High-affinity progesterone-binding sites have been identified, characterized in and purified from porcine liver membranes. They were functionally solubilized by the non-denaturing zwitterionic detergent 3-[(3-~holamidopropyl)dimethylammonio]-l -propanesulfonic acid (Chaps, 20 mM, detergent/protein mass ratio 4 : l ) at a yield of 75-80%. Using [3H]progesterone as radioligand, binding studies showed high-affinity and low-affinity binding sites in microsomal preparations with an apparent Kdl of 11 nM and an apparent Kd2 of 286 nM. In solubilized fractions the high-affinity binding sites were present at an apparent Kd of 69 nM. In both preparations, progesterone binding was time-dependent, saturable, reversible, and showed a similar hierachy of affinities for related steroids. A purification scheme was developed based on anion-exchanger procedures. The purified fraction as identified by maximum specific progesterone-binding activity contained two major polypeptides of apparent molecular masses (SDS/PAGE) of 28 kDa and 56 kDa, respectively. Sequencing of both polypeptides showed an identical amino terminus without significant identity in the amino acid sequence to any known protein primary structure.Keywords: progesterone ; membrane-binding site ; liver; amino acid sequence.For the past decade, nonclassical actions of steroid hormones and related signal transduction pathways have gained increasing scientific interest. Evidence for nongenomic steroid effects are provided for all classes of steroid hormones including the secosteroid vitamin D3 and triiodothyronine (for review see [I, 21).As an example, the sex hormone progesterone has been found to act on oocyte maturation in a nontranscriptional manner in Xenopus laevis 131. An important instant effect of progesterone is the rapid stimulation of ion fluxes in human sperm. Blackmore et al. [4, 51 showed a rapid Ca2+ and Turner and Meizel [6] demonstrated CI-effluxes during the acrosome reaction induced by Progesterone. Moreover, in hepatocytes a rapid progesterone-induced increase of cytosolic CaZ+ was seen resulting from Caz+ influx [7].Another prominent example for a nongenomic steroid effect is the rapid stimulation of Na+/H ' -exchanger in human mononu- So far, none of these membrane steroid-binding proteins has been purified in sufficient amounts to allow molecular analysis and cloning; it appears that the lack of a satisfactory solubilization procedure for these labile proteins is one of the major obstacles in this regard. Here, progesterone-specific binding proteins are identified and characterized in porcine liver microsomes, solubilized, purified and partially sequenced from the N-terminus. Materials and Methods

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The recent identification of the fusicoccin-binding protein (FCBP) in plasma membranes from monocotyledonous and dicotyledonous angiosperms has opened the basis for an elucidation of the toxin’s mechanism(s) of action and indicated a widespread occurrence of the FCBP in plants. Results of a detailed taxonomic survey of fusicoccin-binding sites are reported. Binding sites were not found in prokaryotes, animal tissues, fungi and algae including the most direct extant ancestors of the land plants (Coleochaete). From the Psilotales (Psilophytatae) to the monocotyledonous angiosperms, all taxa analyzed possessed high-affinity microsomal fusicoccin-binding sites. A heterogeneous picture emerged for the Bryophvta. Anthoceros crispulus (Anthocerotae), the only hornwort available to study, lacked fusicoccin binding. Within the Hepaticae as well as the Musci, species lacking and species exhibiting toxin binding were found. The binding site thus seems to have emerged very early in the evolution of the land plants. The tissue distribution of fusicoccin-binding sites was studied in Vicia faba L. shoots. All tissues analyzed showed fusicoccin binding, although not to the same extent. On a per-cell basis, guard cells were found to contain, compared to mesophyll cells, a nine-fold higher number of binding sites. Based on cell surface area, the site density is by a factor of 32 higher in guard cells than in mesophyll cells. Tissue specific expression of the binding sites is suggested by these findings.

SummaryObjectiveWithin its range of therapeutic plasma concentrations, the anticonvulsant retigabine (ezogabine) is believed to selectively act on Kv7 channels. Here, the contribution of specific γ‐aminobutyric acid (GABA)A receptor subtypes to the antiseizure effects of retigabine was investigated.MethodsUsing patch‐clamp recordings, seizure‐like activity, tonic currents, and GABA‐induced currents in hippocampal neurons were tested for their sensitivity toward retigabine, as were recombinant GABA A receptors expressed in tsA 201 cells.ResultsRetigabine reduced seizure‐like activity elicited by low Mg2+ in a concentration‐dependent manner with half maximal inhibition at 1 μm. Seizure‐like activity triggered by blocking either Kv7 channels or GABA A receptors was equally reduced by retigabine, but when these channels/receptors were blocked simultaneously, the inhibition was lost. Retigabine (10 μm) enhanced bicuculline‐sensitive tonic currents in hippocampal neurons, but failed to affect GABA‐evoked currents. However, when receptors involved in phasic GABAergic inhibition were blocked by penicillin, retigabine did enhance GABA‐evoked currents. In tsA 201 cells expressing various combinations of GABA A receptor subunits, 10 μm retigabine enhanced currents through α1β2δ, α4β2δ, α4β3δ, and α6β2δ receptors, but left currents through α1β2γ2S, α4β3γ2S, α5β3γ2S, and α6β2γ2S receptors unaltered. With αβ receptors, retigabine diminished currents through α1β2 and α4β3, but increased currents through α6β2 receptors. The enhancement of currents through α1β2δ receptors by retigabine was concentration dependent and became significant at 1 μm.SignificanceThese results demonstrate that retigabine is a subtype selective modulator of GABA A receptors with preference for extrasynaptic δ‐containing receptors; this property may contribute to its broad antiepileptic effectiveness and explain its lack of effect on absence seizures.