A "minimal" sodium channel construct consisting of ligated S5-P-S6 segments forms a toxin-activatable ionophore.
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.
Chen
Zhenhui
Z
Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA.
Alcayaga
Carmen
C
Suarez-Isla
Benjamin A
BA
O'Rourke
Brian
B
Tomaselli
Gordon
G
Marban
Eduardo
E
D000818:Animals; D002460:Cell Line; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D007476:Ionophores / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D012530:Saxitoxin / Q000493:pharmacokinetics; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D014118:Toxins, Biological / Q000633:toxicity*; D014162:Transfection; D014701:Veratridine / Q000494:pharmacology
D007476:Ionophores; D010446:Peptide Fragments; D015222:Sodium Channels; D014118:Toxins, Biological; D006096:Gramicidin; D012530:Saxitoxin; D014701:Veratridine; D012964:Sodium