Search Results (95)

The family of voltage-dependent anion channels (VDACs) comprises three isoforms (VDAC-1, VDAC-2, VDAC-3). VDACs have been extensively described as localised in the outer mitochondrial membrane where they are involved in the exchange of ions, metabolites, and ATP/ADP between mitochondria and cytosol. The VDAC interacts with disease-specific proteins and thus regulates the mitochondrial function and controls the cellular energy resources, explaining its involvement in cell death and apoptosis. In addition, VDAC-1 and -2 can also be found at other cellular locations such as in the sarcoplasmic reticulum, in the endoplasmic reticulum, as well as in the plasma membrane. Through single-channel pore regulation, oligomerisation, or changed expression levels the VDAC is involved in different neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis, Huntington’s disease, and others. Here, we critically summarise current discussions about the VDAC as a common key player for these diseases. We suggest that the VDAC acts as a transmembrane multifunctional regulatory protein which might serve as a pharmacological target for the development of novel drugs against neurodegenerative diseases such as the application of recombinant antibody technology. Full article

Mammalian spermatozoa undergo a series of physiological and biochemical changes in the oviduct that lead them to acquire the ability to fertilize eggs. During their transit in the oviduct, spermatozoa face a series of environmental changes that can affect sperm viability. A series of ion channels and transporters, as well as the sperm cytoskeleton, allow spermatozoa to remain viable and functional. Cl⁻ channels such as TMEM16A (calcium-activated chloride channel), CFTR (cystic fibrosis transmembrane conductance regulator), and ClC3 (chloride voltage-gated channel 3) are some of the ion transporters involved in maintaining cellular homeostasis. They are expressed in mammalian spermatozoa and are associated with capacitation, acrosomal reaction, and motility. However, little is known about their role in maintaining sperm volume. Therefore, this study aimed to determine the mechanism through which TMEM16A maintains sperm volume during capacitation. The effects of TMEM16A were compared to those of CFTR and ClC3. Spermatozoa were capacitated in the presence of specific TMEM16A, CFTR, and ClC3 inhibitors, and the results showed that only TMEM16A inhibition increased acrosomal volume, leading to changes within the acrosome. Similarly, only TMEM16A inhibition prevented actin polymerization during capacitation. Further analysis showed that TMEM16A inhibition also prevented ERK1/2 and RhoA activation. On the other hand, TMEM16A and CFTR inhibition affected both capacitation and spontaneous acrosomal reaction, whereas ClC3 inhibition only affected the spontaneous acrosomal reaction. In conclusion, during capacitation, TMEM16A activity regulates acrosomal structure through actin polymerization and by regulating ERK1/2 and RhoA activities. Full article

Voltage-gated Ca²⁺ (Ca_V) channels are transmembrane proteins comprising the pore-forming subunit Ca_Vα₁ and the ancillary proteins Ca_Vα₂δ and Ca_Vβ. They are expressed in various tissues, including the nervous system, where they regulate Ca²⁺ entry in response to membrane potential changes. The increase in intracellular Ca²⁺ allows for regulating cell excitability and releasing neurotransmitters, among other cellular events. Leucine-rich repeat kinase 2 (LRRK2) is a serine–threonine kinase involved in vesicular mobilization. Previously, it has been shown that LRRK2 regulates neurotransmission by phosphorylating the Ca_Vβ auxiliary subunit of the Ca_V2.1 (P/Q-type) presynaptic channels. However, it is unknown whether the kinase can regulate the activity of other Ca_V channel subtypes, such as Ca_V1.3 (L-type), which play a significant role in the excitability of dopaminergic neurons in the substantia nigra pars compacta (SNc) and whose dysregulation contributes to neurodegeneration in Parkinson’s disease (PD). Here, we found potential phosphorylation sites for LRRK2 in Ca_Vβ₃ and examined how these molecules interact. We used immunoprecipitation and electrophysiology in HEK-293 cells expressing recombinant Ca_V1.3 channels, both with and without wild-type LRRK2 or its LRRK2^G2019S mutation, which plays a role in familial PD through a possible gain-of-toxic-function mechanism. Our results show that LRRK2^G2019S significantly increases current density through Ca_V1.3 channels, and this effect depends on the presence of Ca_Vβ₃. Site-directed mutagenesis revealed that phosphorylation at S152 in the sequence of Ca_Vβ₃ is necessary and sufficient to explain the abnormal regulation of the channels mediated by LRRK2^G2019S. These data provide new insights into the molecular regulation that mutant LRRK2 may exert on L-type Ca_V1.3 channels, which determine pacemaker activity in dopaminergic neurons of the SNc and may, therefore, play a relevant role in the molecular pathophysiology of PD. Full article

