Pharmacological Targeting of Native CatSper Channels Reveals a Required Role in Maintenance of Sperm Hyperactivation

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca2+ entry evoked by alkaline depolarization. In the absence of external Ca2+, Na+ carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na+]i-reporting probe SBFI in populations of intact sperm. Removal of external Ca2+ increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na+]i is greater for sperm alkalinized with NH4Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na+]i rise is slowed by candidate CatSper blocker HC-056456 (IC50 ∼3 µM). HC-056456 similarly slows the rise of [Ca2+]i that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO3− but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca2+ entry through the CatSper channel is terminated by removal of external Ca2+. Thus, open CatSper channels and entry of external Ca2+ through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception

TATA-Binding Protein (TBP)-Like Factor (TLF) Is a Functional Regulator of Transcription: Reciprocal Regulation of the Neurofibromatosis Type 1 and c-fos Genes by TLF/TRF2 and TBP

The lack of direct targets for TATA-binding protein (TBP)-like factors (TLFs) confounds the understanding of their role in gene expression. Here we report that human TLF (also called TBP-related factor 2 [TRF2]) activates a number of different genes, including the neurofibromatosis type 1 (NF1) gene. The overexpression of TLF increases the amount of NF1 mRNA in cells. In vivo, TLF binds to and upregulates transcription from a fragment of the NF1 promoter. In vitro, purified TLF-TFIIA binds directly to the same NF1 promoter fragment that is required for TLF responsiveness in cells. Furthermore, targeted deletion of TLF in mice reduces NF1 levels. In contrast, TLF inhibits transcription driven by a fragment from the TATA-containing c-fos promoter by sequestering TFIIA. TBP affects the NF1 and c-fos promoters in a manner reciprocal to that of TLF, stimulating the c-fos promoter and inhibiting NF1 transcription. We conclude that TLF is a functional regulator of transcription with targets distinct from those of TBP

TRPC3 channels critically regulate hippocampal excitability and contextual fear memory

Memory formation requires de novo protein synthesis, and memory disorders may result from misregulated synthesis of critical proteins that remain largely unidentified. Plasma membrane ion channels and receptors are likely candidates given their role in regulating neuron excitability, a candidate memory mechanism. Here we conduct targeted molecular monitoring and quantitation of hippocampal plasma membrane proteins from mice with intact or impaired contextual fear memory to identify putative candidates. Here we report contextual fear memory deficits correspond to increased Trpc3 gene and protein expression, and demonstrate TRPC3 regulates hippocampal neuron excitability associated with memory function. These data provide a mechanistic explanation for enhanced contextual fear memory reported herein following knockdown of TRPC3 in hippocampus. Collectively, TRPC3 modulates memory and may be a feasible target to enhance memory and treat memory disorders.Fil: Neuner, Sarah M.. The University of Tennessee Health Science Center. Department of Anatomy and Neurobiology; Estados UnidosFil: Wilmott, Lynda A.. The University of Tennessee Health Science Center. Department of Anatomy and Neurobiology; Estados UnidosFil: Hope, Kevin A.. The University of Tennessee Health Science Center. Department of Anatomy and Neurobiology; Estados UnidosFil: Hoffmann, Brian. Medical College of Wisconsin. Department of Biotechnology and Bioengineering; Estados UnidosFil: Chong, Jayhong A.. MA. Hydra Biosciences, Cambridge; Estados UnidosFil: Abramowitz, Joel. Research Triangle Park. Laboratory of Neurobiology. National Institute of Environmental Health Sciences; Estados UnidosFil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Research Triangle Park. Laboratory of Neurobiology. National Institute of Environmental Health Sciences; Estados UnidosFil: O'Connell, Kristen M.. University of Tennessee Health Science Center. Department of Physiology; Estados UnidosFil: Savio, Chan C.. Northwestern Fienberg School of Medicine. Department of Physiology; Estados UnidosFil: Kaczorowski, Catherine C.. The University of Tennessee Health Science Center. Department of Anatomy and Neurobiology; Estados Unido

