Functional properties of rat brain sodium channels lacking the beta 1 or beta 2 subunit.
The sodium channel purified from rat brain is a heterotrimeric complex of alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000) subunits. alpha and beta 2 are attached by disulfide bonds. Removal of beta 1 subunits by incubation in 1.0 M MgCl2 followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 2 which did not bind [3H]saxitoxin, mediate veratridine-activated 22Na+ influx, or bind the 125I-labeled alpha-scorpion toxin from Leiurus quinquestriatus (LqTx). In contrast, removal of beta 2 subunits by reduction of disulfide bonds with 1.5 mM dithiothreitol followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 1 that retained full sodium channel function. Alpha beta 1 bound [3H]saxitoxin with a KD of 4.1 nM at 36 degrees C. It mediated veratridine-activated 22Na+ influx at a comparable initial rate as intact sodium channels with a K0.5 for veratridine of 46 microM. Tetracaine and tetrodotoxin blocked 22Na+ influx. Like intact sodium channels, alpha beta 1 bound 125I-LqTx in a voltage-dependent manner with a KD of approximately 6 nM at a membrane potential of -60 mV and was specifically covalently labeled by azidonitrobenzoyl 125I-LqTx. When incorporated into planar phospholipid bilayers, alpha beta 1 formed batrachotoxin-activated sodium channels of 24 pS whose voltage-dependent activation was characterized by V50 = -110 mV and an apparent gating charge of 3.3 +/- 0.3. These results indicate that beta 2 subunits are not required for the function of purified and reconstituted sodium channels while a complex of alpha and beta 1 subunits is both necessary and sufficient for channel function in the purified state.
D029845:Amphibian Proteins; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D051381:Rats; D012530:Saxitoxin / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D014701:Veratridine / Q000494:pharmacology
D029845:Amphibian Proteins; D002352:Carrier Proteins; D007473:Ion Channels; D008081:Liposomes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; C027525:saxitoxin-binding protein, Rana catesbeiana; D012530:Saxitoxin; D014701:Veratridine; D012964:Sodium