{"title":"炭疽热钙通道与PA相互作用的电生理证据","authors":"E. Kobrinsky, N. Soldatov","doi":"10.1080/19336950.2016.1172886","DOIUrl":null,"url":null,"abstract":"Tripartite anthrax toxin is composed of nontoxic edema factor (EF, calmodulin/Ca2C-dependent adenylate cyclase), lethal factor (LF, a zinc-dependent metallopeptidase cleaving mitogen activated protein kinases) and non-toxic PA (83 kDa). On release from the anthrax bacillus as monomers, they assemble into toxic complexes on the surface of host cells. According to the established mechanism of toxic effect, PA binds to the cell surface receptors and facilitates the translocation of a “lethal toxin” composed of LF and EF. Two PA receptors have been previously identified, including ATR/TEM8 (anthrax toxin receptor/tumor endothelial marker 8 ) and CMG2 (capillary morphogenesis protein 2 ). Mechanism of toxic effect is not well known, except that PA binds to the cell surface receptors and forms heptameric pores that are involved in translocation of the “lethal toxin.\" The formation of PA channels on the surface of a host target cell is a key step in the pathogenesis of anthrax. PA requires for binding a common von Willebrand factor A (integrin-like) inserted domain that contains MIDAS composed of DxSxSx59–79Tx12–23D, where x is any amino acid. The MIDAS domain is present in the accessory a2d-1 and a2d¡2 subunits of the Cav1 and Cav2 families of the calcium channel. Common features of a2d, ATR/TEM8 and CMG2 include an extracellular von Willebrand factor A domain and a single-pass transmembrane region. Because calcium channels are clustered in the plasma membrane, they suite well to co-localize the PA pores to allow for sufficient entry of anthrax toxin into the cells. Unlike ATR/TEM8 and CMG2, Cav1 and Cav2 calcium channels are present in a wide variety of cells of the body except cells of immune system and blood cells (although a2d is found in lymphocytes). Importantly, they are expressed in endothelial cells, keratinocytes and fibroblasts, which represent the primary locations for bacterial entry. Given the wide distribution of calcium channel a2d proteins, we hypothesize that the a2d subunit may interact with PA. This study presents the first experimental evidence suggesting such an interaction. The patch clamp study was carried out essentially as described earlier 8 with the recombinant human Cav1.2 calcium channels composed of the fluorescently labeled vascular/fibroblast pore-forming ECFPN-a1C,77 (z34815) subunit and accessory vascular b3 (X76555) subunit co-expressed with a2d¡1 (AAA51903) in Cos1 cells. In these studies we used PA-U7, the mutant of PA where the furin cleavage site was deleted. PA-U7 retains ability to bind to receptors but cannot be proteolytically activated. This property of PA-U7 is particularly useful for patch clamp experiments because PA-U7 is unable to form leak channels that would otherwise compromise the specificity of recordings and stability of patch clamp by generating a large non-selective leak current. It was found that PA-U7 inhibited the Ca2C current in a nM range (Fig. 1A). Although there are data that PA-U7 may eventually dissociate from receptors, we did not observe a significant reversal of the calcium channel inhibition after wash-out for 10 min with the PA-free buffer. Longer wash-out was incompatible with the stability of whole-cell patch clamp. Under the same conditions, the inactive PA mutant PA-3M that contains 3 mutations in domain 4 preventing binding to a receptor, did not induce notable inhibition of the Ca2C current (open circles). Slow linear decrease of the","PeriodicalId":9750,"journal":{"name":"Channels","volume":"4 1","pages":"253 - 255"},"PeriodicalIF":3.3000,"publicationDate":"2016-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Electrophysiological evidences of interaction between calcium channels and PA of anthrax\",\"authors\":\"E. Kobrinsky, N. Soldatov\",\"doi\":\"10.1080/19336950.2016.1172886\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Tripartite anthrax toxin is composed of nontoxic edema factor (EF, calmodulin/Ca2C-dependent adenylate cyclase), lethal factor (LF, a zinc-dependent metallopeptidase cleaving mitogen activated protein kinases) and non-toxic PA (83 kDa). On release from the anthrax bacillus as monomers, they assemble into toxic complexes on the surface of host cells. According to the established mechanism of toxic effect, PA binds to the cell surface receptors and facilitates the translocation of a “lethal toxin” composed of LF and EF. Two PA receptors have been previously identified, including ATR/TEM8 (anthrax toxin receptor/tumor endothelial marker 8 ) and CMG2 (capillary morphogenesis protein 2 ). Mechanism of toxic effect is not well known, except that PA binds to the cell surface receptors and forms heptameric pores that are involved in translocation of the “lethal toxin.\\\" The formation of PA channels on the surface of a host target cell is a key step in the pathogenesis of anthrax. PA requires for binding a common von Willebrand factor A (integrin-like) inserted domain that contains MIDAS composed of DxSxSx59–79Tx12–23D, where x is any amino acid. The MIDAS domain is present in the accessory a2d-1 and a2d¡2 subunits of the Cav1 and Cav2 families of the calcium channel. Common features of a2d, ATR/TEM8 and CMG2 include an extracellular von Willebrand factor A domain and a single-pass transmembrane region. Because calcium channels are clustered in the plasma membrane, they suite well to co-localize the PA pores to allow for sufficient entry of anthrax toxin into the cells. Unlike ATR/TEM8 and CMG2, Cav1 and Cav2 calcium channels are present in a wide variety of cells of the body except cells of immune system and blood cells (although a2d is found in lymphocytes). Importantly, they are expressed