WIN 17317-3, a New High-Affinity Probe for Voltage-Gated Sodium Channels

Abstract

The iminodihydroquinoline WIN 17317-3 was previously shown to inhibit selectively the voltage-gated potassium channels, K(v)1.3 and K(v)1.4 [Hill, R. J., et al. (1995) Mol. Pharmacol. 48, 98-104; Nguyen, A., et al. (1996) Mol. Pharmacol. 50, 1672-1679]. Since these channels are found in brain, radiolabeled WIN 17317-3 was synthesized to probe neuronal K(v)1 channels. In rat brain synaptic membranes, [(3)H]WIN 17317-3 binds reversibly and saturably to a single class of high-affinity sites (K(d) 2.2 +/- 0.3 nM; B(max) 5.4 +/- 0.2 pmol/mg of protein). However, the interaction of [(3)H]WIN 17317-3 with brain membranes is not sensitive to any of several well-characterized potassium channel ligands. Rather, binding is modulated by numerous structurally unrelated sodium channel effectors (e.g., channel toxins, local anesthetics, antiarrhythmics, and cardiotonics). The potency and rank order of effectiveness of these agents in affecting [(3)H]WIN 17317-3 binding is consistent with their known abilities to modify sodium channel activity. Autoradiograms of rat brain sections indicate that the distribution of [(3)H]WIN 17317-3 binding sites is in excellent agreement with that of sodium channels. Furthermore, WIN 17317-3 inhibits sodium currents in CHO cells stably transfected with the rat brain IIA sodium channel with high affinity (K(i) 9 nM), as well as agonist-stimulated (22)Na uptake in this cell line. WIN 17317-3 interacts similarly with skeletal muscle sodium channels but is a weaker inhibitor of the cardiac sodium channel. Together, these results demonstrate that WIN 17317-3 is a new, high-affinity, subtype-selective ligand for sodium channels and is a potent blocker of brain IIA sodium channels.

Comparison of the Pharmacological Properties of Rat NaV1.8 with Rat NaV1.2a and Human NaV1.5 Voltage-Gated Sodium Channel Subtypes Using a Membrane Potential Sensitive Dye and FLIPRR

“…However, with the exception of TTX, there were no significant (i.e., ≥tenfold) differences between the potencies of a series of known Na + and Ca 2+ channel blockers to inhibit VGSC activatorevoked depolarization mediated via the three channel subtypes. The pharmacological properties of the VGSC subtypes studied in parallel were consistent with those of rat TTX-R, rNa v 1.2a, and hNa v 1.5 Na + currents previously described in the literature across various studies (Wanner et al 1999;Scholz and Vogel 2000;Brau et al 2001;Xie et al 2001).…”

“…Furthermore, the potencies of TTX for rNa v 1.2a, hNa v 1.5, and rNa v 1.8 VGSC subtypes determined using the FLIPR R assay are in very good agreement with the respective potency estimates obtained using a variety of different techniques, including voltage-clamp (Gellens et al 1992;Scholz and Vogel 2000;Xie et al 2001;present study), [ 22 Na] and [ 14 C]-guanidinium influx (Wanner et al 1999), and Ca 2+ imaging studies using reconstituted synaptosomes (Deffois et al 1996). Low nanomolar IC 50 values for TTX have been reported for both recombinant (Kontis and Goldin 1993) and native TTX-S VGSCs (Francel et al 1987).…”

“…To our knowledge, this is the first study to characterize these three VGSC subtypes in parallel. Previous studies have focused on pharmacological characterization of either native or recombinant TTX-sensitive (TTX-S) VGSCs (Deffois et al 1996;Rose et al 1997;Lingamaneni et al 1999;Xie et al 2001), or recombinant Na v 1.5 currents (Krafte et al 1995;Wanner et al 1999). In contrast, there have been limited studies examining the pharmacology of TTX-resistant (TTX-R) currents (Scholz and Vogel 2000).…”

Targeting Effector Memory T Cells with a Selective Peptide Inhibitor of Kv1.3 Channels for Therapy of Autoimmune Diseases

“…The first small-molecule Kv1.3 blockers with nanomolar affinity that were discovered-iminodihydroquinolines WIN-17317 and CP-339818 and the benzhydryl piperidine UK-78282-also block sodium channels (Wanner et al, 1999) and the neuronal Kv1.4 channel (Hanson et al, 1999). The small-molecule Kv1.3 inhibitors developed by Merck-correolide (Felix et al, 1999;Hanner et al, 1999;Koo et al, 1999;Bao et al, 2005), cyclohexyl-subsituted benzamides (Schmalhofer et al, 2002) and candelalides A-C (Singh et al, 2001)-are poorly selective for Kv1.3.…”

A Novel Drug Binding Site on Voltage-Gated Sodium Channels in Rat Brain

“…Binding sites in brain preparations, deemed to be on sodium channels, have been described for [ 3 H]phenytoin (Francis and Burnham, 1992), [ 3 H]tetracaine (Grima et al, 1986;Reith et al, 1987) (Thomsen et al, 1993), and [ 3 H]WIN 17317-3 (Wanner et al, 1999). The two major differences are with site 1 and 3 toxins, which had no effect on any these previously described binding sites, but, respectively, enhanced and inhibited the binding of (Francis et al, 2000), and doubt has been expressed that the [ 3 H]tetracaine binding site is on sodium channels (Reith et al, 1987).…”