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Channels (Austin). 2016 Jul-Aug; 10(4): 253–255.
Published online 2016 April 5. doi:  10.1080/19336950.2016.1172886
PMCID: PMC4954578

Electrophysiological evidences of interaction between calcium channels and PA of anthrax

Tripartite anthrax toxin is composed of nontoxic edema factor (EF, calmodulin/Ca2+-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.1 Two PA receptors have been previously identified, including ATR/TEM8 (anthrax toxin receptor/tumor endothelial marker 8 2) and CMG2 (capillary morphogenesis protein 2 3). 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 α2δ-1 and α2δ−2 subunits of the Cav1 and Cav2 families of the calcium channel. Common features of α2δ, 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,4 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 α2δ is found in lymphocytes). Importantly, they are expressed in endothelial cells,5 keratinocytes6 and fibroblasts,7 which represent the primary locations for bacterial entry. Given the wide distribution of calcium channel α2δ proteins, we hypothesize that the α2δ 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 ECFPN1C,77 (z34815) subunit and accessory vascular β3 (X76555) subunit co-expressed with α2δ−1 (AAA51903) in Cos1 cells. In these studies we used PA-U7,9 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 Ca2+ 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 Ca2+ current (open circles). Slow linear decrease of the normalized current amplitude in control is due to rundown typically seen with the Cav1.2 calcium channels.

Figure 1.
(A) Inhibition of calcium current by PA in Cos1 cells expressing human recombinant Cav1.2 composed of ECFPN1C,7732δ-1 subunits. Peak calcium currents were elicited by a stepwise depolarization to +30 mV applied ...

α2δ specifically interacts with gabapentin,10-13 a Pfizer’s evolving drug class that is used to treat several clinical disorders, including epilepsy, pain from diabetic neuropathy, postherpetic neuralgia and fibromyalgia, and generalized anxiety disorder. The α2δ-mediated modulation of neurotransmission by these drugs is due to attenuation of the Ca2+ current without changing its kinetics and voltage-dependence. Given the specificity of gabapentin to α2δ, we have tested the effect of this drug at physiological concentrations on PA-induced inhibition of the calcium current. Patch clamp experiments (Fig. 1B) revealed that gabapentin significantly protects the calcium channel current from the inhibition by PA-U7.

Taken together, from the presented patch clamp experiments we conclude that PA interacts with calcium channels with high affinity (K0.5 in a nM range) in an α2δ-specific and gabapentin-sensitive manner. Although the mechanism of the observed effects is not clear, these data show that α2δ represents a new potential molecular target for anthrax.

Abbreviations

MIDAS
metal ion-dependent adhesion site
PA
protective antigen

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We are thankful to Dr. Stephen Leppla (National Institute of Allergy and Infectious Diseases, NIH, USA) for gift of PA mutants.

Funding

This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute on Aging (Z01 AG000294-08 to N.M.S.).

References

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