Abstract
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.
Highlights
Thesodiumchannelpurifiedfromratbrain is a phospholipid vesicles or planar bilayers has shown that these heterotrimericcomplexof a (Mr 260,000), 61
Intact reconstituted brain sodium channels mediate a number of other sodium channel functions including neurotoxin-activated 22Na+influx (7, 8), voltage-dependent LqTx binding, andvoltage-dependent channel proteins (5-10 pmol in 0.1 ml) were incubated with 2 pgof single channel conductance (9)
Our results show that neurotoxin binding and action at three distinct sites on the sodium channel are lost upon removal of 81 (11) (Fig. 1 and Table I), implying that its presence is important for establishing or maintaining the physical integrity of functionally important regions of the purified sodium channel molecule
Summary
Thesodiumchannelpurifiedfromratbrain is a phospholipid vesicles or planar bilayers has shown that these heterotrimericcomplexof a (Mr 260,000), 61 To mediate veratridine-stimulated 22Nafuptake, partially purified sodium channels were incubated in 1.0 M MgC1, t , ~ completely dissociate Dl. The 4 2 complex was isolated by sucrose density gradient sedimentation andreconstituted into phosphatidylcholine vesicles as described under "Experimen-Immunoprecipitated samples (0.2-0.5 pmol) were labeled by resus- tal Procedures." Native sodium channels were carried through pending the protein A-Sepharose pellet in 0.1 ml of[5] mM magnesium chloride, 20 mM Tris-HC1, 5 mM EGTA, 0.1% Triton X-100, pH 7.4.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
