Effect of Na⁺ Flow on Cd²⁺ Block of Tetrodotoxin-resistant Na⁺ Channels

Tetrodotoxin-resistant (TTX-R) Na⁺ channels are 1,000-fold less sensitive to TTX than TTX-sensitive (TTX-S) Na⁺ channels. On the other hand, TTX-R channels are much more susceptible to external Cd²⁺ block than TTX-S channels. A cysteine (or serine) residue situated just next to the aspartate residue of the presumable selectivity filter “DEKA” ring of the TTX-R channel has been identified as the key ligand determining the binding affinity of both TTX and Cd²⁺. In this study we demonstrate that the binding affinity of Cd²⁺ to the TTX-R channels in neurons from dorsal root ganglia has little intrinsic voltage dependence, but is significantly influenced by the direction of Na⁺ current flow. In the presence of inward Na⁺ current, the apparent dissociation constant of Cd²⁺ (∼200 μM) is ∼9 times smaller than that in the presence of outward Na⁺ current. The Na⁺ flow–dependent binding affinity change of Cd²⁺ block is true no matter whether the direction of Na⁺ current is secured by asymmetrical chemical gradient (e.g., 150 mM Na⁺ vs. 150 mM Cs⁺ on different sides of the membrane, 0 mV) or by asymmetrical electrical gradient (e.g., 150 mM Na⁺ on both sides of the membrane, −20 mV vs. 20 mV). These findings suggest that Cd²⁺ is a pore blocker of TTX-R channels with its binding site located in a multiion, single-file region near the external pore mouth. Quantitative analysis of the flow dependence with the flux-coupling equation reveals that at least two Na⁺ ions coexist with the blocking Cd²⁺ ion in this pore region in the presence of 150 mM ambient Na⁺. Thus, the selectivity filter of the TTX-R Na⁺ channels in dorsal root ganglion neurons might be located in or close to a multiion single-file pore segment connected externally to a wide vestibule, a molecular feature probably shared by other voltage-gated cationic channels, such as some Ca²⁺ and K⁺ channels.