Introduction
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.
Pharmacologic action
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).
Conclusion
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.
References:
Jentsch, T. J., Stein, V., Weinreich, F., and Zdebik, A. A. (2002) Physiol. Rev. 82, 503–568.
Jentsch, T. J., Neagoe, I., and Scheel, O. (2005) Curr. Opin. Neurobiol. 15, 319–325.
Zdebik, A. A., Cuffe, J. E., Bertog, M., Korbmacher, C., and Jentsch, T. J. (2004) J. Biol. Chem. 279, 22276–22283.
