The voltage-gated K+ channel, Kv2.1, conducts Na+ in the absence of K+. Ext
ernal tetraethylammonium (TEo) blocks K+ currents through Kv2.1 with an IC5
0 of 5 mM, but is completely without effect in the absence of K+. TEA(o) bl
ock can be titrated back upon addition of low [K+]. This suggested that the
Kv2.1 pore undergoes a cation-dependent conformational rearrangement in th
e external vestibule. Individual mutation of lysine (Lys) 356 and 382 in th
e outer vestibule, to a glycine and a valine, respectively, increased TEA(o
) potency for block of K+ currents by a half log unit. Mutation of Lys 356,
which is located at the outer edge of the external vestibule, significantl
y restored TEA(o) block in the absence of K+ (IC50 = 21 mM). In contrast, m
utation of Lys 382, which is located in the outer vestibule near the TEA bi
nding site, resulted in very weak (extrapolated IC50 = similar to 265 mM) T
EA(o) block in the absence of K+. These data suggest that the cation-depend
ent alteration in pore conformation that resulted in loss of TEA potency ex
tended to the outer edge of the external vestibule, and primarily involved
a repositioning of Lys 356 or a nearby amino acid in the conduction pathway
. Block by internal TEA also completely disappeared in the absence of K+, a
nd could be titrated back with low [K+]. Both internal and external TEA pot
encies were increased by the same low [K+] (30-100 mu M) that blocked Na+ c
urrents through the channel. In addition, experiments that combined block b
y internal and external TEA indicated that the site of K+ action was betwee
n the internal and external TEA binding sites. These data indicate that a K
+-dependent conformational change also occurs internal to the selectivity f
ilter, and that both internal and external conformational rearrangements re
sulted from differences in K+ occupancy of the selectivity filter. Kv2.1 in
activation rate was K+ dependent and correlated with TEA(o) potency; as [K] was raised, TEA(o) became more potent and inactivation became faster. Bot
h TEA(o) potency and inactivation rate saturated at the same [K+ ]. These r
esults suggest that the rate of slow inactivation in Kv2.1 was influenced b
y the conformational rearrangements, either internal to the selectivity fil
ter or near the outer edge of the external vestibule, that were associated
with differences in TEA potency.