Journal of General Physiology最新文献

Numerous elements involved in shear stress-induced signaling have been identified, recognizing their functions as mechanotransducing ion channels situated at cellular membranes. This form of mechanical signaling relies on transmembrane proteins and cytoplasmic proteins that restructure the cytoskeleton, contributing to mechanotransduction cascades. Notably, blood flow generates mechanical forces that significantly impact the structure and remodeling of blood vessels. The primary regulation of blood vessel responses occurs through hemodynamic forces acting on the endothelium. These mechanical events intricately govern endothelial biophysical, biochemical, and genetic responses. Endothelial cells, positioned on the intimal surface of blood vessels, have the capability to express components of the glycocalyx. This endothelial structure emerges as a pivotal factor in mechanotransduction and the regulation of vascular tone. The endothelial glycocalyx assumes diverse roles in both health and disease. Our findings propose a connection between the release of specific enzymes from the rat liver and variations in the hepatic blood flow/mass ratio. Importantly, this phenomenon is not correlated with liver necrosis. Consequently, this review serves as an exploration of the potential involvement of membrane proteins in a hypothetical mechanotransducing phenomenon capable of controlling the release of liver enzymes.

Precise matching of energy substrate delivery to local metabolic needs is essential for the health and function of all tissues. Here, we outline a mechanistic framework for understanding this critical process, which we refer to as electro-metabolic signaling (EMS). All tissues exhibit changes in metabolism over varying spatiotemporal scales and have widely varying energetic needs and reserves. We propose that across tissues, common signatures of elevated metabolism or increases in energy substrate usage that exceed key local thresholds rapidly engage mechanisms that generate hyperpolarizing electrical signals in capillaries that then relax contractile elements throughout the vasculature to quickly adjust blood flow to meet changing needs. The attendant increase in energy substrate delivery serves to meet local metabolic requirements and thus avoids a mismatch in supply and demand and prevents metabolic stress. We discuss in detail key examples of EMS that our laboratories have discovered in the brain and the heart, and we outline potential further EMS mechanisms operating in tissues such as skeletal muscle, pancreas, and kidney. We suggest that the energy imbalance evoked by EMS uncoupling may be central to cellular dysfunction from which the hallmarks of aging and metabolic diseases emerge and may lead to generalized organ failure states-such as diverse flavors of heart failure and dementia. Understanding and manipulating EMS may be key to preventing or reversing these dysfunctions.

JGP study (Bernas et al. 2024. J. Gen. Physiol.https://doi.org/10.1085/jgp.202313436) suggests that, by altering microtubule dynamics, persistent PKA activation promotes the delivery of Nav1.5 channels to intercalated discs.

Neuromuscular acetylcholine receptors (AChRs) are hetero-pentameric, ligand-gated ion channels. The binding of the neurotransmitter acetylcholine (ACh) to two target sites promotes a global conformational change of the receptor that opens the channel and allows ion conduction through the channel pore. Here, by measuring free-energy changes from single-channel current recordings and using molecular dynamics simulations, we elucidate how a constricted hydrophobic region acts as a "gate" to regulate the channel opening in the pore of AChRs. Mutations of gate residues, including those implicated in congenital myasthenia syndrome, lower the permeation barrier of the channel substantially and increase the unliganded gating equilibrium constant (constitutive channel openings). Correlations between hydrophobicity and the observed free-energy changes, supported by calculations of water densities in the wild-type versus mutant channel pores, provide evidence for hydrophobic wetting-dewetting transition at the gate. The analysis of a coupled interaction network provides insight into the molecular mechanism of closed- versus open-state conformational changes at the gate. Studies of the transition state by "phi"(φ)-value analysis indicate that agonist binding serves to stabilize both the transition and the open state. Intersubunit interaction energy measurements and molecular dynamics simulations suggest that channel opening involves tilting of the pore-lining M2 helices, asymmetric outward rotation of amino acid side chains, and wetting transition of the gate region that lowers the barrier to ion permeation and stabilizes the channel open conformation. Our work provides new insight into the hydrophobic gate opening and shows why the gate mutations result in constitutive AChR channel activity.

Voltage-dependent and Ca2+-dependent inactivation (VDI and CDI, respectively) of CaV channels are two biologically consequential feedback mechanisms that fine-tune Ca2+ entry into neurons and cardiomyocytes. Although known to be initiated by distinct molecular events, how these processes obstruct conduction through the channel pore remains poorly defined. Here, focusing on ultrahighly conserved tryptophan residues in the interdomain interfaces near the selectivity filter of CaV1.3, we demonstrate a critical role for asymmetric conformational changes in mediating VDI and CDI. Specifically, mutagenesis of the domain III-IV interface, but not others, enhanced VDI. Molecular dynamics simulations demonstrate that mutations in distinct selectivity filter interfaces differentially impact conformational flexibility. Furthermore, mutations in distinct domains preferentially disrupt CDI mediated by the N- versus C-lobes of CaM, thus uncovering a scheme of structural bifurcation of CaM signaling. These findings highlight the fundamental importance of the asymmetric arrangement of the pseudotetrameric CaV pore domain for feedback inhibition.