Treatment with HC-070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice

<div><p>Background</p><p>Forty million adults in the US suffer from anxiety disorders, making these the most common forms of mental illness. Transient receptor potential channel canonical subfamily (TRPC) members 4 and 5 are non-selective cation channels highly expressed in regions of the cortex and amygdala, areas thought to be important in regulating anxiety. Previous work with null mice suggests that inhibition of TRPC4 and TRPC5 may have anxiolytic effects.</p><p>HC-070 <i>in vitro</i></p><p>To assess the potential of TRPC4/5 inhibitors as an avenue for treatment, we invented a highly potent, small molecule antagonist of TRPC4 and TRPC5 which we call HC-070. HC-070 inhibits recombinant TRPC4 and TRPC5 homomultimers in heterologous expression systems with nanomolar potency. It also inhibits TRPC1/5 and TRPC1/4 heteromultimers with similar potency and reduces responses evoked by cholecystokinin tetrapeptide (CCK-4) in the amygdala. The compound is >400-fold selective over a wide range of molecular targets including ion channels, receptors, and kinases.</p><p>HC-070 <i>in vivo</i></p><p>Upon oral dosing in mice, HC-070 achieves exposure levels in the brain and plasma deemed sufficient to test behavioral activity. Treatment with HC-070 attenuates the anxiogenic effect of CCK-4 in the elevated plus maze (EPM). The compound recapitulates the phenotype observed in both null TRPC4 and TRPC5 mice in a standard EPM. Anxiolytic and anti-depressant effects of HC-070 are also observed in pharmacological in vivo tests including marble burying, tail suspension and forced swim. Furthermore, HC-070 ameliorates the increased fear memory induced by chronic social stress. A careful evaluation of the pharmacokinetic-pharmacodynamic relationship reveals that substantial efficacy is observed at unbound brain levels similar to, or even lower than, the 50% inhibitory concentration (IC₅₀) recorded in vitro, increasing confidence that the observed effects are indeed mediated by TRPC4 and/or TRPC5 inhibition. Together, this experimental data set introduces a novel, high quality, small molecule antagonist of TRPC4 and TRPC5 containing channels and supports the targeting of TRPC4 and TRPC5 channels as a new mechanism of action for the treatment of psychiatric symptoms.</p></div

Structures and activity in fluorometric assays of HC-070 and HC-608.

<p>The chemical structures of (A) HC-070 and (B) HC-608 (Pico145). (C) Inhibition of hTRPC5 by HC-070 and HC-608 in indicator-assisted calcium influx analysis. Concentrations tested ranged from 1 picomolar (pM) to 1 μM. Each data point represents the average of 8 measurements from a 384-well plate. Error bars show the standard deviation. The IC₅₀ values were 9.3 ± 0.9 and 6.2 ± 0.5 nanomolar (nM), respectively. (D) Inhibition of hTRPC4 by HC-070 and HC-608 over the same range of concentrations. The IC₅₀ values were 46.0 ± 3.9 nM and 32.5 ± 1.8 nM, respectively (n = 8). Error bars represent the standard deviation.</p

Pharmacokinetic properties of HC-070.

<p>PK profiles of HC-070 after intravenous (A) and oral (B) administration in C57BL/6 mice. Plasma concentrations were determined by LC-MS/MS. Points represent the individual concentrations at the times indicated. Lines represent mean exposure (n = 12 mice/arm, n = 3 data points per time point). (C) Summary of PK properties. CL = clearance; V_ss = volume of distribution at steady state; MRT_disp = mean residence time of drug molecules after intravascular administration; T_1/2 = half-life. (D) Plasma and brain concentrations measured 2 hours after intravenous or oral administration of 1 or 10 mg/kg HC-070, respectively. C_PL = concentration in plasma, C_BR = concentration in brain, K_P,BR = partitioning coefficient between brain and plasma.</p

HC-070 reduces marble burying behavior.

<p>(A) Mice were administered vehicle or 1, 3 or 10 mg/kg HC-070 orally, 60 minutes prior to testing. The positive control, 10 mg/kg zimelidine, was administered IP 45 minutes prior to testing. At 1, 3, and 10 mg/kg, HC-070 significantly decreased the number of buried marbles compared to vehicle (p <0.05 *, p<0.01**, Dunnett’s post-hoc test following one-way ANOVA, n = 10/group). Zimelidine also decreased the number of buried marbles (p < 0.05, t-test, n = 10/group). Each animal is represented on the graph and the horizontal lines represent the mean. (B) Average plasma exposures from animals that completed the test. Error bars are the standard deviation.</p

HC-070 inhibits recombinantly expressed TRPC4 and TRPC5, as well as TRPC1-containing heteromultimers, in whole-cell manual patch clamp.

<p>HC-070 inhibits recombinantly expressed TRPC4 and TRPC5, as well as TRPC1-containing heteromultimers, in whole-cell manual patch clamp.</p