal network. Expression of a Sar1 dominant negative mutant revealed that endoplasmic reticulum to Golgi transport of Panx1 and Panx3 was mediated via COPII-dependent vesicles. Distinct from Cx43-GFP, fluorescence recovery after photobleaching studies revealed that both Panx1-GFP and Panx3-GFP remained highly mobile at the cell surface. Unlike Cx43, Panx1-GFP exhibited no detectable interrelationship with microtubules. Conversely, cytochalasin B-induced disruption of microfilaments caused a severe loss of cell surface Panx1-GFP, a reduction in the recoverable fraction of Panx1-GFP that remained at the cell surface, and a decrease in Panx1-GFP vesicular transport. Furthermore, co-immunoprecipitation and co-sedimentation assays revealed actin as a novel binding partner of Panx1. Collectively, we conclude that although Panx1 and Panx3 share a common endoplasmic reticulum to Golgi secretory pathway to Cx43, their ultimate cell surface residency appears to be independent of cell contacts and the need for intact microtubules. Importantly, Panx1 has an interaction with actin microfilaments that regulates its cell surface localization and mobility.     

S-Nitrosylation Inhibits Pannexin 1 Channel Function

S-Nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1–1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1^C40A, Panx1^C346A, and Panx1^C426A). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1^C40A/C346A) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.     

The Pannexin 1 Channel Activates the Inflammasome in Neurons and Astrocytes

The inflammasome is a multiprotein complex involved in innate immunity. Activation of the inflammasome causes the processing and release of the cytokines interleukins 1β and 18. In primary macrophages, potassium ion flux and the membrane channel pannexin 1 have been suggested to play roles in inflammasome activation. However, the molecular mechanism(s) governing inflammasome signaling remains poorly defined, and it is undetermined whether these mechanisms apply to the central nervous system. Here we show that high extracellular potassium opens pannexin channels leading to caspase-1 activation in primary neurons and astrocytes. The effect of K⁺ on pannexin 1 channels was independent of membrane potential, suggesting that stimulation of inflammasome signaling was mediated by an allosteric effect. The activation of the inflammasome by K⁺ was inhibited by the pannexin 1 channel blocker probenecid, supporting a role of pannexin 1 in inflammasome activation. Co-immunoprecipitation of neuronal lysates indicates that pannexin 1 associates with components of the multiprotein inflammasome complex, including the P2X7 receptor and caspase-1. Moreover antibody neutralization of the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) blocked ATP-induced cell death in oocytes co-expressing P2X7 receptor and pannexin 1. Thus, in contrast to macrophages and monocytes in which low intracellular K⁺ has been suggested to trigger inflammasome activation, in neural cells, high extracellular K⁺ activates caspase-1 probably through pannexin 1.Pannexin 1 is a vertebrate ortholog of the invertebrate innexin gap junction proteins (1), but it does not appear to form functional gap junctions in vivo. Instead pannexin 1 acts as a membrane channel that carries ions and signaling molecules between the cytoplasm and the extracellular space (2, 3). As such, it is a candidate ATP release channel in various cell types, including erythrocytes, astrocytes, bronchial epithelial cells, and taste cells. Various functional roles have been ascribed to pannexin 1 including local vascular perfusion control and propagation of intercellular calcium waves (4–6). Recently pannexin 1 was also shown to form the large pore of the P2X7 purinergic receptor (7, 8). P2X7 plays a major role in inflammation, and its activation by extracellular ATP results in release of interleukin (IL)²-1β from macrophages, probably involving pannexin 1 as a signaling molecule (7).IL-1β production and maturation are tightly regulated by caspase-1 incorporated into large protein complexes termed inflammasomes (9–11). The molecular composition of the inflammasome depends on the identity of the NOD-like receptor (NLR) family member serving as a scaffold protein in the complex (12). The members of the cytosolic NLR family appear to recognize conserved microbial and viral components termed pathogen-associated molecular patterns in intracellular compartments (13). The bipartite adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) bridges the interaction between NLR proteins and inflammatory caspases and plays a central role in the assembly of inflammasomes and the activation of caspase-1 in response to a broad range of pathogen-associated molecular patterns and intracellular pathogens (14). In addition, the inflammasome can be activated by danger-associated molecular patterns, molecules endogenous to the organism that signal stress or injury, including extracellular ATP acting at ionotropic P2X7 receptors, fibronectin, or monosodium urate crystals (15, 16). Moreover it has been suggested that a rapid K⁺ efflux through ATP-activated P2X7 receptors induces inflammasome assembly (17–20).Despite the recent advances in the understanding of accessory proteins required for full activation of caspase-1, little is known about the signaling pathways that trigger inflammasome activation, particularly in the central nervous system (CNS). Recently we reported that spinal cord neurons contain the NLRP1/NALP1 inflammasome consisting of NLRP1, ASC, caspase-1, caspase-11, and the X-linked inhibitor of apoptosis protein (XIAP) and that spinal cord injury induces rapid activation of the inflammasome, causing processing and secretion of IL-1β and IL-18. Moreover antibody neutralization of ASC reduces caspase-1 activation and IL-1 cytokine processing, leading to significant tissue sparing and functional improvement (21). In this study, we focused on signaling events coupling pannexin 1 and P2X7 receptors to rapid caspase-1 activation in primary neurons and astrocytes. We provide compelling evidence that high extracellular K⁺ opens the pannexin 1 channel and activates inflammasomes in neurons and astrocytes, but not THP-1 cells, thus leading to caspase-1 activation. This signaling pathway in neurons is mediated through protein interactions between pannexin 1 and inflammasome proteins. We also provide evidence that ATP acting on P2X7 induces rapid cell death and that antibody neutralization of ASC blocks ATP-induced cell death. Thus, contrary to the widely accepted view in macrophages and monocytes that low intracellular K⁺ triggers inflammasome activation, high extracellular K⁺ surrounding cells such as neurons and astrocytes opens pannexin 1 channels and induces processing of caspase-1.     

