Block of tetrodotoxin-resistant Na⁺ channel pore by multivalent cations: Gating modification and Na⁺ flow dependence

Tetrodotoxin-resistant (TTX-R) Na⁺ channels are much less susceptible to external TTX but more susceptible to external Cd²⁺ block than tetrodotoxin-sensitive (TTX-S) Na⁺ channels. Both TTX and Cd²⁺ seem to block the channel near the "DEKA" ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La ³⁺, Zn²⁺, Ni²⁺, Co²⁺, and Mn ²⁺ also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd²⁺, the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na⁺ flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na⁺ currents than those in outward Na⁺ current, signaling exit of the blocker along with the Na⁺ flow and a high internal energy barrier for "permeation" of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na⁺ current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na⁺ flow-dependent unblocking of the bound La³⁺ from the open Na⁺ channel, whereas channel inactivation cannot happen with any discernible speed in the La³⁺-blocked channel. Thus, the selectivity filter of Na⁺ channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is "open" to the external solution even if the channel is "closed" ("deactivated"), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.