Human voltage-gated sodium (hNaV) channels are responsible for initiating and propagating action potentials in excitable cells, and mutations have been associated with numerous cardiac and neurological disorders. hNaV1.7 channels are expressed in peripheral neurons and are promising targets for pain therapy. The tarantula venom peptide protoxin-II (PTx2) has high selectivity for hNaV1.7 and is a valuable scaffold for designing novel therapeutics to treat pain. Here, we used computational modeling to study the molecular mechanisms of the state-dependent binding of PTx2 to hNaV1.7 voltage-sensing domains (VSDs). Using Rosetta structural modeling methods, we constructed atomistic models of the hNaV1.7 VSD II and IV in the activated and deactivated states with docked PTx2. We then performed microsecond-long all-atom molecular dynamics (MD) simulations of the systems in hydrated lipid bilayers. Our simulations revealed that PTx2 binds most favorably to the deactivated VSD II and activated VSD IV. These state-specific interactions are mediated primarily by PTx2's residues R22, K26, K27, K28, and W30 with VSD and the surrounding membrane lipids. Our work revealed important protein-protein and protein-lipid contacts that contribute to high-affinity state-dependent toxin interaction with the channel. The workflow presented will prove useful for designing novel peptides with improved selectivity and potency for more effective and safe treatment of pain.

During chronic stress, persistent activation of cAMP-dependent protein kinase (PKA) occurs, which can contribute to protective or maladaptive changes in the heart. We sought to understand the effect of persistent PKA activation on NaV1.5 channel distribution and function in cardiomyocytes using adult rat ventricular myocytes as the main model. PKA activation with 8CPT-cAMP and okadaic acid (phosphatase inhibitor) caused an increase in Na+ current amplitude without altering the total NaV1.5 protein level, suggesting a redistribution of NaV1.5 to the myocytes' surface. Biotinylation experiments in HEK293 cells showed that inhibiting protein trafficking from intracellular compartments to the plasma membrane prevented the PKA-induced increase in cell surface NaV1.5. Additionally, PKA activation induced a time-dependent increase in microtubule plus-end binding protein 1 (EB1) and clustering of EB1 at myocytes' peripheral surface and intercalated discs (ICDs). This was accompanied by a decrease in stable interfibrillar microtubules but an increase in dynamic microtubules along the myocyte surface. Imaging and coimmunoprecipitation experiments revealed that NaV1.5 interacted with EB1 and β-tubulin, and both interactions were enhanced by PKA activation. We propose that persistent PKA activation promotes NaV1.5 trafficking to the peripheral surface of myocytes and ICDs by providing dynamic microtubule tracks and enhanced guidance by EB1. Our proposal is consistent with an increase in the correlative distribution of NaV1.5, EB1, and β-tubulin at these subcellular domains in PKA-activated myocytes. Our study suggests that persistent PKA activation, at least during the initial phase, can protect impulse propagation in a chronically stressed heart by increasing NaV1.5 at ICDs.

Calcium (Ca2+) extrusion is an essential function of the enamel-forming ameloblasts, providing Ca2+ for extracellular mineralization. The plasma membrane Ca2+ ATPases (PMCAs) remove cytosolic Ca2+ (cCa2+) and were recently shown to be efficient when ameloblasts experienced low cCa2+ elevation. Sodium-calcium (Na+/Ca2+) exchange has higher capacity to extrude cCa2+, but there is limited evidence on the function of the two main families of Na+/Ca2+ exchangers in enamel formation. The purpose of this study was to analyze the function of the NCX (coded by SLC8) and the K+-dependent NCKX (coded by SLC24) exchangers in rat ameloblasts and to compare their efficacy in the two main stages of enamel formation: the enamel forming secretory stage and the mineralizing or maturation stage. mRNA expression profiling confirmed the expression of Slc8 and Slc24 genes in enamel cells, Slc24a4 being the most highly upregulated transcript during the maturation stage, when Ca2+ transport increases. Na+/Ca2+ exchange was analyzed in the Ca2+ influx mode in Fura-2 AM-loaded ameloblasts. We show that maturation-stage ameloblasts have a higher Na+/Ca2+ exchange capacity than secretory-stage cells. We also show that Na+/Ca2+ exchange in both stages is dominated by NCKX over NCX. The importance of NCKX function in ameloblasts may partly explain why mutations in the SLC24A4 gene, but not in SLC8 genes, result in enamel disease. Our results demonstrate that Na+/Ca2+ exchangers are fully operational in ameloblasts and that their contribution to Ca2+ homeostasis increases in the maturation stage, when Ca2+ transport need is higher.

TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine-1-phosphate (S1P) receptors. In this study, we test non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo, for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, though this likely occurrs through multiple molecular targets. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct anti-inflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo. Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting which demonstrate potent, polymodal anti-inflammatory activities ex vivo and in vivo.