ABSTRACT
A toxin from the scorpion Tityus serrulatus (TsTX-Kalpha) blocks native squid K(+) channels and their cloned counterpart, sqKv1A, at pH 8 ((native)K(d) approximately 20 nM; (sqKv1A)K(d) approximately 10 nM). In both cases, decreasing the pH below 7.0 significantly diminishes the TsTX-Kalpha effect (pK = 6.6). In the cloned squid channel, the pH dependence of the block is abolished by a single point mutation (H351G), and no change in toxin affinity was observed at higher pH values (pH > or =8.0). To further investigate the TsTX-Kalpha-sqKv1A interaction, the three-dimensional structure of TsTX-Kalpha was determined in solution 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, and K27E) significantly reduced the toxin's affinity for sqKv1A channels. This is consistent with the TsTX-Kalpha-sqKv1A model reported here, which has K27 of the toxin inserted into the ion conduction pathway of the K(+) channel. This toxin-channel model also illustrates a possible mechanism for the pH-dependent block whereby lysine residues from TsTX-Kalpha (K6 and K23) are repelled by protonated H351 on sqKv1A at low pH.
Subject(s)
Neurotoxins/chemistry , Potassium Channels, Voltage-Gated , Potassium Channels/chemistry , Scorpion Venoms/chemistry , Amino Acid Sequence , Animals , Cloning, Molecular , Decapodiformes , Delayed Rectifier Potassium Channels , Ganglia, Invertebrate/chemistry , Ganglia, Invertebrate/drug effects , Ganglia, Invertebrate/physiology , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Neurotoxins/genetics , Neurotoxins/metabolism , Neurotoxins/pharmacology , Nuclear Magnetic Resonance, Biomolecular , Oocytes , Potassium Channel Blockers , Potassium Channels/genetics , Protein Structure, Secondary , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Scorpion Venoms/genetics , Scorpion Venoms/pharmacology , Sequence Homology, Amino Acid , Xenopus laevisABSTRACT
The M(2) integral membrane protein of influenza A virus forms a proton-selective ion channel. We investigated the mechanism for proton transport of the M(2) protein in Xenopus oocytes using a two-electrode voltage clamp and in CV-1 cells using the whole cell patch clamp technique. Membrane currents were recorded while manipulating the external solution to alter either the total or free proton concentration or the solvent itself. Membrane conductance decreased by approximately 50% when D(2)O replaced H(2)O as the solvent. From this, we conclude that hydrogen ions do not pass through M(2) as hydronium ions, but instead must interact with titratable groups that line the pore of the channel. M(2) currents measured in solutions of low buffer concentration (<15 mM in oocytes and <0.15 mM in CV-1 cells) were smaller than those studied in solutions of high buffer concentration. Furthermore, the reversal voltage measured in low buffer was shifted to a more negative voltage than in high buffer. Also, at a given pH, M(2) current amplitude in 15 mM buffer decreased when pH-pK(a) was increased by changing the buffer pK(a). Collectively, these results demonstrate that M(2) currents can be limited by external buffer capacity. The data presented in this study were also used to estimate the maximum single channel current of the M(2) ion channel, which was calculated to be on the order of 1-10 fA.
