Role Of Membrane Potential Research Articles

Cellular communication among smooth muscle cells: The role of membrane potential via connexins

Communication via action potentials among neurons has been extensively studied. However, effective communication without action potentials is ubiquitous in biological systems, yet it has received much less attention in comparison. Multi-cellular communication among smooth muscles is crucial for regulating blood flow, for example. Understanding the mechanism of this non-action potential communication is critical in many cases, like synchronization of cellular activity, under normal and pathological conditions. In this paper, we employ a multi-scale asymptotic method to derive a macroscopic homogenized bidomain model from the microscopic electro-neutral (EN) model. This is achieved by considering different diffusion coefficients and incorporating nonlinear interface conditions. Subsequently, the homogenized macroscopic model is used to investigate communication in multi-cellular tissues. Our computational simulations reveal that the membrane potential of syncytia, formed by interconnected cells via connexins, plays a crucial role in propagating oscillations from one region to another, providing an effective means for fast cellular communication.Statement of Significance: In this study, we investigated cellular communication and ion transport in vascular smooth muscle cells, shedding light on their mechanisms under normal and abnormal conditions. Our research highlights the potential of mathematical models in understanding complex biological systems. We developed effective macroscale electro-neutral bi-domain ion transport models and examined their behavior in response to different stimuli. Our findings revealed the crucial role of connexinmediated membrane potential changes and demonstrated the effectiveness of cellular communication through syncytium membranes. Despite some limitations, our study provides valuable insights into these processes and emphasizes the importance of mathematical modeling in unraveling the complexities of cellular communication and ion transport.

The development of L-type Ca2+ current mediated alternans does not depend on the restitution slope in canine ventricular myocardium

Cardiac alternans have crucial importance in the onset of ventricular fibrillation. The early explanation for alternans development was the voltage-driven mechanism, where the action potential (AP) restitution steepness was considered as crucial determining factor. Recent results suggest that restitution slope is an inadequate predictor for alternans development, but several studies still claim the role of membrane potential as underlying mechanism of alternans. These controversial data indicate that the relationship of restitution and alternans development is not completely understood. APs were measured by conventional microelectrode technique from canine right ventricular papillary muscles. Ionic currents combined with fluorescent measurements were recorded by patch-clamp technique. APs combined with fluorescent measurements were monitored by sharp microelectrodes. Rapid pacing evoked restitution-independent AP duration (APD) alternans. When non-alternating AP voltage command was used, Ca2+i-transient (CaT) alternans were not observed. When alternating rectangular voltage pulses were applied, CaT alternans were proportional to ICaL amplitude alternans. Selective ICaL inhibition did not influence the fast phase of APD restitution. In this study we found that ICaL has minor contribution in shaping the fast phase of restitution curve suggesting that ICaL—if it plays important role in the alternans mechanism—could be an additional factor that attenuates the reliability of APD restitution slope to predict alternans.

A DNA-based voltmeter for organelles.

The role of membrane potential in most intracellular organelles remains unexplored because of the lack of suitable tools. Here, we describe a fluorescent DNA-nanodevice that reports absolute membrane potential and can be targeted to organelles in live cells. This nanodevice, denoted Voltair, bears a voltage-sensitive fluorophore, a reference fluorophore for ratiometry, and acts as an endocytic tracer. Using Voltair we could measure the membrane potential of different organelles in situ in live cells. Voltair can potentially guide the rational design of biocompatible electronics as well as expand our understanding of how membrane potential regulates organelle biology.

Membrane potentials, oxidative stress and the dispersal response of bacterial biofilms to 405 nm light

The majority of chronic infections are caused by biofilms, which have higher levels of antibiotic resistance than planktonic growth. Violet-blue 405 nm light has recently emerged as a novel bactericide, but limited studies have been conducted on its effectiveness against biofilms. We found that in response to 405 nm light both Pseudomonas aeruginosa and Bacillus subtilis biofilms exhibited cell dispersal and membrane potential hyperpolarisations. The response to 405 nm light depended on the stage of biofilm growth. The use of reactive oxygen species scavengers reduced membrane hyperpolarisation and biofilm dispersal in response to 405 nm light. This is the first time that membrane potential hyperpolarisations have been linked with photooxidative stress in bacteria and with biofilm dispersal. These results provide a new insight into the role of membrane potentials in the bacterial stress response and could be used in the development of 405 nm light based biofilm treatments.

Role of Membrane Potential on Entry of Cell-Penetrating Peptide Transportan 10 into Single Vesicles