in endothelial cells, keratinocytes and fibroblasts, which represent the primary locations for bacterial entry. Given the wide distribution of calcium channel a2d proteins, we hypothesize that the a2d subunit may interact with PA. This study presents the first experimental evidence suggesting such an interaction. The patch clamp study was carried out essentially as described earlier 8 with the recombinant human Cav1.2 calcium channels composed of the fluorescently labeled vascular/fibroblast pore-forming ECFPN-a1C,77 (z34815) subunit and accessory vascular b3 (X76555) subunit co-expressed with a2d¡1 (AAA51903) in Cos1 cells. In these studies we used PA-U7, the mutant of PA where the furin cleavage site was deleted. PA-U7 retains ability to bind to receptors but cannot be proteolytically activated. This property of PA-U7 is particularly useful for patch clamp experiments because PA-U7 is unable to form leak channels that would otherwise compromise the specificity of recordings and stability of patch clamp by generating a large non-selective leak current. It was found that PA-U7 inhibited the Ca2C current in a nM range (Fig. 1A). Although there are data that PA-U7 may eventually dissociate from receptors, we did not observe a significant reversal of the calcium channel inhibition after wash-out for 10 min with the PA-free buffer. Longer wash-out was incompatible with the stability of whole-cell patch clamp. Under the same conditions, the inactive PA mutant PA-3M that contains 3 mutations in domain 4 preventing binding to a receptor, did not induce notable inhibition of the Ca2C current (open circles). 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Electrophysiological evidences of interaction between calcium channels and PA of anthrax
Tripartite anthrax toxin is composed of nontoxic edema factor (EF, calmodulin/Ca2C-dependent adenylate cyclase), lethal factor (LF, a zinc-dependent metallopeptidase cleaving mitogen activated protein kinases) and non-toxic PA (83 kDa). On release from the anthrax bacillus as monomers, they assemble into toxic complexes on the surface of host cells. According to the established mechanism of toxic effect, PA binds to the cell surface receptors and facilitates the translocation of a “lethal toxin” composed of LF and EF. Two PA receptors have been previously identified, including ATR/TEM8 (anthrax toxin receptor/tumor endothelial marker 8 ) and CMG2 (capillary morphogenesis protein 2 ). Mechanism of toxic effect is not well known, except that PA binds to the cell surface receptors and forms heptameric pores that are involved in translocation of the “lethal toxin." The formation of PA channels on the surface of a host target cell is a key step in the pathogenesis of anthrax. PA requires for binding a common von Willebrand factor A (integrin-like) inserted domain that contains MIDAS composed of DxSxSx59–79Tx12–23D, where x is any amino acid. The MIDAS domain is present in the accessory a2d-1 and a2d¡2 subunits of the Cav1 and Cav2 families of the calcium channel. Common features of a2d, ATR/TEM8 and CMG2 include an extracellular von Willebrand factor A domain and a single-pass transmembrane region. Because calcium channels are clustered in the plasma membrane, they suite well to co-localize the PA pores to allow for sufficient entry of anthrax toxin into the cells. Unlike ATR/TEM8 and CMG2, Cav1 and Cav2 calcium channels are present in a wide variety of cells of the body except cells of immune system and blood cells (although a2d is found in lymphocytes). Importantly, they are expressed in endothelial cells, keratinocytes and fibroblasts, which represent the primary locations for bacterial entry. Given the wide distribution of calcium channel a2d proteins, we hypothesize that the a2d subunit may interact with PA. This study presents the first experimental evidence suggesting such an interaction. The patch clamp study was carried out essentially as described earlier 8 with the recombinant human Cav1.2 calcium channels composed of the fluorescently labeled vascular/fibroblast pore-forming ECFPN-a1C,77 (z34815) subunit and accessory vascular b3 (X76555) subunit co-expressed with a2d¡1 (AAA51903) in Cos1 cells. In these studies we used PA-U7, the mutant of PA where the furin cleavage site was deleted. PA-U7 retains ability to bind to receptors but cannot be proteolytically activated. This property of PA-U7 is particularly useful for patch clamp experiments because PA-U7 is unable to form leak channels that would otherwise compromise the specificity of recordings and stability of patch clamp by generating a large non-selective leak current. It was found that PA-U7 inhibited the Ca2C current in a nM range (Fig. 1A). Although there are data that PA-U7 may eventually dissociate from receptors, we did not observe a significant reversal of the calcium channel inhibition after wash-out for 10 min with the PA-free buffer. Longer wash-out was incompatible with the stability of whole-cell patch clamp. Under the same conditions, the inactive PA mutant PA-3M that contains 3 mutations in domain 4 preventing binding to a receptor, did not induce notable inhibition of the Ca2C current (open circles). Slow linear decrease of the
期刊介绍:
Channels is an open access journal for all aspects of ion channel research. The journal publishes high quality papers that shed new light on ion channel and ion transporter/exchanger function, structure, biophysics, pharmacology, and regulation in health and disease.
Channels welcomes interdisciplinary approaches that address ion channel physiology in areas such as neuroscience, cardiovascular sciences, cancer research, endocrinology, and gastroenterology. Our aim is to foster communication among the ion channel and transporter communities and facilitate the advancement of the field.