Molecular Pharmacology最新文献

Brain excitability is dysfunctional in epilepsy and overlapping neuropsychiatric conditions including autism spectrum disorder (ASD). Epilepsy and ASD are often attributed to malfunctioning coordination between synaptic excitation and inhibition. Dravet syndrome (DS) is a severe form of epilepsy arising from haploinsufficiency of the SCN1A gene that encodes the voltage-gated sodium channel Nav1.1. A DS mouse model (Scn1a^+/-) recapitulated essential features of DS and revealed that sodium current density was profoundly reduced in GABAergic inhibitory interneurons while pyramidal cells were spared, suggesting that DS is an "interneuronopathy." Further studies from the Catterall group and others have expanded this picture: DS symptoms, which include recurrent seizures, ataxia, cognitive impairment, ASD, and premature death, could be assigned in part to brain region-specific effects; the Nav1.1 mutations cause dysfunction in some subtypes of interneurons, not others, and are temporally restricted; DS-causing sodium channel mutations were found throughout SCN1A as well as in SCN1B, encoding the β1 subunit. Interest in therapeutic approaches was sparked by preclinical studies of cannabidiol (CBD) that led to the 2018 US Food and Drug Administration approval for treatment of seizures in patients with DS. Independent evidence showed that CBD antagonized GPR55, a G protein-coupled receptor activated by the lipid signaling molecule lysophosphatidylinositol (LPI). We summarized evidence from our group and others that CBD has a dual mechanism of action, targeting both ion channels and GPR55. CBD quells an epileptogenic vicious cycle: seizures strengthen LPI-GPR55 signaling while LPI-GPR55 signaling elevates the synaptic excitatory-inhibitory ratio, thereby promoting further seizures. SIGNIFICANCE STATEMENT: Modern medicine relies on ion channels and G protein-coupled receptors (GPCRs) as key targets. In studies of Dravet syndrome, a devastating genetic disorder with features of epilepsy and autism, William Catterall connected NaV1.1 mutations to deficient excitability of inhibitory neurons. He and his colleagues explored preclinical interventions using cannabidiol (CBD) and clobazam, opening the way to a current understanding of CBD's therapeutic mechanism. CBD affects both ion channels and GPR55, a GPCR activated by lysophosphatidylinositol, an activity-dependent lipid messenger, readjusting the synaptic excitatory-inhibitory ratio.

Ritonavir (RTV) is an important drug for anti-human immunodeficiency virus treatment and is mainly metabolized by cytochrome P450 (CYP) 3A4. Clinically, the most common side effect of RTV treatment is hepatoxicity. We previously showed that the long noncoding RNA hepatocyte nuclear factor 4 alpha (HNF4A) antisense 1 (HNF4A-AS1) negatively regulated CYP3A4 expression and participated in RTV-induced hepatotoxicity in vitro, but the mechanism has not been well understood. In this study, similar results were observed in the mouse, where liver-specific knockdown of Hnf4aos (homolog of human HNF4A-AS1) led to increased serum aspartate (∼1.8-fold) and alanine transaminase (∼2.4-fold) levels and enlarged and degenerated hepatocytes 24 hours after RTV administration. Meanwhile, endoplasmic reticulum stress markers GRP78, PDI, and XBP-1 increased about 2.4-fold, 2.1-fold, and 2.7-fold, respectively. The aggravated liver injury correlated with Hnf4aos knockdown, attributable to heightened Cyp3a11 (homolog of human CYP3A4) expression (mRNA and protein levels were 1.8-fold and 2.5-fold, respectively). Importantly, in vitro studies revealed the underlying mechanism that HNF4A-AS1 mediated the interaction between heterogeneous nuclear ribonucleoprotein C and HNF4A, whereas heterogeneous nuclear ribonucleoprotein C promoted HNF4A degradation through the ubiquitination pathway, thereby decreasing CYP3A4 expression and alleviating RTV-induced liver injury. Overall, our findings unveil a novel mechanism by which HNF4A-AS1 regulates CYP3A4 expression to influence RTV-induced liver injury. SIGNIFICANCE STATEMENT: HNF4A-AS1 negatively regulates the expression of CYP3A4, whose overexpression is highly correlated with ritonavir (RTV)-induced liver injury. In this study, the role of Hnf4aos (homolog of human HNF4A-AS1) in RTV-induced hepatotoxicity was confirmed in mice. We found that HNF4A-AS1 and HNRNPC form a complex and facilitate the ubiquitination and degradation of HNF4A protein, thereby decreasing CYP3A4 expression and alleviating RTV hepatotoxicity.

