Sodium channel selectivity filter regulates antiarrhythmic drug binding

Abstract

Local anesthetic antiarrhythmic drugs block Na ؉ channels and have important clinical uses. However, the molecular mechanism by which these drugs block the channel has not been established. The family of drugs is characterized by having an ionizable amino group and a hydrophobic tail. We hypothesized that the charged amino group of the drug may interact with charged residues in the channel's selectivity filter. Mutation of the putative domain III selectivity filter residue of the adult rat skeletal muscle Na ؉ channel (1) K1237E increased resting lidocaine block, but no change was observed in block by neutral analogs of lidocaine. An intermediate effect on the lidocaine block resulted from K1237S and there was no effect from K1237R, implying an electrostatic effect of Lys. Mutation of the other selectivity residues, D400A (domain I), E755A (domain II), and A1529D (domain IV) allowed block by externally applied quaternary membrane-impermeant derivatives of lidocaine (QX314 and QX222) and accelerated recovery from block by internal QX314. Neo-saxitoxin and tetrodotoxin, which occlude the channel pore, reduced the amount of QX314 bound in D400A and A1529D, respectively. Block by outside QX314 in E755A was inhibited by mutation of residues in transmembrane segment S6 of domain IV that are thought to be part of an internal binding site. The results demonstrate that the Na ؉ channel selectivity filter is involved in interactions with the hydrophilic part of the drugs, and it normally limits extracellular access to and escape from their binding site just within the selectivity filter. Participation of the selectivity ring in antiarrhythmic drug binding and access locates this structure adjacent to the S6 segment.

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

“…Block with a saturating concentration of R13Q also significantly reduced QX222 block of the 1-Y401C current (not shown). These results are similar to the reports stating that TTX (16,27) and neo-saxitoxin (27) protected the channels from outside QX314 block.…”

“…To compare recovery from QX222 block in 1-WT and 1-Y401C, QX222 was microinjected into oocytes by using the same procedure as before (27). Consistent with our previous report on recovery of the 1 channels from QX314 block (27), recovery of QX222 in 1-WT was also slow, with 22.5 Ϯ 3.0% (n ϭ 6) of the current recovering during a 30-min period (Fig. 3C).…”

“…Extensive studies have shown that local anesthetic drugs typically access their binding site within the Na ϩ channel pore from the inside (22), and consequently, membrane-impermeant quaternary analogs fail to block nerve and skeletal muscle Na ϩ channels from the outside (16,19,20,27, and this report) (Fig. 7A).…”

“…Oocytes were incubated at 16°C for 1 to 6 days before examination. Recordings were made in the two-electrode voltage clamp configuration by using a CA-1 voltage clamp (Dagan Corp., Minneapolis, MN), as reported (27). All recordings were made at room temperature (20-22°C) in a bathing solution that consisted of 90 mM NaCl, 2.5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM Hepes (pH 7.2).…”

“…If there is indeed an access pathway to the internal site, it would also provide an alternative path for dissociation of the bound drug (15,16,27). To compare recovery from QX222 block in 1-WT and 1-Y401C, QX222 was microinjected into oocytes by using the same procedure as before (27).…”