Voltage-gated sodium (Nav) channels play a crucial role in initiating and propagating action potentials throughout the heart, muscles and nervous systems, making them targets for a number of drugs and toxins. While patch-clamp electrophysiology is considered the gold standard for measuring ion channel activity, its labor-intensive and time-consuming nature highlights the need for fast screening strategies to facilitate a preliminary selection of potential drugs or hazards. In this study, a high-throughput and cost-effective biosensing method was developed to rapidly identify specific agonists and inhibitors targeting the human Nav1.1 (hNav1.1) channel. It combines a red fluorescent dye sensitive to transmembrane potentials with CHO cells stably expressing the hNav1.1 α-subunit (hNav1.1-CHO). In the initial screening mode, the tested compounds were mixed with pre-equilibrated hNav1.1-CHO cells and dye to detect potential agonist effects via fluorescence enhancement. In cases where no fluorescence enhancement was observed, the addition of a known agonist veratridine allowed the indication of inhibitor candidates by fluorescence reduction, relative to the veratridine control without test compounds. Potential agonists or inhibitors identified in the initial screening were further evaluated by measuring concentration–response curves to determine EC₅₀/IC₅₀ values, providing semi-quantitative estimates of their binding strength to hNav1.1. This robust, high-throughput biosensing assay was validated through comparisons with the patch-clamp results and tested with 12 marine toxins, yielding consistent results. It holds promise as a low-cost, rapid, and long-term stable approach for drug discovery and non-target screening of neurotoxins. Full article

Somatic mutations are common in cancer, with only a few driving the progression of the disease, while most are silent passengers. Some mutations may hinder or even reverse cancer progression. The voltage-gated proton channel (H_V1) plays a key role in cellular pH homeostasis and shows increased expression in several malignancies. Inhibiting H_V1 in cancer cells reduces invasion, migration, proton extrusion, and pH recovery, impacting tumor progression. Focusing on HVCN1, the gene coding for the human voltage-gated proton channel (hH_V1), 197 mutations were identified from three databases: 134 missense mutations, 51 sense mutations, and 12 introducing stop codons. These mutations cluster in two hotspots: the central region of the N-terminus and the region coding for the S4 transmembrane domain, which contains the channel’s voltage sensor. Five somatic mutations within the S4 segment (R205W, R208W, R208Q, G215E, and G215R) were selected for electrophysiological analysis and MD simulations. The findings reveal that while all mutants remain proton-selective, they all exhibit reduced effective charge displacement and proton conduction. The mutations differentially affect hH_V1 kinetics, with the most pronounced effects observed in the two Arg-to-Trp substitutions. Mutation of the first voltage-sensing arginine (R1) to tryptophan (R205W) causes proton leakage in the closed state, accelerates channel activation, and diminishes the voltage dependence of gating. Except for R205W, the mutations promote the deactivated channel configuration. Altogether, these data are consistent with impairment of hH_V1 function by mutations in the S4 transmembrane segment, potentially affecting pH homeostasis of tumor cells. Full article

Pathogenic variants in KCNC3, which encodes the voltage-gated potassium channel Kv3.3, are associated with spinocerebellar ataxia type 13. SCA13 is a neurodegenerative disease characterized by ataxia, dysarthria and oculomotor dysfunction, often in combination with other signs and symptoms such as cognitive impairment. Known disease-causing variants are localized in the protein coding regions and predominantly in the transmembrane and voltage sensing domains. In a patient with an ataxic movement disorder and progressive cognitive decline, the c.-6C>A variant was detected in the Kozak sequence of KCNC3. The Kozak sequence is responsible for efficient initiation of translation. Functional analysis of the new c.-6C>A variant and the upstream 5’-UTR region of KCNC3 by luciferase assays, quantitative PCR and methylation analysis shows increased protein expression but no effect on transcription rate. Therefore, increased translation initiation of KCNC3 transcripts compared to wild-type Kozak sequence seems to be the cause of the increased expression. Variants in the regulatory elements of disease-causing genes probably play an underestimated role. Full article