LIM and Src homology 3 (SH3) protein 2 (LASP2) is a small focal adhesion protein first identified as a splice variant of the nebulette gene (Nebl). As the newest member of the nebulin protein family, the regulation and function of LASP2 remain largely unknown. Our previous RNA-sequencing results identified Nebl as one of the most highly induced genes in the mouse liver in response to the activation of pregnane X receptor (PXR). In this study, we investigated this phenomenon further and show that PXR induces Lasp2 instead of Nebl, which partially use the same exons. Lasp2 was found to be induced in response to PXR ligand pregnenolone 16α-carbonitrile (PCN) treatment in mouse liver in vivo both after 4-day treatment and after long-term, 28-day treatment and in both male and female mice. Interestingly, the Lasp2 induction was more efficient in high-fat diet-fed mice (103-fold after 4-day PCN treatment) than in the normal chow-fed mice (32-fold after 4-day PCN treatment). Lasp2 induction was abolished in PXR knockout mice but could be rescued by re-expression of PXR, indicating that Lasp2 induction is PXR mediated. In mouse primary hepatocytes cycloheximide did not inhibit Lasp2 induction by PCN and a PXR binding site could be recognized upstream of the mouse Lasp2 gene suggesting direct regulation of Lasp2 by PXR. In human 3D hepatocytes, rifampicin induced only a modest increase in LASP2 expression. This study shows for the first time that PXR activation strongly induces Lasp2 expression in mouse liver and establishes Lasp2 as a novel PXR target gene. SIGNIFICANCE STATEMENT: RNA-sequencing results have previously identified nebulette (Nebl) to be efficiently induced by pregnane X receptor activating compounds. This study shows that instead of Nebl, LIM and Src homology 3 (SH3) protein 2 (Lasp2) coding for a small focal adhesion protein and partly sharing exons with the Nebl gene is a novel target of pregnane X receptor in mouse liver.

NS309 (6,7-dichloro-1H-indole-2,3-dione-3-oxime) is widely used as a pharmacological tool to increase the activity of small- and intermediate-conductance calcium-activated potassium channels. NS309 is assumed to function as a positive allosteric gating modulator. However, its binding site and the molecular details of its action remain unknown. Here, we show that NS309 has a profound effect on the calcium-dependent gating of the intermediate-conductance Ca²⁺-activated K⁺ channel K_Ca3.1. In inside-out experiments, 10 μM NS309 shifted the calcium EC₅₀ from 430 to 31 nM. In whole-cell experiments, changing free intracellular calcium from 250 nM to 3 μM decreased the EC₅₀ of NS309 from 74 to 8.6 nM. We further observed that NS309 could elicit greater responses than saturating calcium, making it a "superagonist." Molecular modeling suggested 2 possible binding sites for NS309 in K_Ca3.1, which we probed by mutagenesis and determined that NS309 is binding in the interface between the S₄₅A segment of the intracellular S4-S5 linker and the N-lobe of the channel-associated calmodulin. Molecular dynamic simulations revealed that NS309 pushes several water molecules out of the interface pocket, establishes stable contacts with S181 and L185 in the S₄₅A segment of K_Ca3.1 and E54 in calmodulin, and promotes longer sustained widening of the inner gate of K_Ca3.1 at V282 in the S6 segment. Polar substitutions of the hydrophobic-gating residues V282 and A279 resulted in constitutively open channels that could not be further potentiated by NS309, suggesting that NS309 produces its agonistic effects by increasing the open probability of the inner gate of K_Ca3.1. SIGNIFICANCE STATEMENT: The publication of the full-length cryo-electron microscopy structure of the intermediate-conductance Ca²⁺-activated K⁺ channel K_Ca3.1 suggested that the previously reported binding site of NS309 (6,7-dichloro-1H-indole-2,3-dione-3-oxime) was a crystallization artifact because this structure only included the C-terminus and the channel-associated calmodulin. This study demonstrates that the true binding site of NS309 is located between the S4 and S5 linker of K_Ca3.1 and the N-lobe of calmodulin. NS309 acts as a stabilizing force within the gating interface and increases the open probability of the inner hydrophobic gate.

