ClC-2 chloride channels are voltage-gated ion channels that are expressed in neuronal and epithelial cells where they are critical mediators for the passive diffusion of Cl across the plasma membrane. Georgia anion toxin 2 ( GaTx2) is a 3.2 kDa peptide composed of 29 residues with three disulfide bonds. GaTx2 has an apparent dissociation constant of about 12 µM for CIC-2 at -100 mV. GaTx2 inhibits CIC-2 with higher affinity than any other available drugs and, in fact, is the best inhibitor of any chloride channel. The high affinity and specificity exhibited by GaTx2 will make it a very powerful pharmacological tool for detecting ClC-2 structure/function.
The basic pharmacological characteristics of the inhibitory activity of GaTx2 on ClC-2 include affinity, inhibition mechanism and specificity. Using two-electrode voltage-clamp, researchers created a dose-response curve for inhibition of ClC-2 by GaTx2 at VM = -100 mV, and calculated a KD of 22 pM. This value was very similar to the value obtained from the dose-response curves produced by the multi-channel plaque, which gives a KD value of 12 pM. Additionally, it is reported that kon = 43 x 106 M-1s-1, and koff = 0.0034 s-1 from TEVC recordings, which is consistent with the rate constants for other peptide inhibitors. GaTx2 is able to increase the latency of the first opening by nearly eight-fold to slow down ClC-2 activation. Also, outside-out macropatches revealed that GaTx2 is unable to inhibit open ClC-2 channels. Thus, this toxin may act as a gating modifier. GaTx2 is specific for ClC-2, being unable to inhibit other ClC channels or transporters, other major classes of Cl channels, or voltage-dependent K+1 channels. This high affinity, specificity of ClC-2 inhibitor will provide an excellent tool for studies designed to understand the function and regulation of this channel, and will help define its physiological role(s).
Mutations in ClC-2 are associated with epilepsy, whereas insufficient activity of wild-type ClC-2 is associated with constipation-associated inflammatory bowel disease. GaTx2 will help determine the role of ClC-2 in these cells and may help to determine the membrane localization of ClC-2 in specific cell types. Therefore, GaTx2 may act as a lead compound for peptide drugs targeting ClC-2.
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