Document Detail

A "minimal" sodium channel construct consisting of ligated S5-P-S6 segments forms a toxin-activatable ionophore.
MedLine Citation:
PMID:  11973330     Owner:  NLM     Status:  MEDLINE    
The large size (six membrane-spanning repeats in each of four domains) and asymmetric architecture of the voltage-dependent Na+ channel has hindered determination of its structure. With the goal of determining the minimum structure of the Na+ channel permeation pathway, we created two stable cell lines expressing the voltage-dependent rat skeletal muscle Na+ channel (micro1) with a polyhistidine tag on the C terminus (muHis) and pore-only micro1 (muPore) channels with S1-S4 in all domains removed. Both constructs were recognized by a Na+ channel-specific antibody on a Western blot. muHis channels exhibited the same functional properties as wild-type micro1. In contrast, muPore channels did not conduct Na+ currents nor did they bind [3H]saxitoxin. Veratridine caused 40 and 54% cell death in muHis- and muPore-expressing cells, respectively. However, veratridine-induced cell death could only be blocked by tetrodotoxin in cells expressing muHis, but not muPore. Furthermore, using a fluorescent Na+ indicator, we measured changes in intracellular Na+ induced by veratridine and a brevotoxin analogue, pumiliotoxin. When calibrated to the maximum signal after addition of gramicidin, the maximal percent increases in fluorescence (deltaF) were 35 and 31% in cells expressing muHis and muPore, respectively. Moreover, in the presence of 1 microm tetrodotoxin, deltaF decreased significantly to 10% in muHis- but not in muPore-expressing cells (43%). In conclusion, S5-P-S6 segments of micro1 channels form a toxin-activable ionophore but do not reconstitute the Na+ channel permeation pathway with full fidelity.
Zhenhui Chen; Carmen Alcayaga; Benjamin A Suarez-Isla; Brian O'Rourke; Gordon Tomaselli; Eduardo Marban
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.     Date:  2002-04-24
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  277     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2002 Jul 
Date Detail:
Created Date:  2002-07-01     Completed Date:  2002-08-27     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  24653-8     Citation Subset:  IM    
Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA.
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MeSH Terms
Cell Line
Gramicidin / pharmacology
Ionophores / metabolism*
Membrane Potentials / physiology
Models, Molecular
Muscle, Skeletal / physiology
Peptide Fragments / metabolism
Protein Conformation
Saxitoxin / pharmacokinetics
Sodium / metabolism*
Sodium Channels / chemistry,  drug effects,  physiology*
Spectrometry, Fluorescence
Toxins, Biological / toxicity*
Veratridine / pharmacology
Grant Support
Reg. No./Substance:
0/Ionophores; 0/Peptide Fragments; 0/Sodium Channels; 0/Toxins, Biological; 1405-97-6/Gramicidin; 35523-89-8/Saxitoxin; 71-62-5/Veratridine; 7440-23-5/Sodium

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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