Summary Acid sensing ion channels (ASICs) detect extracellular protons produced during inflammation or ischemic injury and belong to the super family of degenerin/epithelial sodium channels. Here, we determine the cocrystal structure of chicken ASIC1a with MitTx, a pain-inducing toxin from the Texas coral snake, to define the structure of the open state of ASIC1a. In the MitTx-bound open state and in the previously determined low pH desensitized state, TM2 is a discontinuous α-helix in which the Gly-Ala-Ser selectivity filter adopts an extended, belt-like conformation, swapping the cytoplasmic one-third of TM2 with an adjacent subunit. Gly 443 residues of the selectivity filter provide a ring of 3 carbonyl oxygen atoms with a radius of ~3.6 Å, presenting an energetic barrier for hydrated ions. The ASIC1a-MitTx complex illuminates the mechanism of MitTx action, defines the structure of the selectivity filter of voltage-independent, sodium-selective ion channels and captures the open state of an ASIC.
Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in over-expression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol we show how to use small-scale transient transfection and fluorescence-detection, size-exclusion chromatography (FSEC) experiments using a GFP-His8 tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI− (N-acetylglucosaminyltransferase I-negative) cells in suspension culture, and over-express the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl), for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.
Acid sensing ion channels (ASICs) are voltage-independent, amiloride-sensitive channels implicated in diverse physiological processes ranging from nociception to taste. Despite the importance of ASICs in physiology, we know little about the mechanism of channel activation. Here we show that psalmotoxin activates non- and sodium-selective currents in chicken ASIC1a at pH 7.25 and 5.5, respectively. Crystal structures of ASIC1a – psalmotoxin complexes map the toxin binding site to the extracellular domain and illuminate how toxin binding triggers an expansion of the extracellular vestibule and stabilization of the open channel pore. At pH 7.25 the pore is ~10 Å in diameter whereas at pH 5.5 the pore is largely hydrophobic and elliptical in cross section with dimensions of ~5 by ~7 Å, consistent with a barrier mechanism for ion selectivity. These studies define mechanisms for activation of ASICs, illuminate the basis for dynamic ion selectivity and provide the blueprints for new therapeutic agents.
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