8 research outputs found
The Na+ Channel Inactivation Gate Is a Molecular Complex: A Novel Role of the COOH-terminal Domain
Electrical activity in nerve, skeletal muscle, and heart requires finely tuned activity of voltage-gated Na+ channels that open and then enter a nonconducting inactivated state upon depolarization. Inactivation occurs when the gate, the cytoplasmic loop linking domains III and IV of the α subunit, occludes the open pore. Subtle destabilization of inactivation by mutation is causally associated with diverse human disease. Here we show for the first time that the inactivation gate is a molecular complex consisting of the III-IV loop and the COOH terminus (C-T), which is necessary to stabilize the closed gate and minimize channel reopening. When this interaction is disrupted by mutation, inactivation is destabilized allowing a small, but important, fraction of channels to reopen, conduct inward current, and delay cellular repolarization. Thus, our results demonstrate for the first time that physiologically crucial stabilization of inactivation of the Na+ channel requires complex interactions of intracellular structures and indicate a novel structural role of the C-T domain in this process
Location of modulatory β subunits in BK potassium channels
Large-conductance voltage- and calcium-activated potassium (BK) channels contain four pore-forming α subunits and four modulatory β subunits. From the extents of disulfide cross-linking in channels on the cell surface between cysteine (Cys) substituted for residues in the first turns in the membrane of the S0 transmembrane (TM) helix, unique to BK α, and of the voltage-sensing domain TM helices S1–S4, we infer that S0 is next to S3 and S4, but not to S1 and S2. Furthermore, of the two β1 TM helices, TM2 is next to S0, and TM1 is next to TM2. Coexpression of α with two substituted Cys’s, one in S0 and one in S2, and β1 also with two substituted Cys’s, one in TM1 and one in TM2, resulted in two αs cross-linked by one β. Thus, each β lies between and can interact with the voltage-sensing domains of two adjacent α subunits
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Extraction of junctional complexes from triad junctions of rabbit skeletal muscle
Triadin in skeletal muscle exists as a disulfide linked oligomer. Triadin is solubilized in CHAPS after reduction to its monomer. Purified reduced triadin is not retained on a hydroxylapatite (HAPT) column in the presence of 30 mM potassium phosphate (KP\sb{\rm i}), while the junctional foot protein (JFP) and dihydropyridine receptor (DHPr) purified in the absence of triadin are retained. In contrast, triadin solubilized as a detergent extract of reduced triadic vesicles is retained by the HAPT column and elutes concomitantly with the JFP and DHPr.Triadin derived from a detergent extract of reduced vesicles is retained by HAPT in the presence of 180 mM KP\sb{\rm i} which elutes a portion of the JFP and DHPr. Triadin elutes with the remaining portion of JFP and DHPr upon the addition of KCl (820 mM) to the 180 mM KP\sb{\rm i} medium. Several lines of evidence support the existence of a complex between triadin, JFP and DHPr in the high salt extract: (1) All three proteins co-elute in the void volume of molecular sieve column; (2) a portion of triadin co-migrates with the DHPr but separates from the JFP upon rate zonal centrifugation; and (3) the complex is immunoprecipitated by monoclonal antibodies directed against the individual proteins. These results demonstrate a role for triadin as the linkage in forming a ternary complex between the JFP and DHPr at the triad junction.\lbrack\sp{125}IJFP binds to triadin in a saturable manner. The binding becomes weaker in hypertonic salt concentrations. Autoradiography of the overlays confirm that binding still occurs even in 0.5 M salt. Scatchard analysis determined that \lbrack\sp{125}IJFP bound to triadin with a B\sb{\rm max} of 100 pmol mg\sp{-1} and a K\sb{\rm d} of 40 nM.The influence of various ligands on binding was investigated using \lbrack\sp{125}I) JFP and triadin immobilized on nitrocellulose. Ca\sp{2+} inhibits binding whereas, Mg\sp{2+} produces a dose dependent increase in binding. Furthermore, ATP and ruthenium red inhibits binding. These data indicate that the potency of binding between triadin and the JFP is controlled in a complex manner by activators or inhibitors of channel opening.</p