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 ( 200 M) is 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. key words: ion permeation • flux-coupling • selectivity filter • multiion pore • single-file region I N T R O D U C T I O N Tetrodotoxin (TTX)* is a well-known blocker of voltage-gated Na channels. In mammalian central neurons many Na channels are selectively inhibited by nanomolar or subnanomolar external TTX. These channels are referred to as TTX-sensitive (TTX-S) channels. However, some other Na channels are much less sensitive to TTX, and require hundreds of nanomoles to hundreds of micromoles of TTX to produce the inhibition (TTX-resistant or TTX-R channels; Kleinhaus and Pritchard, 1976; Cohen et al., 1981; Lombet et al., 1982; Roy and Narahashi, 1992). In the nervous system, the dorsal root ganglion neurons contain abundant TTX-R channels (Kostyuk et al., 1981; Roy and Narahashi 1992; Akopian et al., 1996; Rush et al., 1998) which have been implicated to play an important role in the physiology and pathophysiology of pain transmission (Akopian et al., 1999; Kral et al., 1999). In addition to TTX sensitivity, TTX-R and TTX-S channels are also different in the pore-blocking effect of transitional metal ions such as Cd 2 and Zn 2 (Frelin et al., 1986; Backx et al., 1992; Sheets and Hanck, 1992). In dorsal root ganglion neurons, 5 mM Cd 2 inhibits 85% of TTX-R currents yet inhibits only 30% of TTX-S currents (at 0 mV; Roy and Narahashi, 1992). In cardiac myocytes or Purkinje cells, which contain almost only TTX-R channels (the TTX-R channels in heart and in dorsal root ganglion neurons are distinct but closely related molecular clones, for reviews see Goldin et al., 2000; Goldin, 2001), 0.1–0.3 mM Cd 2 caused 50% inhibition of the Na current (IC50 0.1–0.3 mM; Visentin et al., 1990; Ravindran et al., 1991; Sheets and Hanck, 1992). But the IC50 of Cd 2 block of Na current in rat skeletal muscle, which contains almost only TTX-S channels, is 17 mM (Ravindran et al., 1991). It has been shown that one single amino acid at position 374 of the channel protein plays a critical role in both TTX and Cd 2 sensitivity. In TTX-S channels this amino acid is tyrosine (Y374), but in TTX-R channels it is cysteine or serine. TTX-R channels with a point mutation at this position (C374Y) show markedly decreased affinity to Cd 2 , but increased affinity to TTX (Satin et al., 1992). Also, mutant (Y374C) TTX-S channels show markedly increased affinity to Cd 2 , but decreased affinity to TTX (Backx et al., 1992). The selectivity filter of the Na channel has been implicated to involve the DEKA ring in the pore (highly Address correspondence to: Chung-Chin Kuo, Department of Physiology, National Taiwan University, College of Medicine No. 1, Jen-Ai Rd., 1st Section Taipei, 100, Taiwan. Fax: (886) 2-2396-4350; E-mail: [email protected] * Abbreviations used in this paper: BTX, batrachotoxin; TTX, tetrodotoxin. on Jne 0, 2017 D ow nladed fom Published July 15, 2002