Journal of General Physiology最新文献

Lopez-Mateos et al. show that AlphaFold 2 can generate structural ensembles of NaV channels and that β-subunits and calmodulin reshape these ensembles, while emphasizing that these are testable structural hypotheses, not actual thermodynamic populations.

Cholesterol, abundantly present in distinct plasma membrane pools, is a critical modulator of ion channel function, including hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that regulate the excitability of dorsal root ganglion (DRG) nociceptor neurons. Depletion of membrane cholesterol potentiated HCN channel opening and accelerated activation kinetics, whereas cholesterol supplementation reduced channel opening and slowed activation kinetics. However, the relative contributions of cholesterol that organizes ordered membrane domains (OMDs) versus freely accessible cholesterol pools to HCN channel modulation remain unknown. Using fluorescence lifetime imaging microscopy, FRET and fluorescence anisotropy techniques, we examined how supplementing cholesterol alters plasma membrane properties and HCN gating in nociceptor DRG neurons. We uncovered a process of sequential, stepwise membrane remodeling: an initial phase with OMD expansion and a rapid rise in free cholesterol, followed by continued accumulation of free cholesterol without further OMD expansion. Notably, the slope factor of the HCN G-V relationship is sensitive to OMD expansion but remains unaffected by changes in free cholesterol. Other gating parameters, including open probability and activation kinetics, were affected by elevating free cholesterol. In a rat model of nerve injury, where DRG neurons exhibit reduced free cholesterol levels and smaller OMDs, HCN channel modulation by cholesterol involves contributions from both OMD expansion and free cholesterol accumulation. In contrast, in naïve DRG neurons-characterized by high cholesterol and large OMDs-modulation occurs mostly via increased free cholesterol. These findings provide mechanistic insights into cholesterol-dependent modulation of ion channels and its role in neuropathic pain.

Regulation of the agonist sensitivity of neurotransmitter receptors is critical for proper functioning of neuronal circuits and is, therefore, conserved across evolutionary time. Mutations that alter agonist sensitivity are often pathological in humans. A brain-expressing nicotinic acetylcholine receptor (nAChR) from the frog Xenopus tropicalis shows ∼20× greater sensitivity to ACh as orthologs from human, chickens, and other frogs prompt us to examine the molecular basis for this extreme sensitivity. We identified a single amino acid substitution in the third transmembrane domain (M3) of the X. tropicalis α4 nAChR subunit, F294 (S in other vertebrate orthologs), that confers the high sensitivity. Surprisingly, we noted variation at this site in sequences deposited in NCBI, suggesting either allelic variation or RNA editing. By sequencing genomic DNA and mRNA (cDNA) from the same individuals from two different colonies of X. tropicalis, we determined that a possible source of this variation is RNA editing. The unedited receptor from X. tropicalis (S294) has a similar ACh sensitivity as those from other vertebrates. Further work must be done to examine possible adaptations of edited receptors and if the frog's brain compensates for an increase in sensitivity since increases in agonist sensitivity lead to pathology in humans.

Voltage-gated sodium channels (Navs) selectively conduct Na+ to generate action potentials. Na+ permeates Navs with significantly higher efficiency than many other cations, but Li+ can also permeate Navs to an extent comparable with Na+. Li+ in the blood is known to enter cells via Navs and to have a beneficial effect on various neuropathies. However, the molecular basis of the high Li+ selectivity of Navs was unclear. In this study, using a prokaryotic Nav, we successfully created the first Nav mutant to be more selective for Li+ than for Na+. Electrophysiological and crystallographic analyses suggested the critical determinants of high Li+ selectivity: the strong electrostatic interaction between the ion pathway and hydrated ions, and the smaller number of hydration water exchanges within the ion pathway. Additionally, the extensive interactions around the ion pathway were shown to support monovalent cation selectivity. New drug directions based on the molecular basis for Li+ permeation may target various neurological disorders and could clarify the broader biological effects of lithium.

Molecular oxygen exists in three electronic states: the triplet ground state and two singlet (1O2) excited states. Under physiological conditions, 1O2 can be produced either through photodynamic processes, which require light, photosensitizer, and oxygen, or via metabolic reactions involving enzymes and other reactive oxygen species (ROS). 1O2 readily reacts with biomacromolecules, however, its volatile chemical nature and the lack of precise working models hamper the study of its molecular mechanism and physiological significance. Here we report that human CNG channels from rod photoreceptors are very sensitive to the process of photodynamic modification (PDM). Multiple lines of evidence indicate 1O2 is the major player in PDM, including the application of a genetically encoded photosensitizer, a popularly used photosensitizer to produce 1O2, and two known quenchers for 1O2. The 1O2-mediated modification increases the opening of hCNGA1 in the absence or under subsaturating concentrations of cyclic guanosine monophosphate (cGMP), and in conjunction with ligand gating, acts synergistically on channel opening. Mutagenesis and mass spectroscopy (MS) analysis reveal key residues affecting the PDM process. Taken together, through tackling the PDM of rod photoreceptor CNG channels, this study provides essential insights into the modification of protein molecules by 1O2, a ubiquitous and potentially critical signaling molecule.

