Requirement for the N-Terminal Coiled-Coil Domain for Expression and Function, but not Subunit Interaction of, the ADPR-Activated TRPM2 Channel

Transient receptor potential melastatin 2 (TRPM2) proteins form multiple-subunit complexes, most likely homotetramers, which operate as Ca2+-permeable, nonselective cation channels activated by intracellular ADP-ribose (ADPR) and oxidative stress. Each TRPM2 channel subunit is predicted to contain two coiled-coil (CC) domains, one in the N-terminus and the other in the C-terminus. Our recent study has shown that the C-terminal CC domain plays an important, but not exclusive, role in the TRPM2 channel assembly. This study aimed to examine the potential role of the N-terminal CC domain. Domain deletion dramatically reduced protein expression and abolished ADPR-evoked currents but did not alter the subunit interaction. Deletion of both CC domains strongly attenuated the subunit interaction, confirming that the C-terminal CC domain is critical in the subunit interaction. Glutamine substitutions into individual hydrophobic residues at positions a and d in the heptad repeats to disrupt the CC formation had no effect on protein expression, subunit interaction, or ADPR-evoked currents. Mutation of Ile658 to glutamine, which did not perturb the CC formation, decreased ADPR-evoked currents without affecting protein expression, subunit interaction, or membrane trafficking. These results collectively suggest the requirement for the N-terminal CC domain for protein expression and function, but not subunit interaction, of the TRPM2 channel

Regulation of MMP2 and MMP9 metalloproteinases by FSH and growth factors in bovine granulosa cells

Matrix metalloproteinases (MMP) are key enzymes involved in tissue remodeling. Within the ovary, they are believed to play a major role in ovulation, and have been linked to follicle atresia. To gain insight into the regulation of MMPs, we measured the effect of hormones and growth factors on MMP2 and MMP9 mRNA levels in non-luteinizing granulosa cells in serum-free culture. FSH and IGF1 both stimulated estradiol secretion and inhibited MMP2 and MMP9 mRNA abundance. In contrast, EGF and FGF2 both inhibited estradiol secretion but had no effect on MMP expression. At physiological doses, none of these hormones altered the proportion of dead cells. Although we cannot link MMP expression with apoptosis, the specific down regulation by the gonadotropic hormones FSH and IGF1 in vitro suggests that excess MMP2 and MMP9 expression is neither required nor desired for follicle development

Molecular mechanism for 3:1 subunit stoichiometry of rod cyclic nucleotide-gated ion channels

Molecular determinants of ion channel tetramerization are well characterized, but those involved in heteromeric channel assembly are less clearly understood. The heteromeric composition of native channels is often precisely controlled. Cyclic nucleotide-gated (CNG) channels from rod photoreceptors exhibit a 3:1 stoichiometry of CNGA1 and CNGB1 subunits that tunes the channels for their specialized role in phototransduction. Here we show, using electrophysiology, fluorescence, biochemistry, and X-ray crystallography, that the mechanism for this controlled assembly is the formation of a parallel 3-helix coiled-coil domain of the carboxy-terminal leucine zipper region of CNGA1 subunits, constraining the channel to contain three CNGA1 subunits, followed by preferential incorporation of a single CNGB1 subunit. Deletion of the carboxy-terminal leucine zipper domain relaxed the constraint and permitted multiple CNGB1 subunits in the channel. The X-ray crystal structures of the parallel 3-helix coiled-coil domains of CNGA1 and CNGA3 subunits were similar, suggesting that a similar mechanism controls the stoichiometry of cone CNG channels

Plasma _unbound and brain_unbound concentrations of atomoxetine in absence or presence of morphine; esreboxetine in absence or presence of morphine and/or fluoxetine; fluoxetine in absence or presence of morphine and/or esreboxetine; and duloxetine in absence or presence of morphine and ondansetron - at 75 min post-dosing.

<p>n = 3–6 for each group. Data are presented as mean ± standard deviation.</p

Atomoxetine exhibited antinociceptive synergy with morphine using a fixed-ratio design in the rat formalin model.

