Nicotinic acetylcholine receptors (nAChRs) are primary targets of insecticides. While most mammalian nAChRs form functional channels in heterologous systems, insect nAChRs remain notoriously difficult to reconstitute. Here, we co-expressed insect/human subunit chimeras to enable functional analysis of heteromeric insect nAChR ligand‑binding sites in Xenopus laevis oocytes. First, EGFP-tagged human/cockroach α7 chimeras (Hα7/Pα7) were generated by exchanging the signal peptide (SP), extracellular (ECD), transmembrane (TMD), and intracellular (L3-4) domains. While all constructs were translated into protein, Hα7-like acetylcholine-evoked currents were only observed when human N- and C-terminal ECD and ICD were present, indicating that these domains impose insect-specific constraints on homomeric nAChR formation. Second, we generated heteromeric chimeric receptors by fusing insect-derived ECDs onto hα7 subunits. Co-expression of Pα6/Hα7L3-4 and Pα7ECD/Hα7 chimeras resulted in current amplitudes < 1 µA. Co-expression of the respective bee Aα5ECD/Hα7, Aα6ECD/Hα7, and Aα7ECD, CT/Hα7 chimeras yielded currents > 1 µA. α-Bungarotoxin inhibited diheteromeric Pα6α7 chimeras (IC50 49 nM) and triheteromeric Aα5/α6/α7 chimeras (IC50 51.2 nM). Third, this approach was applied to cockroach Pα2 and Pβ1 subunits. Replacement of the Pα2 ICD and the Pβ1 TMD/ICD with homologous regions from Hα2 and Hβ2 subunits and co-expression with Pα1 resulted in a ~ 100-fold current increase (2.6 µA) compared to the wild‑type Pα1α2β1 receptor. The chimeric receptor was activated by neonicotinoids (EC50 clothianidin: 24.1 nM, EC50 thiacloprid: 13.6 nM) and α-bungarotoxin-sensitive (IC50: 36.6 nM). In conclusion, we demonstrate that replacement of insect ICD or ICD and TMD enables formation of functional insect-like heteromeric nAChRs without co-expression of chaperones.
Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome in which diastolic dysfunction develops against a background of interacting mechanisms: inflammation, oxidative stress, impaired calcium homeostasis, myocardial fibrosis, coronary microvascular dysfunction, mitochondrial impairment, and neurohumoral activation. Effective mechanism-targeted therapies remain limited. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed in cardiac sensory nerves, vascular endothelial cells, immune cells, and possibly other cardiovascular cell populations. It responds to diverse physical and chemical stimuli and may influence calcium handling, inflammatory signaling, oxidative stress, myocardial fibrosis, mitophagy, and ferroptosis. Preclinical studies have linked altered TRPV1 expression or activity to myocardial remodeling, impaired relaxation, microvascular dysfunction, and heart failure progression, but direct evidence in HFpEF is still sparse. This review summarizes the biological properties of TRPV1, considers how its signaling might intersect with HFpEF pathophysiology, reviews experimental TRPV1-targeted interventions, and identifies the evidence needed before TRPV1 can be evaluated as a potential therapeutic target.
KV7.4 channels are important regulators of membrane potential and excitability in multiple tissues and are emerging as key effectors of steroid hormone signaling. Here, 17β-estradiol (E2) was examined as a modulator of recombinant KV7.4 channels expressed in HEK293 cells. Whole-cell patch-clamp recordings showed that acute application of E2 reduced KV7.4 current density. At nanomolar concentrations, both E2 and the alternate GPER1 agonist aldosterone inhibited KV7.4 currents only when G protein-coupled estrogen receptor 1 (GPER1) was co-expressed, indicating a receptor-dependent mechanism. Pharmacological inhibition showed that phospholipase C (PLC) and protein kinase C (PKC), but not adenylyl cyclase, contribute to GPER1-mediated inhibition of KV7.4. In contrast, equivalent experiments performed with KV7.5 channels showed no significant effect of E2 despite GPER1 co-expression, suggesting subtype-selective regulation within the KV7 family. Surface biotinylation experiments suggest that GPER1 co-expression reduced surface KV7.4 expression, with a further reduction observed following E2 treatment. Finally, both E2 and aldosterone negatively regulate the sensitivity of KV7.2-5 activator ML213-mediated relaxation in arteries from female rats. Together, these findings identify KV7.4 as a downstream effector of rapid GPER1-dependent estrogen signaling and suggest that steroid status may influence KV7-targeted therapeutic responses.
Store-operated calcium entry (SOCE) mediated by STIM and Orai proteins is a fundamental Ca2+ influx mechanism that critically regulates intracellular calcium homeostasis and participates in cardiovascular pathophysiology. Upon endoplasmic reticulum Ca2+ store depletion, STIM1/2 activate plasma membrane Orai1/3 channels, initiating Ca2+ entry that drives vasoconstriction, smooth muscle proliferation, platelet activation, and cardiac hypertrophy. Dysregulated SOCE is closely associated with hypertension, atherosclerosis, pulmonary hypertension, and thromboembolic disorders. However, SOCE is not a simple binary pathway but operates within a complex regulatory network. Beyond the core STIM-Orai axis, auxiliary proteins including transient receptor potential canonical 1 (TRPC1), tetraspanin 18 (Tspan18), tropomyosin 3 (TPM3), SOCE-associated regulatory factor (SARAF), and A-kinase anchoring protein 79/150 (AKAP79/150) modulate SOCE amplitude, kinetics, and downstream signaling in a cell- and context-dependent manner. Moreover, the functional consequences of SOCE are highly heterogeneous: Orai1 protects adult cardiomyocytes but promotes pathological hypertrophy in neonatal cells, posing a therapeutic dilemma. Although preclinical studies have shown efficacy of SOCE inhibitors, clinical translation remains hindered by poor isoform selectivity, suboptimal pharmacokinetics, lack of tissue-specific delivery, disease-stage-dependent effects, and absence of validated biomarkers. Importantly, recent evidence has definitively ruled out amlodipine-induced CRAC channel activation at therapeutic concentrations, confirming it as an experimental artifact. This review systematically summarizes the molecular complexity, functional diversity, and translational barriers of STIM/Orai-mediated SOCE, aiming to inform precision therapeutic strategies for cardiovascular diseases.
Hyperexcitability is a biomarker of early-stage Alzheimer’s Disease (AD) and hastens cognitive decline later in its course. Mechanistic target of rapamycin (mTOR) signaling contributes to the slope of this trajectory, as evidenced by early increased brain expression and the rescue of hyperexcitability by genetic deletion of mTOR complex 2 (mTORC2); however, a molecular mechanism directly linking mTOR signaling to membrane hyperexcitability in early-stage AD remains elusive. Here, we show that hyperactive mTOR signaling stimulates the voltage-gated Na+ channel 1.2 (Nav1.2), a previously identified downstream phosphorylation target of mTORC2 and a key regulator of membrane electrogenesis. Augmented Nav1.2 channel function induced by hyperactive mTOR signaling requires the action of mTORC2 and is selective among major brain Nav channel isoforms. In a murine AD model, neocortical pyramidal neurons display augmented Nav1.2 channel function and hyperexcitability through a mechanism that requires mTORC2 activity. These results highlight the mTORC2-Nav1.2 interaction as a therapeutic target for attenuating pathogenic hyperexcitability in early-stage AD.