Pannexin1离子通道的结构与功能研究进展

Pannexin1 (PANX1)是一个可以通透包括ATP在内的多种化合物以及离子的离子通道蛋白.它可以在细胞凋亡、炎症等多种生理现象中起到重要的作用,被认为是一个具有潜力的治疗靶点.若要将其当作治疗靶点,需要对其作为离子通道的特性有较为深刻的认识.此前针对该离子通道的研究有诸多限制,随着近几年冷冻电镜技术的发展,对离子通道的研究发展到了一个新的阶段.受益于冷冻电镜技术,PANX1蛋白的结构被解析出来,包括激活机制、离子通路以及小分子抑制剂的阻断机制在内的多个问题有了阶段性的解答.本文旨在从结构生物学的角度,介绍该离子通道的一些基础特性,并分析目前该蛋白质研究上所遇到的问题,为将来的工作提出重点研究目标.     

Contribution of Pannexin1 to Experimental Autoimmune Encephalomyelitis

Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.     

Intrinsic properties and regulation of Pannexin 1 channel

Pannexin 1 (Panx1) channels are generally represented as non-selective, large-pore channels that release ATP. Emerging roles have been described for Panx1 in mediating purinergic signaling in the normal nervous, cardiovascular, and immune systems, where they may be activated by mechanical stress, ionotropic and metabotropic receptor signaling, and via proteolytic cleavage of the Panx1 C-terminus. Panx1 channels are widely expressed in various cell types, and it is now thought that targeting these channels therapeutically may be beneficial in a number of pathophysiological contexts, such as asthma, atherosclerosis, hypertension, and ischemic-induced seizures. Even as interest in Panx1 channels is burgeoning, some of their basic properties, mechanisms of modulation, and proposed functions remain controversial, with recent reports challenging some long-held views regarding Panx1 channels. In this brief review, we summarize some well-established features of Panx1 channels; we then address some current confounding issues surrounding Panx1 channels, especially with respect to intrinsic channel properties, in order to raise awareness of these unsettled issues for future research.     

Pannexin 1 activity in astroglia sets hippocampal neuronal network patterns

Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.

Astrocytes have mostly been shown to boost neuronal activity. This study reveals that activity-dependent activation of astroglial pannexin 1 channels inhibits hippocampal neuronal networks by decreasing neuronal excitability via purinergic signaling, uncovering a novel astroglial negative feedback loop mechanism.     

Carbenoxolone inhibits Pannexin1 channels through interactions in the first extracellular loop

Pannexin1 (Panx1) is an ATP release channel important for controlling immune responses and synaptic strength. Various stimuli including C-terminal cleavage, a high concentration of extracellular potassium, and voltage have been demonstrated to activate Panx1. However, it remains unclear how Panx1 senses and integrates such diverse stimuli to form an open channel. To provide a clue on the mechanism underlying Panx1 channel gating, we investigated the action mechanism of carbenoxolone (CBX), the most commonly used small molecule for attenuating Panx1 function triggered by a wide range of stimuli. Using a chimeric approach, we discovered that CBX reverses its action polarity and potentiates the voltage-gated channel activity of Panx1 when W74 in the first extracellular loop is mutated to a nonaromatic residue. A systematic mutagenesis study revealed that conserved residues in this loop also play important roles in CBX function, potentially by mediating CBX binding. We extended our experiments to other Panx1 inhibitors such as probenecid and ATP, which also potentiate the voltage-gated channel activity of a Panx1 mutant at position 74. Notably, probenecid alone can activate this mutant at a resting membrane potential. These data suggest that CBX and other inhibitors, including probenecid, attenuate Panx1 channel activity through modulation of the first extracellular loop. Our experiments are the first step toward identifying a previously unknown mode of CBX action, which provide insight into the role of the first extracellular loop in Panx1 channel gating.     