Widely distributed in all kingdoms of life, voltage sensors in the membrane serve important functions via their movements driven by changes in voltage across the membrane (membrane potential). A voltage sensor domain contains 4 transmembrane segments (S1-S4). The S1-S3 helices form a hydrophobic constriction site (HCS, also known as the gating charge transfer center) that spans roughly one-third of the membrane thickness. Flanked by aqueous vestibules connected to the extracellular solution above the HCS or cytoplasmic solution below the HCS, the HCS forms a gating pore for the S4 segment bearing multiple basic residues. Membrane potential changes cause S4 to move through the HCS in a 3₁₀ helical conformation. This S4 translocation generates a gating current as the positively charged S4 basic residues traverse the membrane electric field, transferring these gating charges from one aqueous vestibule to the other. For voltage-gated ion channels with their voltage sensor domains connected to pore domains, the HCS in the voltage sensor domain allows S4 but not ions to go through, while the channel pore formed by the pore domains mediates ion permeation. Voltage sensor mutations could result in ω currents that are conducted through the gating pore of mutant voltage-gated ion channels. These ω currents may cause pathological consequences in patients with periodic paralysis. Besides voltage-gated ion channels, the sperm-specific Na⁺/H⁺ exchanger and voltage-sensing phosphatases contain voltage sensors for membrane potential regulation. Notably, voltage-gated proton channels that are important for pH homeostasis are formed solely by the voltage sensor domain, which mediates proton permeation. SIGNIFICANCE STATEMENT: Voltage sensors mediate voltage regulation of ion channels, transporters, and phosphatases. The voltage sensor domain composed of 4 transmembrane segments (S1-S4) focuses the membrane electric field to the hydrophobic constriction site. To mediate voltage regulation, S4 basic residues within a 3₁₀ helix move across the hydrophobic constriction site without concurrent ion flow through this gating pore. As a counterexample, voltage-gated proton channels are formed by the voltage sensor to mediate proton permeation. These ingeniously engineered voltage sensors are conserved throughout evolution.

Action potentials of individual nerve axons are the electrical signals that propagate nervous information quickly around the brain and the body. This essay discusses milestones, from the definition of electricity in 1600 to the recent elucidation of the molecular structures of ion channels and membrane proteins that underlie action potential initiation and propagation. There were several key steps. The theory of electricity and electromagnetism had to be developed enough to allow discovery and measurement of animal electricity by biophysically minded physiologists. The theory of ions and electrochemistry had to be developed enough to allow prediction and verification of an ionic basis for animal electricity. Methods to amplify electrical signals with vacuum tubes and transistors were required for quantitative measurement and display of the action potentials and currents. Physiologists had to move from extracellular recording using nerve trunks to intracellular recording using single nerve fibers. Electronic feedback and mathematical modeling were needed to recognize the conductance changes of nerve membranes during activity. Pharmacology with neurotoxins allowed recognition of underlying voltage-gated ion channels. Protein purification, cloning, and sequencing identified the molecular basis of ion channels, and atomic structures showed in graphic detail how they work. SIGNIFICANCE STATEMENT: This is a brief scientific history of the action potential, the quintessential electrical message of our nerves. As with other histories in biology, this one reiterates that major scientific advances depend on advances in physics and physical chemistry, development of the right preparations and instruments, and the experimental genius and conceptual insights of clever scientists and their students.

