Effect of Na(+) flow on Cd(2+) 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 Cd(2+) 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 Cd(2+). In this study we demonstrate that the binding affinity of Cd(2+) 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 Cd(2+) ( approximately 200 microM) is approximately 9 times smaller than that in the presence of outward Na(+) current. The Na(+) flow-dependent binding affinity change of Cd(2+) 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 Cd(2+) 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 Cd(2+) 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 Ca(2+) and K(+) channels.
Kuo
Chung-Chin
CC
Department of Physiology, National Taiwan University College of Medicine. Department of Neurology, National Taiwan University Hospital, Taiwan. cckuo@ha.mc.ntu.edu.tw
Lin
Ting-Jiun
TJ
Hsieh
Chi-Pan
CP
D000818:Animals; D001667:Binding, Competitive; D002104:Cadmium / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D002586:Cesium / Q000494:pharmacology; D004351:Drug Resistance; D004553:Electric Conductivity; D004594:Electrophysiology; D009994:Osmolar Concentration; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D015222:Sodium Channels / Q000187:drug effects* / Q000502:physiology*; D013779:Tetrodotoxin / Q000494:pharmacology*
D015222:Sodium Channels; D002104:Cadmium; D002586:Cesium; D013779:Tetrodotoxin; D012964:Sodium; D002118:Calcium