H-Y Antigen in the Teleost

H-Y antigen, presumably the product of mammalian testis-determining genes, has been detected in three species of teleost fish, Xiphophorus maculatus, Haplochromis burtoni , and Oryzias latipes , and in hybrids of the genus Tilapia . In X. maculatus H-Y was most readily detected in YY males, suggesting that a genetic determinant of H-Y antigen expression may exist on the teleost Y-chromosome. Although H-Y was detected in males and not in females in each of the species that we studied, male heterogamety has not been firmly established for H. burtoni . Thus despite the extreme phlyogenetic conservation of H-Y genes and their association with the Y-chromosome, it remains open to question whether H-Y will always be found in the heterogametic sex, and whether serologically defined H-Y antigen plays any part in the differentiation of the teleost gonad.     

Pannexin1 Channel Proteins in the Zebrafish Retina Have Shared and Unique Properties

In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.     

Pannexin1 and Pannexin3 Exhibit Distinct Localization Patterns in Human Skin Appendages and are Regulated during Keratinocyte Differentiation and Carcinogenesis

Having shown that Panx1 and Panx3 are expressed in the epidermis, we investigated their distribution in human skin adnexal structures and skin cancer. Both proteins were found in hair follicles, sebaceous and eccrine glands, as well as blood vessels. Panx1 was detected as punctate or diffuse intracellular labeling, while Panx3 was only observed as diffuse intracellular staining, suggesting different functions. We also identified the Panx3 immunoreactive ～70 kD species modulated during keratinocyte differentiation as Panx3. Since our data indicate that pannexins are regulated during keratinocyte differentiation, we assessed whether their levels are altered under circumstances in which keratinocyte differentiation is compromised. We found that Panx1 and Panx3 levels are highly reduced in human keratinocyte tumors, thus showing for the first time that both pannexins are dysregulated in human cancers. Altogether, these data suggest that Panx1 and Panx3 have distinct and unique functions within the skin in health and disease.     

Pannexin1 and Pannexin2 Channels Show Quaternary Similarities to Connexons and Different Oligomerization Numbers from Each Other

Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other.     

Pannexin 1 constitutes the large conductance cation channel of cardiac myocytes

A large conductance (～300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca(2+) release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca(2+) release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.     

Pannexin1 and Pannexin3 Exhibit Distinct Localization Patterns in Human Skin Appendages and are Regulated during Keratinocyte Differentiation and Carcinogenesis

Having shown that Panx1 and Panx3 are expressed in the epidermis, we investigated their distribution in human skin adnexal structures and skin cancer. Both proteins were found in hair follicles, sebaceous and eccrine glands, as well as blood vessels. Panx1 was detected as punctate or diffuse intracellular labeling, while Panx3 was only observed as diffuse intracellular staining, suggesting different functions. We also identified the Panx3 immunoreactive ～70 kD species modulated during keratinocyte differentiation as Panx3. Since our data indicate that pannexins are regulated during keratinocyte differentiation, we assessed whether their levels are altered under circumstances in which keratinocyte differentiation is compromised. We found that Panx1 and Panx3 levels are highly reduced in human keratinocyte tumors, thus showing for the first time that both pannexins are dysregulated in human cancers. Altogether, these data suggest that Panx1 and Panx3 have distinct and unique functions within the skin in health and disease.     

Dopaminergic Mechanisms in the Teleost Retina

A specific dopamine-sensitive adenylate cyclase has been identified in homogenates of the teleost (carp) retina. Maximal stimulation by 100 microM-dopamine resulted in a 5--10-fold increase in adenylate cyclase activity with half-maximal stimulation occurring at a concentration of 1 microM. l-Noradrenaline and l-adrenaline were some 10 times less potent than dopamine whilst the alpha- and beta-adrenoreceptor agonists, l-phenylephrine and dl-isoprenaline were inactive. Apomorphine elicited a partial stimulation of adenylate cyclase activity whilst various ergot alkaloids produced mixed agonist/antagonist responses. Dopamine-stimulated adenylate cyclase activity was potently antagonised by various neuroleptic drugs including fluphenazine, alpha-flupenthixol and alpha-piflutixol, and to a lesser extent by the butyrophenone derivatives haloperidol and spiperone. The benzamide derivatives, metoclopramide and sulpiride, together with the alpha- and beta-adrenoreceptor blocking agents, phentolamine and propranolol respectively were essentially inactive at blocking dopamine-stimulated adenylate cyclase activity. These data suggest the presence of a highly specific dopamine-sensitive adenylate cyclase in homogenates of teleost retina possessing similar pharmacological properties to the dopamine-sensitive adenylate cyclase observed in the mammalian central nervous system.