1. Ch 3. Superfamily of Voltage-gated channels
Voltage-gated Na+ Channel (Nav)
K+(Kv)
Ca2+ (Cav)
~ 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

2. [Model for Nav]

3. < 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

4. [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)

5. 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

6. [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