The pregnane X receptor (PXR), a ligand-activated nuclear receptor, regulates the transcription of several genes that encode many enzymes and transporters related to drug metabolism. PXR also performs an important role as a physiological sensor in the modulation of endobiotic metabolism for hormones, bile acids, cholesterol, fatty acids, and glucose. Dysregulation of these PXR-mediated pathways is implicated in the progression of metabolic dysfunction-associated steatohepatitis (MASH), contributing to the complex interplay of factors involved in chronic liver disease development and exacerbation affecting millions worldwide. This review highlights the current knowledge of PXR expression and its role in endobiotic metabolism related to MASH development, which is associated with diverse causes and dire outcomes. This review focuses on elucidating the molecular pathways associated with PXR activation directly or indirectly and PXR interaction with other regulatory factors. Although there is still much to comprehend about the intricate details of these pathways, the conclusion is drawn that PXR exerts a crucial role in the pathological and physiological pathways of hepatic cellular processes, which holds promise as a potential pharmacological target for exploring novel therapeutic approaches for MASH treatment and/or prevention. SIGNIFICANCE STATEMENT: The pregnane X receptor (PXR) plays a fundamental role in regulating gene expression involved in xenobiotic and endobiotic metabolism. Dysregulation of PXR-mediated pathways is related to the development of metabolic dysfunction-associated steatohepatitis. The ligand-independent pathways regulating PXR hepatic functions through phosphorylation shed light on possible indirect molecular mechanisms and pathways that regulate PXR activity and function. Understanding these pathways may provide insight into new pharmaceutical interventions for metabolic dysfunction-associated steatohepatitis development.

Organic anion transporting polypeptide (OATP) 1B1 is crucial for hepatic uptake of many drugs and endogenous substrates. The clinically relevant OATP1B1 c.521 T>C (V174A) polymorphism exhibits reduced transport activity in vitro and in vivo in humans. Previously, we reported increased total phosphorylation of V174A-OATP1B1 compared to wild-type (WT)-OATP1B1, although the differentially phosphorylated sites remain to be identified. Lysine-acetylation, a key posttranslational modification (PTM), has not been investigated in OATP1B1. This study aimed to identify differential PTMs of WT-OATP1B1 and V174A-OATP1B1 by quantitatively comparing the relative abundance of modified peptides using liquid chromatography-tandem mass spectrometry-based proteomics and to assess the impact of these PTMs on OATP1B1 transport function using [³H]-estradiol-17-β-D-glucuronide as substrate in transporter-expressing human embryonic kidney 293 cells. We discovered that OATP1B1 is lysine-acetylated at 13 residues. Compared to WT-OATP1B1, V174A-OATP1B1 has increased concurrent phosphorylation at S659 and S663 and concurrent phosphorylation (at S659 and S663) and lysine-acetylation (at K650) (P < .05). Variants mimicking concurrent phosphorylation (S659E-S663E-OATP1B1) and concurrent phosphorylation and acetylation (K650Q-659E-S663E-OATP1B1) both demonstrated reduced substrate transport by 0.86 ± 0.055-fold and 0.65 ± 0.047-fold of WT-OATP1B1 (both P < .05), respectively. Single-site mimics of phosphorylation or lysine-acetylation at K650, S659, and S663 did not affect OATP1B1 transport function, indicating cooperative effects on OATP1B1 by concurrent PTMs. All variants and WT-OATP1B1 were primarily localized to the plasma membrane and colocalized with plasma membrane protein Na/K-ATPase as determined by immunofluorescent staining and confocal microscopy. The current study elucidates a novel mechanism in which concurrent serine-phosphorylation and lysine-acetylation impair OATP1B1-mediated transport, suggesting potential interplay between these PTMs in regulating OATP1B1. SIGNIFICANCE STATEMENT: Understanding organic anion transporting polypeptide (OATP1B1) regulation is key to predicting altered drug disposition. The Val174Ala-OATP1B1 polymorphism exhibits reduced transport activity and is the most effective predictor of statin-induced myopathy. Val174Ala-OATP1B1 was found to be associated with increased serine-phosphorylation at Ser659 and Ser663 and lysine-acetylation at Lys650; concurrent PTMs at these sites reduce OATP1B1 function. These findings revealed a novel mechanism involved in transporter regulation, suggesting potential interplay between these PTMs in governing hepatic drug transport and response.