Temperature-dependent contractility of rat tunica dartos muscle: Contribution of cold, menthol-sensitive TRPM8.

Tunica dartos smooth muscle (TDSM) lies beneath the scrotal skin, and its contraction leads to scrotum wrinkling upon cooling. However, neither the nature of TDSM cold-sensitivity nor the underlying molecular sensors are well understood. Here we have investigated the role of cold/menthol-sensitive TRPM8 channel in TDSM temperature-dependent contractility. The contraction of isolated male rat TDSM strips was studied by tensiometry. TRPM8 expression was assayed by RT-PCR and fluorescence immunochemistry. Isolated TDSM strips responded to cooling from 33 °C to 20 °C by enhancement of basal tension, and increase of the amplitude and duration of electric field stimulated (EFS) contractions. The effects of cold on basal tension, but not on EFS-contractions, could be 80% inhibited by TRPM8 blockers, capsazepine and BCTC [N-(4­tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide], and could be partially mimicked by menthol. RT-PCR and immunolabeling showed TRPM8 mRNA and protein expression in TDSM cells with protein labelling being predominantly localized to intracellular compartments. Chemical castration of male rats consequent to the treatment with androgen receptor blocker, flutamide, led to the abrogation of cold effects on TDSM basal tension, but not on EFS-contractions, and to the disappearance of TRPM8 protein expression. We conclude that TRPM8 is involved in the maintenance of basal cold-induced TDSM tonus, but not in sympathetic nerve-mediated contractility, by acting as endoplasmic reticulum Ca2+ release channel whose expression in TDSM cells requires the presence of a functional androgen receptor. Thus, TRPM8 plays a crucial role in scrotal thermoregulation which is important for maintaining normal spermatogenesis and male fertility.

Complex modulation of Ca(v)3.1 T-type calcium channel by nickel.

Nickel is considered to be a selective blocker of low-voltage-activated T-type calcium channel. Recently, the Ni(2+)-binding site with critical histidine-191 (H191) within the extracellular IS3-IS4 domain of the most Ni(2+)-sensitive Cav3.2 T-channel isoform has been identified. All calcium channels are postulated to also have intrapore-binding site limiting maximal current carried by permeating divalent cations (PDC) and determining the blockade by non-permeating ones. However, the contribution of the two sites to the overall Ni(2+) effect and its dependence on PDC remain uncertain. Here we compared Ni(2+) action on the wild-type "Ni(2+)-insensitive" Cav3.1w/t channel and Cav3.1Q172H mutant having glutamine (Q) equivalent to H191 of Cav3.2 replaced by histidine. Each channel was expressed in Xenopus oocytes, and Ni(2+) blockade of Ca(2+), Sr(2+), or Ba(2+) currents was assessed by electrophysiology. Inhibition of Cav3.1w/t by Ni(2+) conformed to two sites binding. Ni(2+) binding with high-affinity site (IC50 = 0.03-3 µM depending on PDC) produced maximal inhibition of 20-30% and was voltage-dependent, consistent with its location within the channel's pore. Most of the inhibition (70-80%) was produced by Ni(2+) binding with low-affinity site (IC50 = 240-700 µM). Q172H-mutation mainly affected low-affinity binding (IC50 = 120-160 µM). The IC50 of Ni(2+) binding with both sites in the Cav3.1w/t and Cav3.1Q172H was differentially modulated by PDC, suggesting a varying degree of competition of Ca(2+), Sr(2+), or Ba(2+) with Ni(2+). We conclude that differential Ni(2+)-sensitivity of T-channel subtypes is determined only by H-containing external binding sites, which, in the absence of Ni(2+), may be occupied by PDC, influencing in turn the channel's permeation.