Negative gating modulation by (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) depends on residues in the inner pore vestibule: pharmacological evidence of deep-pore gating of K(Ca)2 channels.

Acting as a negative gating modulator, (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) shifts the apparent Ca(2+)-dependence of the small-conductance Ca(2+)-activated K(+) channels K(Ca)2.1-2.3 to higher Ca(2+) concentrations. Similar to the positive K(Ca) channel-gating modulators 1-ethyl-2-benzimidazolinone (1-EBIO) and cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methylpyrimidin-4-yl]-amine (CyPPA), the binding site for NS8593 has been assumed to be located in the C-terminal region, in which these channels interact with their Ca(2+) sensor calmodulin. However, by using a progressive chimeric approach, we were able to localize the site-of-action of NS8593 to the K(Ca)2 pore. For example, when we transferred the C terminus from the NS8593-insensitive intermediate-conductance K(Ca)3.1 channel to K(Ca)2.3, the chimeric channel remained as sensitive to NS8593 as wild-type K(Ca)2.3. In contrast, when we transferred the K(Ca)2.3 pore to K(Ca)3.1, the channel became sensitive to NS8593. Using site-directed mutagenesis, we subsequently identified two specific residues in the inner vestibule of K(Ca)2.3 (Ser507 and Ala532) that determined the effect of NS8593. Mutation of these residues to the corresponding residues in K(Ca)3.1 (Thr250 and Val275) made K(Ca)2.3 insensitive to NS8593, whereas introduction of serine and alanine into K(Ca)3.1 was sufficient to render this channel highly sensitive to NS8593. It is noteworthy that the same two residue positions have been found previously to mediate sensitivity of K(Ca)3.1 to clotrimazole and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). The location of Ser507 in the pore-loop near the selectivity filter and Ala532 in an adjacent position in S6 are within the region predicted to contain the K(Ca)2 channel gate. Hence, we propose that NS8593-mediated gating modulation occurs via interaction with gating structures at a position deep within the inner pore vestibule.

Insensitivity of NMRI mice to selective serotonin reuptake inhibitors in the tail suspension test can be reversed by co-treatment with 5-hydroxytryptophan.

RATIONALE: Exploring differences between mouse strains in drug effects in models of antidepressant-like activity may provide clues to the neurobiology of antidepressant responses. OBJECTIVES: The objective of this study was to explore whether insensitivity to selective serotonin reuptake inhibitors (SSRIs) in NMRI mice in the tail suspension test can be related to 5-hydroxytryptamine (5-HT) function. MATERIALS AND METHODS: We compared NMRI and C57Bl/6 mice, a SSRI-sensitive strain, in the tail suspension test following citalopram, paroxetine, or fluoxetine and determined 5-HT transporter (5-HTT) densities, 5-HT tissue and extracellular levels, 5-HT synthesis, tryptophan hydroxylase 2 (TPH2) genotypes and hypothermia induced by the 5-HT(1A) agonist 8-OH-DPAT. In NMRI mice, we tested if co-treatment with 5-HTP would increase 5-HT levels and confer SSRI sensitivity in the tail suspension test. RESULTS: C57Bl/6, but not NMRI, mice responded to SSRIs in the tail suspension test. 5-HTT densities in the frontal cortex and hippocampus were similar between the strains. NMRI mice had lower tissue 5-HT levels in these regions and decreased extracellular 5-HT in the frontal cortex at baseline and following citalopram. C57Bl/6 mice were more sensitive to 8-OH-DPAT-induced hypothermia. Both strains had the 1473C TPH2 genotype and similar 5-HT synthesis. In NMRI mice, 5-HTP co-treatment restored the tail suspension and extracellular 5-HT responses to SSRIs to levels equivalent to those seen in C57Bl/6 mice. CONCLUSION: Low 5-HT function in NMRI mice may account for their insensitivity to SSRIs in the tail suspension test. As the tail suspension test is a predictor of clinical efficacy, the current data suggest that 5-HTP adjunct treatment may benefit SSRI treatment refractory patients.