Local anesthetic anchoring to cardiac sodium channels - Implications into tissue-selective drug targeting

Local anesthetics inhibit Na+ channels ina variety of tissues, leading to p otentially serious side effects when used clinically. We have created a-ser ies of novel local anesthetics by connecting benzocaine (BZ) to the sulfhyd ryl-reactive group methanethiosulfonate (MTS) via variable-length polyethyl ether linkers (L) (MTS-LX-BZ [X represents 0, 3, 6, or 9]). The application of MTS-LX-BZ agents modified native rat cardiac as well as heterologously expressed human heart (hH1) and rat skeletal muscle (rSkM1): Na+ channels i n a manner resembling that of free BZ. Like BZ, the effects of MTS-LX-BZ on rSkM1 channels were completely reversible. In contrast, MTS-LX-BZ modifica tion of heart and mutant rSkM1 channels, containing a pore Cysteine at the equivalent location as cardiac Na+ channels (ie, Y401C), persisted after dr ug washout unless treated with DTT, which suggests anchoring to the pore vi a a disulfide bond. Anchor-ed MTS-LX-BZ competitively reduced the affinity of cardiac Naf channels for lidocaine but had minimal effects on mutant cha nnels with disrupted local anesthetic modification properties. These result s establish that anchored MTS-LX-BZ compounds interact with the local anest hetic binding site (LABS). Variation in the linker length altered the poten cy of channel modification by the;anchored drugs, thus providing informatio n on the spatial relationship between the anchoring site and the LABS. Our observations demonstrate that local anesthetics can be anchored to the extr acellular pore cysteine in cardiac Na+ channels and dynamically interact wi th the intracellular LABS. These results suggest that nonselective agents, such as local anesthetics, might be made more selective by linking these ag ents to target-specific anchors.