Pharmacological and nutritional targeting of voltage-gated sodium channels in the treatment of cancers.

Voltage-gated sodium (NaV) channels, initially characterized in excitable cells, have been shown to be aberrantly expressed in non-excitable cancer tissues and cells from epithelial origins such as in breast, lung, prostate, colon, and cervix, whereas they are not expressed in cognate non-cancer tissues. Their activity was demonstrated to promote aggressive and invasive potencies of cancer cells, both in vitro and in vivo, whereas their deregulated expression in cancer tissues has been associated with metastatic progression and cancer-related death. This review proposes NaV channels as pharmacological targets for anticancer treatments providing opportunities for repurposing existing NaV-inhibitors or developing new pharmacological and nutritional interventions.

Amino acid substitutions in the human homomeric ß3 GABAA receptor that enable activation by GABA.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The α1ß2γ2 receptor is the major subtype in the brain; GABA binds at the ß2(+)α1(-) interface. The structure of the homomeric ß3 GABAAR, which is not activated by GABA, has been solved. Recently, four additional heteromeric structures were reported, highlighting key residues required for agonist binding. Here, we used a protein engineering method, taking advantage of knowledge of the key binding residues, to create a ß3(+)α1(-) heteromeric interface in the homomeric human ß3 GABAAR that enables GABA-mediated activation. Substitutions were made in the complementary side of the orthosteric binding site in loop D (Y87F and Q89R), loop E (G152T), and loop G (N66D and A70T). The Q89R and G152T combination enabled low-potency activation by GABA and potentiation by propofol but impaired direct activation by higher propofol concentrations. At higher concentrations, GABA inhibited gating of ß3 GABAAR variants containing Y87F, Q89R, and G152T. Reversion of Phe87 to tyrosine abolished GABA's inhibitory effect and partially recovered direct activation by propofol. This tyrosine is conserved in homomeric GABAARs and in the Erwinia chrysanthemi ligand-gated ion channel and may be essential for the absence of an inhibitory effect of GABA on homomeric channels. This work demonstrated that only two substitutions, Q89R and G152T, in ß3 GABAAR are sufficient to reconstitute GABA-mediated activation and suggests that Tyr87 prevents inhibitory effects of GABA.

A role for loop G in the ß1 strand in GABAA receptor activation.

KEY POINTS: The role of the ß1 strand in GABAA receptor function is unclear. It lies anti-parallel to the ß2 strand, which is known to participate in receptor activation. Molecular dynamics simulation revealed solvent accessible residues within the ß1 strand of the GABAA ß3 homopentamer that might be amenable to analysis using the substituted Cys accessibility method. Cys substitutions from Asp43 to Thr47 in the GABAA α1 subunit showed that D43C and T47C reduced the apparent potency of GABA. F45C caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Cys43 and Cys47 were accessible to 2-aminoethyl methanethiosulphonate (MTSEA) modification, whereas Cys45 was not. Both GABA and the allosteric agonist propofol reduced MTSEA modification of Cys43 and Cys47. By contrast, modification of Cys64 in the ß2 strand loop D was impeded by GABA but unaffected by propofol. These data reveal movement of ß1 strand loop G residues during agonist activation of the GABAA receptor. ABSTRACT: The GABAA receptor α subunit ß1 strand runs anti-parallel to the ß2 strand, which contains loop D, known to participate in receptor activation and agonist binding. However, a role for the ß1 strand has yet to be established. We used molecular dynamics simulation to quantify the solvent accessible surface area (SASA) of ß1 strand residues in the GABAA ß3 homopentamer structure. Residues in the complementary interface equivalent to those between Asp43 and Thr47 in the α1 subunit have an alternating pattern of high and low SASA consistent with a ß strand structure. We investigated the functional role of these ß1 strand residues in the α1 subunit by individually replacing them with Cys residues. D43C and T47C substitutions reduced the apparent potency of GABA at α1ß2γ2 receptors by 50-fold and eight-fold, respectively, whereas the F45C substitution caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Receptors with D43C or T47C substitutions were sensitive to 2-aminoethyl methanethiosulphonate (MTSEA) modification. However, GABA-evoked currents mediated by α1(F45C)ß2γ2 receptors were unaffected by MTSEA, suggesting that this residue is inaccessible. Both GABA and the allosteric agonist propofol reduced MTSEA modification of α1(D43C)ß2γ2 and α1(T47C)ß2γ2 receptors, indicating movement of the ß1 strand even during allosteric activation. This is in contrast to α1(F64C)ß2γ2 receptors, where only GABA, but not propofol, reduced MTSEA modification. These findings provide the first functional evidence for movement of the ß1 strand during gating of the receptor and identify residues that are critical for maintaining GABAA receptor function.

Common determinants of single channel conductance within the large cytoplasmic loop of 5-hydroxytryptamine type 3 and alpha4beta2 nicotinic acetylcholine receptors.

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (gamma) below the resolution of single channel recording (966 +/- 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range gamma values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4'), 436 (0'), and 440 (4') residues by 5-HT3B (-4'Gln, 0'Asp, and 4'Ala) residues increases gamma to 36.5 +/- 1.0 pS. The 0' residue makes the most substantial contribution to gamma of the 5-HT3AR. Replacement of 0'Arg by aspartate, glutamate (alpha7 nAChR subunit MA 0'), or glutamine (beta2 subunit MA 0') increases gamma to the resolvable range (>6 pS). By contrast, replacement of 0'Arg by phenylalanine (alpha4 subunit MA 0') reduced gamma to 416 +/- 107 fS. In reciprocal experiments with alpha4beta2 nAChRs (gamma = 31.3 +/- 0.8 pS), replacement of MA 0' residues by arginine in alpha4beta2(Q443R) and alpha4(F588R)beta2 reduced gamma slightly. By contrast, the gamma of double mutant alpha4(F588R)beta2(Q443R) was halved. The MA -4' and 4' residues also influenced gamma of 5-HT3ARs. Replacement of nAChR alpha4 or beta2 MA 4' residues by arginine made current density negligible. By contrast, replacement of both -4' residues by arginine produced functional nAChRs with substantially reduced gamma (11.4 +/- 0.5 pS). Homology models of the 5-HT3A and alpha4beta2 nAChRs against Torpedo nAChR revealed MA -4', 0', and 4' residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family.