Ch 3. Superfamily of Voltage-gated channels

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Presentation transcript:

Ch 3. Superfamily of Voltage-gated channels Voltage-gated Na+ Channel (Nav) K+(Kv) Ca2+ (Cav) 1. 1960~ pharmacological tools ( blocker ) define discrete entities TTX (tetrodotoxin) : puffer fish, inward current completely blocked 1970 Toshio Narahashi: Northwestern U. TEA (tetraethylammonium) : K+ channel blocker: outward current blocked STX (saxitoxin) : Na+ channel blocker Dinoflagella (적조), contaminated shell fish 2. Blockers separate current of K+ & Na+ → K+ channel, Na+ channel : procain, lidocain : K+ channel, Na+ channel blocker

[Model for Nav]

< single channel recording> Patch-clamp technique: Bert Sakmann & Erwin Neher Giga seal Sylgarding (resin) Patch Configurations Cell attached patch → Inside-out patch (pull) ↓suck Whole cell patch → Outside-out patch (pull slowly) [ Voltage-gated Na+ channel ] [ Voltage-gated K+ channel ] fast activation - fast activation fast in activation - slow inactivation

[Cloning of Channels] Ist Chanel Cloning: n Ach R of myocyte : Shosaku Numa (Kyoto U) Purify with a-bungarotoxin ↓ Solubilization of cell membrane Affinity fraction by TTX Amino acid sequencing Nucleotide oligo synthesizing Hybridization Full sequencing Voltage-gated Na+ channel cloning : Shoshaku Numa Cloning Strategy: Same as nAcR cloning but purify with antibody to Nav Cloning of voltage-gated K+ channel: positional cloning > - Drosophila, mutant (Lily Jan, UCSF)

α-subunit : pore forming subunit -subunit: membrane targeting pore : ion selectivity voltage sensor : at S4 region, (+)-charged amino acid (Arg, Lys) Inactivation theory P-type inactivation : ball peptide, fast inactivation N-type inactivation : phosphorylation or intracellular signal, slow inactivation

[Cloning of Voltage-gated K+ channel] Drosophila mutant  positional cloning : “Shaker” 4 types of voltage-gated K channels in Drosophila Shaker: a mutant fly(Drosophila (-)) Ether → leg shaking Shaker Shal Shab Show