Potassium-dependent changes in the conformation of the Kv2.1 potassium channel pore

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.