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 (μ1) with a polyhistidine tag on the C terminus (μHis) and pore-only μ1 (μPore) channels with S1-S4 in all domains removed. Both constructs were recognized by a Na⁺ channel-specific antibody on a Western blot. μHis channels exhibited the same functional properties as wild-type μ1. In contrast, μPore channels did not conduct Na⁺ currents nor did they bind [³H]saxitoxin. Veratridine caused 40 and 54% cell death in μHis- and μPore-expressing cells, respectively. However, veratridine-induced cell death could only be blocked by tetrodotoxin in cells expressing μHis, but not μPore. 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 (AF) were 35 and 31% in cells expressing μHis and μPore, respectively. Moreover, in the presence of 1 μM tetrodotoxin, ΔF decreased significantly to 10% in μHis- but not in μPore-expressing cells (43%). In conclusion, S5-P-S6 segments of μ1 channels form a toxin-activable ionophore but do not reconstitute the Na⁺ channel permeation pathway with full fidelity.