Regulation of transient Na+ conductance by intra- and extracellular K+ in the human delayed rectifier K+ channel Kv1.5

1. Significant Na+ conductance has been described in only a few native and cloned K+ channels, but has been used to characterize inactivation and K+ b inding within the permeation pathway, and to refine models of K+ flux throu gh multi-ion pores. Here we use Na+ permeation of the delayed rectifier Kchannel Kv1.5 to study extra- and intracellular K+ (K-o(+) and K-i(+), resp ectively) regulation of conductance and inactivation, using whole-cell reco rding from human embryonic kidney (HEK)-293 cells. 2. Kv1.5 Na+ currents in the absence of K-o(+) and K-i(+) were confirmed by : (i) resistance of outward Na+ currents to dialysis by K+-free solutions; (ii) tail current reversal potential changes with Na-o(+) with a slope of 5 5.8 mV per decade; (iii) block by 4-aminopyridine (50 % at 50 mu M), and re sistance to Cl- channel inhibition. 3. Na+ currents were transient followed by a small sustained current. An en velope test confirmed that activated Kv1.5 channels conducted Na+, and that rapid current decay reflected C-type inactivation. Sustained currents (sim ilar to 13% of peak) represented Na+ flux through inactivated Kv1.5 channel s. 4. K-o(+) could modulate the maximum available Na+ conductance in the stabl e cell line while channels were closed. Before the first pulse of a train, increasing K-o(+) concentration increased the subsequent Na+ conductance fr om similar to 15 (0 mM K-o(+)) to 30 nS (5 mM K-o(+)), with a K-d Of 23 mu M Repeated low rate depolarizations in Na-i(+)/Na-o(+) solutions induced a use-dependent loss of Kv1.5 channel Na+ conductance, distinct from that cau sed by C-type inactivation. K-o(+) binding that sensed little of the electr ic field could prevent this secondary loss of available Kv1.5 channels with a K-d of 230 mu M. These two effects on conductance were both voltage inde pendent, and had no effect on channel inactivation rate. 5. K-o(+) concentrations greater than or equal to 0.3 mM slowed the inactiv ation rate in a strongly voltage-dependent manner. This suggested it could compete for binding at a K+ site or sites deeper in the pore, as well as re storing the Na+ conductance. K-i(+) was able to modulate the inactivation r ate but was unable to affect conductance. 6. Mutation of arginine 487 in the outer pore region of the channel to vali ne (R487V) greatly reduced C-type inactivation in Na+ solutions, caused los s of channel use dependence, and prevented any conductance increase upon th e addition of 0.1 mM K-o(+). Our results confirm the existence of a high af finity binding site at the selectivity filter that regulates inactivation, and also reveals the presence of at least one additional high affinity oute r mouth site that predominantly regulates conductance of resting channels, and protects channels activated by depolarization when they conduct Na+.