Aims: Atrial fibrillation (AF) is the most common chronic/recurrent arrhythmia, which significantly impairs quality of life and increases cardiovascular morbidity and mortality. Therefore, the aim of the present study was to investigate the properties of three repolarizing potassium currents which were shown to contribute to AF-induced electrical remodeling, i.e., the transient outward (I_to), inward rectifier (I_K1) and acetylcholine-sensitive (I_K,ACh) potassium currents in isolated atrial myocytes obtained from dogs either with sinus rhythm (SR) or following chronic atrial tachypacing (400/min)-induced AF. Methods: Atrial remodeling and AF were induced by chronic (4–6 weeks of) right atrial tachypacing (400/min) in dogs. Transmembrane ionic currents were measured by applying the whole-cell patch-clamp technique at 37 °C. Results: The I_to current was slightly downregulated in AF cells when compared with that recorded in SR cells. This downregulation was also associated with slowed inactivation kinetics. The I_K1 current was found to be larger in AF cells; however, this upregulation was not statistically significant in the voltage range corresponding with atrial action potential (−80 mV to 0 mV). I_K,ACh was activated by the cholinergic agonist carbachol (CCh; 2 µM). In SR, CCh activated a large current either in inward or outward directions. The selective I_K,ACh inhibitor tertiapin (10 nM) blocked the outward CCh-induced current by 61%. In atrial cardiomyocytes isolated from dogs with AF, the presence of a constitutively active I_K,ACh was observed, blocked by 59% with 10 nM tertiapin. However, in “AF atrial myocytes”, CCh activated an additional, significant ligand-dependent and tertiapin-sensitive I_K,ACh current. Conclusions: In our dog AF model, I_to unlike in humans was downregulated only in a slight manner. Due to its slow inactivation kinetics, it seems that I_to may play a more significant role in atrial repolarization than in ventricular working muscle myocytes. The presence of the constitutively active I_K,ACh in atrial myocytes from AF dogs shows that electrical remodeling truly developed in this model. The I_K,ACh current (both ligand-dependent and constitutively active) seems to play a significant role in canine atrial electrical remodeling and may be a promising atrial selective drug target for suppressing AF. Full article

Arterial hypertension is a highly prevalent chronic disease worldwide, with several etiologies and treatments that may eventually have side effects or result in patients developing tolerance. There is growing interest in traditional medicine and functional foods to isolate biomolecules that could be useful as coadjuvants for treating several aliments. Pitaya, a desert fruit endemic in Mexico, is a rich source of bioactive molecules (betalains and phenolic compounds). In this work, the vasorelaxation properties of pitaya juice concentrate and fraction one were investigated using aortic and mesenteric rings from rats. The incubation of rings with pitaya juice concentrate or fraction one induced significant vasorelaxation, independent of the endothelium, and showed resistance to potassium channel blockers. This vasorelaxation was associated with the transmembrane influx of extracellular calcium through the vascular smooth muscle cells, with an inhibitory effect on the voltage-dependent calcium channel currents. Also, 400 mg/mL of pitaya juice concentrate in spontaneous hypertensive rats reduced their blood pressure for 48 h. Phytochemical analyses showed that the primary compounds in F1 were glycosidic in nature, and could be a complex mixture of disaccharides, dimeric disaccharides, or even tetrasaccharides. The glycosidic compounds found in F1 primarily contributed to vasodilatation, establishing a voltage-dependent calcium channel inhibition as a possible molecular target. Full article

In situ electroporation, a non-invasive technique for enhancing the permeability of cell membranes, has emerged as a powerful tool for intracellular delivery and manipulation. This method allows for the precise introduction of therapeutic agents, such as nucleic acids, drugs, and proteins, directly into target cells within their native tissue environment. Herein, we introduce an innovative electroporation strategy that employs a Janus particle (JP)-based microelectrode to generate a localized and controllable electric field within a microfluidic chip. The microfluidic device is engineered with an indium tin oxide (ITO)-sandwiched microchannel, where the electric field is applied, and suspended JP microelectrodes that induce a stronger localized electric field. The corresponding simulation model is developed to better understand the dynamic electroporation process. Numerical simulations for both single-cell and chain-assembled cell electroporation have been successfully conducted. The effects of various parameters, including pulse voltage, duration medium conductivity, and radius of Janus microelectrode, on cell membrane permeabilization are systematically investigated. Our findings indicate that the enhanced electric intensity near the poles of the JP microelectrode significantly contributes to the electroporation process. In addition, the distribution for both transmembrane voltage and the resultant nanopores can be altered by conveniently adjusting the relative position of the JP microelectrode, demonstrating a selective and in situ electroporation technique for spatial control over the delivery area. Moreover, the obtained differences in the distribution of electroporation between chain cells can offer insightful directives for the electroporation of tissues or cell populations, enabling the precise and targeted modulation of specific cell populations. As a proof of concept, this work can provide a robust alternative technique for the study of complex and personalized cellular processes. Full article

Connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells are of paramount importance for intercellular communication. In physiological conditions, HCs can form gap junction (GJ) channels, providing a direct diffusive path between neighbouring cells. In addition, unpaired HCs provide conduits for the exchange of solutes between the cytoplasm and the extracellular milieu, including messenger molecules involved in paracrine signalling. The synergistic action of membrane potential and Ca²⁺ ions controls the gating of the large and relatively unselective pore of connexin HCs. The four orders of magnitude difference in gating sensitivity to the extracellular ([Ca²⁺]_e) and the cytosolic ([Ca²⁺]_c) Ca²⁺ concentrations suggests that at least two different Ca²⁺ sensors may exist. While [Ca²⁺]_e acts as a spatial modulator of the HC opening, which is most likely dependent on the cell layer, compartment, and organ, [Ca²⁺]_c triggers HC opening and the release of extracellular bursts of messenger molecules. Such molecules include ATP, cAMP, glutamate, NAD⁺, glutathione, D-serine, and prostaglandins. Lost or abnormal HC regulation by Ca²⁺ has been associated with several diseases, including deafness, keratitis ichthyosis, palmoplantar keratoderma, Charcot–Marie–Tooth neuropathy, oculodentodigital dysplasia, and congenital cataracts. The fact that both an increased and a decreased Ca²⁺ sensitivity has been linked to pathological conditions suggests that Ca²⁺ in healthy cells finely tunes the normal HC function. Overall, further investigation is needed to clarify the structural and chemical modifications of connexin HCs during [Ca²⁺]_e and [Ca²⁺]_c variations. A molecular model that accounts for changes in both Ca²⁺ and the transmembrane voltage will undoubtedly enhance our interpretation of the experimental results and pave the way for developing therapeutic compounds targeting specific HC dysfunctions. Full article

In this report we present seven lines of bioinformatic evidence supporting the conclusion that the Pentameric Ligand-gated Ion Channel (pLIC) Family is a member of the Voltage-gated Ion Channel (VIC) Superfamily. In our approach, we used the Transporter Classification Database (TCDB) as a reference and applied a series of bioinformatic methods to search for similarities between the pLIC family and members of the VIC superfamily. These include: (1) sequence similarity, (2) compatibility of topology and hydropathy profiles, (3) shared domains, (4) conserved motifs, (5) similarity of Hidden Markov Model profiles between families, (6) common 3D structural folds, and (7) clustering analysis of all families. Furthermore, sequence and structural comparisons as well as the identification of a 3-TMS repeat unit in the VIC superfamily suggests that the sixth transmembrane segment evolved into a re-entrant loop. This evidence suggests that the voltage-sensor domain and the channel domain have a common origin. The classification of the pLIC family within the VIC superfamily sheds light onto the topological origins of this family and its evolution, which will facilitate experimental verification and further research into this superfamily by the scientific community. Full article

Mitochondria are most likely descendants of strictly aerobic prokaryotes from the class Alphaproteobacteria. The mitochondrial matrix is surrounded by two membranes according to its relationship with Gram-negative bacteria. Similar to the bacterial outer membrane, the mitochondrial outer membrane acts as a molecular sieve because it also contains diffusion pores. However, it is more actively involved in mitochondrial metabolism because it plays a functional role, whereas the bacterial outer membrane has only passive sieving properties. Mitochondrial porins, also known as eukaryotic porins or voltage-dependent anion-selective channels (VDACs) control the permeability properties of the mitochondrial outer membrane. They contrast with most bacterial porins because they are voltage-dependent. They switch at relatively small transmembrane potentials of 20 to 30 mV in closed states that exhibit different permeability properties than the open state. Whereas the open state is preferentially permeable to anionic metabolites of mitochondrial metabolism, the closed states prefer cationic solutes, in particular, calcium ions. Mitochondrial porins are encoded in the nucleus, synthesized at cytoplasmatic ribosomes, and post-translationally imported through special transport systems into mitochondria. Nineteen beta strands form the beta-barrel cylinders of mitochondrial and related porins. The pores contain in addition an α-helical structure at the N-terminal end of the protein that serves as a gate for the voltage-dependence. Similarly, they bind peripheral proteins that are involved in mitochondrial function and compartment formation. This means that mitochondrial porins are localized in a strategic position to control mitochondrial metabolism. The special features of the role of mitochondrial porins in apoptosis and cancer will also be discussed in this article. Full article

