• Interactions between members of the alpha-K family of scorpion toxins and voltage-gated potassium channels

      Ellis, Karen Catherine; Weber, David J., Ph.D. (2002)
      The interaction of the alpha-K toxins with their target K+ channels has been the subject of intense study to help identify elements of their sequence and structure that are responsible for their varied selectivity and specficity. The Pandinotoxins, PiTX-Kalpha and PiTX-Kbeta, differ from each other by one residue (P10E). Yet, the affinity of PiTX-Kbeta for voltage-gated, rapidly inactivating K+ channels in dorsal root ganglia (DRG) neurons is 800-fold lower than that of PiTX-Kalpha. (Kalpha-IC 50 = 8.0 nM versus Kbeta-IC50 = 6500 nM). The 3D structure of PiTX-Kbeta was determined by NMR spectroscopy and compared to that of PiTX-Kalpha to understand this difference. This comparison shows that structural differences between the two toxins occur at a residue that is critical for blocking K+ channels (K27) as well as at the site of the natural mutation (P10E). In PiTX-Kbeta, the negatively charged carboxylate oxygen of E10 can approach the positive charge of K27 and presumably reduces the net positive charge in this region of the toxin. This is likely the reason why PiTX-Kbeta binds K+ channels from DRG neurons with a much lower affinity than does PiTX-Kalpha. Another member of the alpha-K family from the scorpion Tityus serrulatus (TsTX-Kalpha) blocks native squid K+ channels and their cloned counterpart, sqKv1A (nativeKd ≈ 20 nM; sqKv1AKd ≈ 10 nM) in a pH dependent manner (pK = 6.6). To further investigate the TsTX-Kalpha-sqKv1A interaction, the 3D structure of TsTX-Kalpha was determined by NMR spectroscopy, and a model of the TsTX-Kalpha-sqKv1A complex was generated. As found for other alpha-K toxins such as charybdotoxin (CTX), site-directed mutagenesis at toxin residue K27 (K27A, K27R, K27E) significantly reduced the toxins affinity for sqKv1A channels. The toxin-channel model illustrates a possible mechanism for the pH-dependent block whereby lysine residues from TsTX-Kalpha (K6, K23) are repelled by protonated H351 on sqKvlA at low pH. Finally, attempts to recombinantly express and purify the first member of a new family of scorpion toxins (beta-K toxins) are presented.