Since the discovery of the cardiac isoform of myosin-binding protein-C (cMyBP-C), there has been continued interest in how cMyBP-C impacts cardiac function in both health and disease. cMyBP-C is a regulatory protein in the sarcomere that controls beat-to-beat changes in contractility in response to dynamic environmental demands placed upon the heart. Changes in force production during the contractile cycle are modulated through interactions of cMyBP-C with myosin and actin. Post-translational modifications (PTMs) of cMyBP-C, of which phosphorylation has received the most attention, are critical to the function of cMyBP-C in the healthy heart and is affected in many disease states. While each of the PTMs that will be discussed in this review have known and often widespread effects on important cellular processes spanning transcriptional regulation, cell signaling, and metabolism, their impact on cMyBP-C function remains poorly understood and in some cases unverified. This Review focuses on the current understanding of cMyBP-C PTMs, namely phosphorylation, S-glutathionylation, S-nitrosylation, acetylation, citrullination, carbonylation, and O-GlcNAcylation. The potential for PTMs to exert wide ranging and likely nuanced effects may influence the range of cMyBP-C's response to varied conditions and may offer opportunities to identify novel therapeutic paradigms in the setting of disease.

The sarcomeric protein titin plays a central role in thick filament structure and function through its modular A-band domains, including the understudied P-zone, which links the C-zone to the M-band. To investigate the first four domains of titin's P-zone (A164-A167), we deleted them in a mouse model (TtnΔA164-167). Echocardiography and cardiomyocyte mechanics revealed mild changes to diastolic function and enlargement of the heart, but preserved contractility. The EDL muscle showed contractile deficits at the whole muscle level and increased passive stiffness at the myofiber level. Immunoelectron and super-resolution microscopy revealed altered thick filament architecture, including a ∼40-nm shift of titin and myosin binding protein-C epitopes toward the M-band, disruption of titin's α and β conformations, and shorter thick filaments. The structural changes are consistent with the loss of a myosin helical repeat. These findings establish a key structural role of titin's P-zone domains A164-A167 in templating thick filament protein arrangement, including the importance of titin's α and β conformations.

The effect of a potentiating drug on ion channel function is typically evaluated by comparing current responses to the control agonist in the presence and absence of the potentiator. Differences in ratios of responses are then taken as proof of distinct potentiation properties when comparing modulation by different compounds. In these experiments, the concentration of the agonist is typically kept low to generate a small fractional control response. The precise relative magnitude of the control response is, however, not standardized among labs and can range from a concentration producing a response equal to just 2% of maximal (EC2) to over EC25 in different studies. Here, we have investigated the relationship between the magnitude of the control response and the expected response ratio. As the EC value of the control response increases, the ratio of responses to agonist in the presence and absence of the potentiator decreases. We provide equations to calculate the expected response ratios at different levels of control responses and free energy changes at different response ratios. Lastly, we discuss the effect of the value of EC of the control response on the efficacy of negative allosteric inhibitors.

Claudin-15 (CLDN15) molecules form channels that directly regulate cation and water transport. In the gastrointestinal tract, this transport indirectly impacts nutrient absorption. However, the mechanisms governing ion transport through these channels remain poorly understood. We addressed this question by building on our previous cell culture studies and an all-atom molecular dynamics simulation model of CLDN15. By mutating D55 to a bulkier glutamic acid or neutral amino acid asparagine, our in vitro measurements showed that the D55E mutation decreased charge selectivity and favored small ion permeability, while the D55N mutation led to reduced charge selectivity without markedly altering size selectivity. By establishing a simplified (reduced) CLDN15 molecular dynamics model that excludes nonessential transmembrane regions, we were able to probe how D55 modified cation dehydration, charge interaction, and permeability. These results provide novel insight into organization of the CLDN15 selectivity filter and suggest that D55 plays a dual role in shaping both electrostatic and steric properties of the pore, but its electrostatic role is more prominent in determining CLDN15 cation permeability. This knowledge can be used toward the development of effective strategies to modulate CLDN15 function. The experimental approach established can be further extended to study the function of other claudin channels. Together, these advancements will help us to modulate tight junctions to promote human health.

Permeabilized muscle fibers have a chemically disturbed sarcolemma that allows for the mixing of the extra- and intracellular environments and is important for a large variety of experimental methods. The experimental tools and skillsets used to study muscle mechanics vary widely between groups and are often underreported in published methodologies. More accessible details help improve the transparency of the method and provide primary reference material. To that end, we use our firsthand experiences to provide a guide for the preparation and use of permeabilized fibers. We focus on tissue collection, experimental apparatus design and function, practical considerations for handling preparations during an experiment, and detail some key changes to the structure of permeabilized samples. We further suggest ways scientists can take advantage of emerging technologies to increase experimental throughput, decrease experimental error, and support (or improve) data quality.