KCNE3 is a single-pass integral membrane protein that regulates numerous voltage-gated potassium channel functions such as KCNQ1. Previous solution NMR studies suggested a moderate degree of curved α-helical structure in the transmembrane domain (TMD) of KCNE3 in lyso-myristoylphosphatidylcholine (LMPC) micelles and isotropic bicelles with the residues T71, S74 and G78 situated along the concave face of the curved helix. During the interaction of KCNE3 and KCNQ1, KCNE3 pushes its transmembrane domain against KCNQ1 to lock the voltage sensor in its depolarized conformation. A cryo-EM study of KCNE3 complexed with KCNQ1 in nanodiscs suggested a deviation of the KCNE3 structure from its independent structure in isotropic bicelles. Despite the biological significance of KCNE3 TMD, the conformational properties of KCNE3 are poorly understood. Here, all atom molecular dynamics (MD) simulations were utilized to investigate the conformational dynamics of the transmembrane domain of KCNE3 in a lipid bilayer containing a mixture of POPC and POPG lipids (3:1). Further, the effect of the interaction impairing mutations (V72A, I76A and F68A) on the conformational properties of the KCNE3 TMD in lipid bilayers was investigated. Our MD simulation results suggest that the KCNE3 TMD adopts a nearly linear α helical structural conformation in POPC-POPG lipid bilayers. Additionally, the results showed no significant change in the nearly linear α-helical conformation of KCNE3 TMD in the presence of interaction impairing mutations within the sampled time frame. The KCNE3 TMD is more stable with lower flexibility in comparison to the N-terminal and C-terminal of KCNE3 in lipid bilayers. The overall conformational flexibility of KCNE3 also varies in the presence of the interaction-impairing mutations. The MD simulation data further suggest that the membrane bilayer width is similar for wild-type KCNE3 and KCNE3 containing mutations. The Z-distance measurement data revealed that the TMD residue site A69 is close to the lipid bilayer center, and residue sites S57 and S82 are close to the surfaces of the lipid bilayer membrane for wild-type KCNE3 and KCNE3 containing interaction-impairing mutations. These results agree with earlier KCNE3 biophysical studies. The results of these MD simulations will provide complementary data to the experimental outcomes of KCNE3 to help understand its conformational dynamic properties in a more native lipid bilayer environment. Full article

Presenilin 1 (PS1) is a transmembrane proteolytic subunit of γ-secretase that cleaves amyloid precursor proteins. Mutations in PS1 (mPS1) are associated with early-onset familial Alzheimer’s disease (AD). The link between mutated PS1, mitochondrial calcium regulation, and AD has been studied extensively in different test systems. Despite the wide-ranging role of mPS1 in AD, there is a paucity of information on the link between PS1 and neuronal cell death, a hallmark of AD. In the present study, we employed the selective mitochondrial uncoupler carbonyl cyanide chlorophenylhydrazone (CCCP) and compared the reactivity of mPS1-transfected cultured rat hippocampal neurons with PS1 and control neurons in a situation of impaired mitochondrial functions. CCCP causes a slow rise in cytosolic and mitochondrial calcium in all three groups of neurons, with the mPS1 neurons demonstrating a faster rise. Consequently, mPS1 neurons were depolarized by CCCP and measured with TMRM, a mitochondrial voltage indicator, more than the other two groups. Morphologically, CCCP produced more filopodia in mPS1 neurons than in the other two groups, which were similarly affected by the drug. Finally, mPS1 transfected neurons tended to die from prolonged exposure to CCCP sooner than the other groups, indicating an increase in vulnerability associated with a lower ability to regulate excess cytosolic calcium. Full article

The majority of voltage-gated ion channels contain a defined voltage-sensing domain and a pore domain composed of highly conserved amino acid residues that confer electrical excitability via electromechanical coupling. In this sense, the voltage-gated proton channel (Hv1) is a unique protein in that voltage-sensing, proton permeation and pH-dependent modulation involve the same structural region. In fact, these processes synergistically work in concert, and it is difficult to separate them. To investigate the process of Hv1 voltage sensor trapping, we follow voltage-sensor movements directly by leveraging mutations that enable the measurement of Hv1 channel gating currents. We uncover that the process of voltage sensor displacement is due to two driving forces. The first reveals that mutations in the selectivity filter (D160) located in the S1 transmembrane interact with the voltage sensor. More hydrophobic amino acids increase the energy barrier for voltage sensor activation. On the other hand, the effect of positive charges near position 264 promotes the formation of salt bridges between the arginines of the voltage sensor domain, achieving a stable conformation over time. Our results suggest that the activation of the Hv1 voltage sensor is governed by electrostatic–hydrophobic interactions, and S4 arginines, N264 and selectivity filter (D160) are essential in the Ciona-Hv1 to understand the trapping of the voltage sensor. Full article