ABSTRACT
Cysteine-rich secretory proteins (CRISPs) are widely distributed, and notably occur in the mammalian reproductive tract and in the salivary glands of venomous reptiles. Most CRISPs can inhibit ion channels, such as the cyclic nucleotide-gated ion channel, potassium channel, and calcium channel. Natrin is a CRISP that has been purified from snake venom. Its targets include the calcium-activated potassium channel, the voltage-gated potassium channel, and the calcium release channel/ryanodine receptor (RyR). Immunoprecipitation experiments showed that natrin binds specifically to type 1 RyR (RyR1) from skeletal muscle. Natrin was found to inhibit both the binding of ryanodine to RyR1, and the calcium-channel activity of RyR1. Cryo-electron microscopy and single-particle image reconstruction analysis revealed that natrin binds to the clamp domains of RyR1. Docking of the crystal structure of natrin into our cryo-electron microscopy density map of the RyR1 + natrin complex suggests that natrin inhibits RyR1 by stabilizing a domain-domain interaction, and that the cysteine-rich domain of natrin is crucial for binding. These findings help reveal how natrin toxin inhibits the RyR calcium release channel, and they allow us to posit a generalized mechanism that governs the interaction between CRISPs and ion channels.
Subject(s)
Elapid Venoms/chemistry , Elapid Venoms/metabolism , Ryanodine Receptor Calcium Release Channel/chemistry , Ryanodine Receptor Calcium Release Channel/metabolism , Animals , Calcium Channel Blockers/chemistry , Calcium Channel Blockers/metabolism , Calcium Channel Blockers/toxicity , Calcium Channels/metabolism , Catalytic Domain , Cryoelectron Microscopy , Crystallography, X-Ray , Cysteine , Elapid Venoms/toxicity , Models, Molecular , Molecular Conformation , Protein Binding/drug effects , Protein Structure, Tertiary , Ryanodine/metabolism , Substrate SpecificityABSTRACT
Cysteine-rich secretory proteins (CRISPs) are secreted single-chain proteins found in different sources. Natrin is a member of the CRISP family purified from the snake venom of Naja naja atra, which has been reported as a BKca channel blocker. In our study, crystals of natrin were obtained in two different crystal forms and the structure of one of them was solved at a resolution of 1.68A. Our electrophysiological experiments indicated that natrin can block the ion channel currents of the voltage-gated potassium channel Kv1.3. Docking analyses of the interaction between natrin and Kv1.3 revealed a novel interaction pattern different from the two previously reported K(+) channel inhibition models termed "functional dyad" and "basic ring". These findings offered new insights into the function of natrin and how the specific interactions between CRISPs and different ion channels can be achieved.
