Gating mechanisms and pharmacology of voltage-activated ion channels

National Institute Of Neurological Disorders And Stroke

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Abstract

1) Conservation of the cooling agent binding pocket within the TRPM subfamily Transient Receptor Potential (TRP) channels are a large and diverse family of tetrameric cation selective channels that are activated by many different types of stimuli, including noxious heat or cold, organic ligands such as vanilloids or cooling agents, or intracellular Ca2+. Structures available for all subtypes of TRP channels reveal that the transmembrane domains are closely related despite their unique sensitivity to activating stimuli. Here we use computational and electrophysiological approaches to explore the conservation of the cooling agent binding pocket identified within the S1-S4 domain of the Melastatin subfamily member TRPM8, the mammalian sensor of noxious cold, with other TRPM channel subtypes. We find that a subset of TRPM channels, including TRPM2, TRPM4 and TRPM5, contain pockets very similar to the cooling agent binding pocket in TRPM8. We then show how the cooling agent icilin modulates activation of TRPM4 to intracellular Ca2+, enhancing the sensitivity of the channel to Ca2+ and diminishing outward-rectification to promote opening at negative voltages. Mutations known to promote or diminish activation of TRPM8 by cooling agents similarly alter activation of TRPM4 by icilin, suggesting that icilin binds to the cooling agent binding pocket to promote opening of the channel. These findings demonstrate that TRPM4 and TRPM8 channels share related binding pockets that are allosterically coupled to opening of the pore. 2) Action potentials are generated by opening of voltage-activated sodium (Nav) and potassium (Kv) channels1, which can rapidly inactivate to shape the nerve impulse and contribute to synaptic facilitation and short-term memory. The mechanism of fast inactivation was proposed to involve an intracellular domain that blocks the internal pore in both Nav and Kv channels; however, recent studies in Nav and Kv channels support a mechanism in which the internal pore closes during inactivation. Here we investigate the mechanism of fast inactivation in the Shaker Kv channel using cryo-electron microscopy, mass spectrometry and electrophysiology. We resolved structures of a fully inactivated state in which the non-polar end of the N terminus plugs the internal pore in an extended conformation. The N-terminal methionine is deleted, leaving an alanine that is acetylated and interacts with a pore-lining isoleucine residue where RNA editing regulates fast inactivation14. Opening of the internal activation gate is required for fast inactivation because it enables the plug domain to block the pore and repositions gate residues to interact with and stabilize that domain. We also show that external K+ destabilizes the inactivated state by altering the conformation of the ion selectivity filter rather than by electrostatic repulsion. These findings establish the mechanism of fast inactivation in Kv channels, revealing how it is regulated by RNA editing and N-terminal acetylation, and providing a framework for understanding related mechanisms in other voltage-activated channels.