Initial Opening Steps of K+ Channels Probed by Extracellular Multivalent Cations

Abstract

The structural model of the open state of the Kv1.2-2.1 chimeric channel (Long et al., PDB 2R9R) has 4 negative residues distributed in S1, S2 and S3, all bound to positive residues in S4. Extrapolating the model to the resting state, the two outermost negative residues, E183 in S1 and E226 in S2, are exposed to the extracellular fluid and, in theory, not bound to S4 residues; E183 and E226 should thus be available to bind external cations. We have examined the effects on opening kinetics of multivalent cations using Shaker channels expressed in HEK cells. Addition of La3+ (50 μM) to the extracellular solution (in mM: 145 NaCl, 2 KCl, 10 CaCl2, pH 7.0) markedly prolonged the sigmoidal delay and slowed the rising phase of K+ current (IK) at all voltages tested. The closing kinetics of IK at negative voltages remained unaltered. Gating currents (Ig) recorded from a non-conducting mutant (W434F) showed that La3+ (50 μM) reduced the initial amplitude of Ig nearly twofold. We postulate that La3+ binds to the unoccupied negative side chains of E183 and E226, hindering outward S4 motion, thus increasing the lag and slowing the rise of IK. We further postulate that La3+, in the activated “up” state of S4, is not bound to E183 or E266, which are occupied by S4 positive charges; La3+ therefore has no effect on closing. Similar measurements using Ca2+ showed that at high concentrations it has an effect similar to that of La3+ on the activation time course. This Ca2+-dependent prolongation of the activation lag and slowing of the rising phase of IK is noticeably enhanced by complete removal of extracellular K+, perhaps because K+ and Ca2+ compete for E183 and E226 in the deactivated state.