<p>(A) The dose-response curve of a fixed-ratio of 3 parts atomoxetine (Atx, IP) to 1 part morphine (Mor, SC) leftward shifted relative to the atomoxetine dose-response curve alone (n = 6–12). All data points are shown as mean ± SEM for each group and are expressed as percentage of controls. (B) An isobologram for the combined effects of atomoxetine and morphine in a fixed ratio combination 3∶1. The ED₅₀ value for morphine is plotted on the abscissa, and the ED₅₀ value for atomoxetine is plotted on the ordinate. The solid line represents the line of additivity and the isobol point (observed ED₅₀ value) is located to the left and below the theoretical additive ED₅₀ value (with non-overlapping 95% CI). (C) The dose-response curve of a fixed-ratio of concomitant administration of 10 part atomoxetine (IP) to 1 part morphine (SC) leftward shifted relative to the atomoxetine dose-response curve alone (n = 6–16). All data points are shown as mean ± SEM for each group and are expressed as percentage of controls. (D) An isobologram for the combined effects of atomoxetine and morphine in a fixed ratio combination 10∶1. The isobol point (observed ED₅₀ value) is located to the left and below the theoretical additive ED₅₀ value (without overlapping 95% CI).</p

Atomoxetine exhibited antinociceptive synergy with morphine using a fixed-dose design in the rat formalin model.

<p>(A) Both 3 and 10 mg/kg atomoxetine (Atx, IP) shifted the morphine (Mor) dose-response curve leftward in the rat formalin model (n = 6–16). Morphine alone: ED₅₀ = 2.3 mg/kg (95% CI: 2.0–2.5); morphine+atomoxetine (IP, 3 mg/kg): ED₅₀ = 1.1 mg/kg (95% CI: 0.8–1.6); and morphine+atomoxetine (IP, 10 mg/kg): ED₅₀ = 0.6 mg/kg (95% CI: 0.4–0.8). All data points are shown as mean ± SEM for each group and are expressed as percentage of controls. Inset (A) Atomoxetine (IP) at 3 and 10 mg/kg was associated with 67±10% and 84±3% for NET and 35±9% and 64±5% for SERT occupancy measured <i>ex vivo</i> at 75 min post-dose, respectively. All occupancy data represent mean (± SEM) for each group. (B) A subefficacious dose of morphine 1 mg/kg (SC) left-shifted the atomoxetine dose-response curve (n = 6–16). Atomoxetine alone: ED₅₀ = 27.8 mg/kg (95% CI: 22–36); and atomoxetine+morphine (SC, 1 mg/kg): ED₅₀ = 2.5 mg/kg (95% CI: 1.3–4.7). (C) A fixed combination of NET selective inhibitor esreboxetine (Esrbx, IP, 10 mg/kg) and SERT selective inhibitor fluoxetine (Flx, IP, 1 mg/kg) left-shifted the morphine dose-response curve (n = 6–12). Morphine alone: ED₅₀ = 2.3 mg/kg (95% CI: 2.0–2.5); morphine+esreboxetine (IP, 10 mg/kg)+fluoxetine (IP, 1 mg/kg): ED₅₀ = 0.3 mg/kg (95% CI: 0.2–0.7).</p

Antinociceptive synergy between atomoxetine and morphine did not reflect impaired motor coordination.

<p>The white bars represent the % reduction in the flinching behavior compared to vehicle-treatment in the rat formalin model (n = 10–22), and the grey bars represent the change in latency for rats to fall from an accelerating rotating rod compared to vehicle treatment in the rat RotaRod test (n = 8). All data points are shown as mean ± SEM for each group and are expressed as percentage of controls. Data from one-way ANOVA are as follows: rat formalin model: F _{(4, 53)} = 36.12, p<0.0001; RotaRod: F _{(4, 34)} = 4.604, p = 0.004. Data from the <i>post hoc</i> Dunnett’s test follows: **p<0.01, q = 3.265; ***p<0.001, q = 9.258–9.370, compared to vehicle treatment.</p