A critical residue for isoform difference in tetrodotoxin affinity is a molecular determinant of the external access path for local anesthetics in the cardiac sodium channel

Abstract

Membrane-impermeant quaternary derivatives of lidocaine (QX222 and QX314) block cardiac Na ؉ channels when applied from either side of the membrane, but they block neuronal and skeletal muscle channels poorly from the outside. To find the molecular determinants of the cardiac external QX access path, mutations of adult rat skeletal muscle ( 1) and rat heart (rH1) Na ؉ channels were studied by two-electrode voltage clamp in Xenopus oocytes. Mutating the 1 domain I P-loop Y401, which is the critical residue for isoform differences in tetrodotoxin block, to the heart sequence (Y401C) allowed outside QX222 block, but its mutation to brain type (Y401F) showed little block. 1-Y401C accelerated recovery from block by internal QX222. Block by external QX222 in 1-Y401C was diminished by chemical modification with methanethiosulfonate ethylammonium (MTSEA) to the outer vestibule or by a double mutant ( 1-Y401C͞F1579A), which altered the putative local anesthetic binding site. The reverse mutation in heart rH1-C374Y reduced outside QX314 block and slowed dissociation of internal QX222. Mutation of 1-C1572 in IVS6 to Thr, the cardiac isoform residue (C1572T), allowed external QX222 block, and accelerated recovery from internal QX222 block, as reported. Blocking efficacy of outside QX222 in 1-Y401C was more than that in 1-C1572T, and the double mutant ( 1-Y401C͞C1572T) accelerated internal QX recovery more than 1-Y401C or 1-C1572T alone. We conclude that the isoform-specific residue (Tyr͞Phe͞Cys) in the P-loop of domain I plays an important role in drug access as well as in tetrodotoxin binding. Isoform-specific residues in the IP-loop and IVS6 determine outside drug access to an internal binding site.V oltage-gated Na ϩ channels are membrane proteins responsible for generation of action potentials in nerve, skeletal muscle, and heart. The Na ϩ channel ␣-subunit consists of four homologous domains (I-IV), each containing six transmembrane segments (S1-S6) (1, 2). P-loops connecting S5 and S6 in each domain form the Na ϩ channel outer vestibule (3-7). Na ϩ channel isoforms from various tissues are highly homologous (2, 8). However, cardiac channels are relatively insensitive to tetrodotoxin (TTX) compared with brain and skeletal muscle channels (9, 10). This isoform difference is explained by an isoformspecific residue (Tyr͞Phe͞Cys) in the P-loop of domain I (IP-loop) (11-14) (Fig. 1). An aromatic residue at this position renders the channel TTX sensitive.The Na ϩ channel is also the site of action of local anesthetic antiarrhythmic drugs. Two conserved aromatic residues (Phe and Tyr) in IVS6 are involved in the local anesthetic binding site (15)(16)(17), and isoform differences in local anesthetic affinity are much less than in toxin affinity (18). In contrast, access of local anesthetic drugs differs markedly between Na ϩ channel isoforms. Quaternary membrane-impermeant derivatives of lidocaine such as QX314 and QX222 do not block nerve channels when applied to the outside (19,20), but outside QX314 blocks the cardiac chann...

The Selectivity Filter of the Voltage-gated Sodium Channel Is Involved in Channel Activation

“…This notion is supported by a recent report demonstrating that the residue DIV-S6 Val 1583 is implicated in BTX binding and in channel gating (64). According to a recent model of the Na ϩ channel, the DIV-S6 residue Val 1583 may be in reasonable proximity to the DIV-P-loop residue Ala 1529 to allow for interaction between the two residues during a rearrangement associated with channel gating (70,71). In this context it is noteworthy that the DIV P-loop may not only be extremely flexible, as discussed above, but may protrude farther into the pore than P-loops of domains I-III, based on electrical distance measurements (19).…”

“…A mutual relationship appears to exist between the local anesthetic block and the conformation of the outer channel vestibule because outer pore mutations influence the access of local anesthetics to their binding pocket (71,72), and local anesthetic binding is associated with a structural rearrangement of the outer channel vestibule (73). Furthermore, recent studies suggest that the mechanism of action of the local anesthetic lidocaine involves transitions along the activation pathway (74 -76).…”

Cardiac-Specific External Paths for Lidocaine, Defined by Isoform-Specific Residues, Accelerate Recovery From Use-Dependent Block

“…The isoform-specific residue Cys373 in the outer vestibule of hH1a, which permits external QX access, 14 is thought to be in the hydrophilic path for ion permeation because it interacts well with guanidium toxins and with external Cd 2ϩ . 24,25 If the QX path defined by C373 in the heart channel provides an egress path for lidocaine not present in 1, mutation to the 1-specific residue h-C373Y would be expected to slow the recovery from UDB compared with wild-type hH1a.…”

“…The 1 ␣-subunit (provided by Dr J.R. Moorman, University of Virginia, Charlottesville, Va) and the P-loop mutant 1-Y401C were prepared as described previously. 7,14 Stage V and stage VI Xenopus oocytes were isolated and injected with Ϸ50 to 100 nanograms of cRNA, in vitro-synthesized by a T7 or SP6 transcription system (Ambion), using hH1a or rat skeletal muscle (1) ␣-subunit clones. An equimolar cRNA of the human ␤1 subunit was included in all injections.…”

“…2,7,14 In contrast, external QX is effective in blocking cardiac Na ϩ channels, 15 and isoform-specific residues determine this external access path. Qu et al 16 identified an isoform-specific residue in the upper part of D4S6 (Thr in heart, Val in brain, and Cys in 1) that is a determinant for external QX block, and we identified an isoform-specific residue within the P-loop of domain 1 (Cys in heart, Phe in brain, and Tyr in 1).…”