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 Cd 2 block than tetrodotoxin-sensitive (TTX-S) Na channels. Both TTX and Cd 2 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 , Zn 2 , Ni 2 , Co 2 , and Mn 2 also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd 2 , 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 La 3 from the open Na channel, whereas channel inactivation cannot happen with any discernible speed in the La 3 -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. key words: ion permeation • flux-coupling • single-file region • inactivation • activation I N T R O D U C T I O N Voltage-gated Na channels are important membrane proteins essential for the activity of many excitable cells. In mammalian neurons some Na channels are effectively inhibited by nanomolar or subnanomolar external tetrodotoxin (TTX) and are classified as the TTX-sensitive (TTX-S) channels. However, the other Na channels are inhibited only when there is micromolar or submicromolar TTX and are classified as the TTX-resistant (TTX-R) channels (Kleinhaus and Pritchard, 1976; Cohen et al., 1981; Lombet et al., 1982; Roy and Narahashi, 1992). 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). For example, IC50 of Cd 2 block of the TTX-R channels in cardiac myocytes or Purkinje cells is 100-fold lower than that of the TTX-S channels in skeletal muscle cells (Visentin et al., 1990; Ravindran et al., 1991; Sheets and Hanck, 1992). Interestingly, it has been shown that the 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 phenylalanine or tyrosine, but in TTX-R channels it is cysteine or serine. TTX-R channels with a point mutation C374Y show markedly decreased affinity to Cd 2 but increased affinity to TTX (Satin et al., 1992), whereas Y374C mutant TTX-S channels show markedly increased affinity to Cd 2 but decreased affinity to TTX (Backx et al., 1992). The highly conserved aspartate, glutamate, lysine, and alanine in the pore loops of domain I, II, III, and IV in the Na channel protein, respectively (the “DEKA” ring), have been implicated to form the selectivity filter of the channel, because mutations in the ring significantly change ionic selectivity (Heinemann et al., 1992; Favre et al., 1996; Pérez-Garcia et al., 1997; Sun et al., 1997). As position 374 is located next to the aspartate residue in the DEKA ring (D373), the Cd 2 blocking site very likely is contiguous to the DEKA ring 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) 2-23964350; email: [email protected] Abbreviation used in this paper: TTX, tetrodotoxin. on July 1, 2017 jgp.rress.org D ow nladed fom