Evidence for a Multivalent Interaction of Symmetrical, N-Linked, Lidocaine Dimers with Voltage-Gated Na⁺ Channels

Abstract

The interaction of symmetrical lidocaine dimers with voltagegated Na ϩ channels (VGSCs) was examined using a FLIPR membrane potential assay and voltage-clamp. The dimers, in which the tertiary amines of the lidocaine moieties are linked by an alkylene chain (two to six methylene units), inhibited VGSC activator-evoked depolarization of cells heterologously-expressing rat (r) Na v 1.2a, human (h) Na v 1.5, and rNa v 1.8, with potencies 10-to 100-fold higher than lidocaine (compound 1). The rank order of potency (C 4 (compound 4) Ͼ C 3 (compound 3) Ն C 2 (compound 2) ϭ C 5 (compound 5) ϭ C 6 (compound 6) Ͼ Ͼ compound 1) was similar at each VGSC. Compound 4 exhibited strong use-dependent inhibition of hNa v 1.5 with pIC 50 values Ͻ4.5 and 6.0 for tonic and phasic block, respectively. Coincubation with local anesthetics but not tetrodotoxin attenuated compound 4-mediated inhibition of hNa v 1.5. These data suggest that the compound 4 binding site(s) is identical, or allosterically coupled, to the local anesthetic receptor. The dissociation rate of the dimers from hNa v 1.5 was dependent upon the linker length, with a rank order of compound 1 Ͼ compound 5 ϭ compound 6 Ͼ compound 2 Ͼ Ͼ compound 3. The observation that both the potency and dissociation rate of the dimers was dependent upon linker length is consistent with a multivalent interaction at VGSCs. hNa v 1.5 VGSCs did not recover from inhibition by compound 4. However, "chase" with free local anesthetic site inhibitors increased the rate of dissociation of compound 4. Together, these data support the hypothesis that compound 4 simultaneously occupies two binding sites on VGSCs, both of which can be bound by known local anesthetic site inhibitors.

A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M2 Muscarinic Receptor

“…9C). These results are consistent with a multivalent interaction (Kramer and Karpen, 1998;Rao et al, 1998;Smith et al, 2006). Likewise, the presence of orthosteric or allosteric muscarinic ligands accelerated the rate of dissociation of [ 3 H]THRX-160209 over the rates determined in the absence of competing ligand (Fig.…”

“…However, this is not necessarily true for multivalent ligands and differences in dissociation rate would be expected if a competing monovalent ligand alters the affinity of the multivalent ligand for the receptor. Certain systems allow the entry and binding of a fragment to a binding pocket with a partially bound multivalent ligand-this is typical of some, but not necessarily all, multivalent systems (Rao et al, 1998;Smith et al, 2006). From our data, it seems that fragmentinduced acceleration is a feature of the THRX-160209 interaction with the M 2 receptor.…”

“…Atropine and other orthosteric ligands had no effect on the dissociation rate of the monovalent ligand [ 3 H]NMS, but atropine did accelerate the slower dissociation phase of [ 3 H]THRX-160209, consistent with multivalent interaction. A high concentration of monovalent ligand is required for fragment-induced acceleration because the monovalent ligand must compete with high effective local concentrations of the multivalent ligand (Kramer and Karpen, 1998;Smith et al, 2006). Thus, in the present study, high concentrations of atropine or obidoxime were required to accelerate the dissociation rate of [ 3 H]THRX-160209.…”

“…From our data, it seems that fragmentinduced acceleration is a feature of the THRX-160209 interaction with the M 2 receptor. This acceleration is of particular interest because this phenomenon distinguishes multivalent from monovalent systems (Rao et al, 1998;Smith et al, 2006). It is noteworthy that using the "infinite" dilution protocol, the shape of the [ 3 H]THRX-160209 dissociation curve generated in the absence of competing monovalent ligand is biphasic.…”

Preferential block of inactivation-deficient Na+ currents by capsaicin reveals a non-TRPV1 receptor within the Na+ channel

“…For example, prenylamine blocks the Na + channel effectively via a receptor that is different from the local anesthetic receptor (Mujtaba et al, 2002). Furthermore, symmetrical N-linked lidocaine dimers appear to have a significantly higher affinity with the Na + channel than lidocaine, indicating an additional receptor situated adjacent to the local anesthetic site (Smith et al, 2006). An adjacent binding site for capsaicin near the local anesthetic receptor is also consistent with the observation that batrachotoxinmodified Na + channels are resistant to capsaicin block.…”