Cell-penetrating peptides (CPPs) can translocate across plasma membranes to enter the cytosol of eukaryotic cells without decreasing cell viability. We revealed the mechanism underlying this translocation by examining the effect of membrane potential, φm, on the entry of a CPP, transportan 10 (TP10), into the lumen of single giant unilamellar vesicles (GUVs). For this purpose, we used the single GUV method to detect the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into the lumen of single GUVs. First, we used various K+ concentration differences to apply different negative membrane potentials on single GUVs containing gramicidin A in their membrane and confirmed these potentials using the φm-sensitive fluorescent probe 3,3′-dihexyloxacarbocyanine iodine. The fluorescence intensity of the GUV membranes (i.e., the rim intensity) due to 3,3′-dihexyloxacarbocyanine iodine increased with |φm| up to 118 mV, and its dependence on |φm| less than 28 mV agreed with a theoretical estimation (i.e., the dye concentration in the inner leaflet of a GUV is larger than that in the outer leaflet according to the Boltzmann distribution). We then examined the effect of φm on the entry of CF-TP10 into GUVs using single GUVs containing small GUVs or large unilamellar vesicles inside the mother GUV lumen. We found that CF-TP10 entered the GUV lumen without pore formation and the rate of entry of CF-TP10 into the GUV lumen, Ventry, increased with an increase in |φm|. The rim intensity due to CF-TP10 increased with an increase in |φm|, indicating that the CF-TP10 concentration in the inner leaflet of the GUV increased with |φm|. These results indicate that the φm-induced elevation in Ventry can be explained by the increase in CF-TP10 concentration in the inner leaflet with |φm|. We discuss the mechanism underlying this effect of membrane potential based on the pre-pore model of the translocation of CF-TP10 across a GUV membrane.

Erythrocyte plasma membrane potential: past and current methods for its measurement.

The plasma membrane functions both as a natural insulator and a diffusion barrier to the movement of ions. A wide variety of proteins transport and pump ions to generate concentration gradients that result in voltage differences, while ion channels allow ions to move across the membrane down those gradients. Plasma membrane potential is the difference in voltage between the inside and the outside of a biological cell, and it ranges from ~- 3 to ~- 90 mV. Most of the most significant discoveries in this field have been made in excitable cells, such as nerve and muscle cells. Nevertheless, special attention has been paid to some events controlled by changes in membrane potential in non-excitable cells. The origins of several blood disorders, for instance, are related to disturbances at the level of plasma membrane in erythrocytes, the structurally simplest red blood cells. The high simplicity of erythrocytes, in particular, made them perfect candidates for the electrophysiological studies that laid the foundations for understanding the generation, maintenance, and roles of membrane potential. This article summarizes the methodologies that have been used during the past decades to determine Δψ in red blood cells, from seminal microelectrodes, through the use of nuclear magnetic resonance or lipophilic radioactive ions to quantify intra and extracellular ions, to continuously renewed fluorescent potentiometric dyes. We have attempted to highlight the advantages and disadvantages of each methodology, as well as to provide a description of the technical aspects involved.

Modulating Calcium Entry into Microvascular Endothelium by Controlling Membrane Potential During Submaximal Muscarinic Receptor Activation

Vascular smooth muscle cell relaxation and vasodilation are coupled to a rise in endothelial cell [Ca2+]i and hyperpolarization. However, the role of membrane potential (Vm) in regulating endothelial cell [Ca2+]i remains controversial. To test the hypothesis that Vm can govern Ca2+ entry into endothelial cells in accord with its electrochemical gradient, we evaluated [Ca2+]i using Fura-2 photometry (F340/F380) while controlling Vm using intracellular current injection. Intact endothelial tubes (width: 60 μm, length: ≤ 1 mm) were freshly isolated from superior epigastric arteries of C57BL/6 mice (n≥7; 32°C, pH 7.4). Endothelial cells were electrically coupled to each other through gap junctions and lacked inherent voltage-sensitive ion channel activity as indicated by linearity (R2 = 0.990±0.002) of the current-voltage relationship throughout the Vm range of ≈-80 mV to +10 mV during current injection (±0.5 to 5 nA). In the absence of receptor activation, Vm was −37±1 mV and [Ca2+]i did not change with hyperpolarization or depolarization. In contrast, during submaximal activation of muscarinic (Gq protein-coupled) receptors (GPCRs) with acetylcholine (100 nM; ∼EC50), [Ca2+]i increased with hyperpolarization to −60±1 mV and decreased with depolarization to −28±2 mV. The effect of membrane hyperpolarization on [Ca2+]i was abolished following removal of [Ca2+]o (control [Ca2+]o = 2 mM) and reduced by half in endothelium isolated from TRPV4-/- mice during Vm > −50 mV. Thus, submaximal stimulation of GPCRs enables physiological changes in Vm to modulate Ca entry through voltage-insensitive ion channels in the plasma membrane of native microvascular endothelial cells. (Support: NIH grants R37-HL041026, 5T32-AR048523, 1K99-AG047198)

Introducing Bacterial Electrophysiology

An optical bioprobe reveals action potential–like electrical spikes in individual bacteria.

Role of membrane potential on the control of cytochrome c oxidase over respiration in intact hepatoma HepG2 cells

Role of membrane potential on the control of cytochrome c oxidase over respiration in intact hepatoma HepG2 cells

Kv1.3 is the exclusive voltage‐gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count

A delayed rectifier voltage-gated K+ channel (Kv) represents the largest ionic conductance of platelets and megakaryocytes, but is undefined at the molecular level. Quantitative RT-PCR of all known Kv α and ancillary subunits showed that only Kv1.3 (KCNA3) is substantially expressed in human platelets. Furthermore, megakaryocytes from Kv1.3−/− mice or from wild-type mice exposed to the Kv1.3 blocker margatoxin completely lacked Kv currents and displayed substantially depolarised resting membrane potentials. In human platelets, margatoxin reduced the P2X1- and thromboxaneA2 receptor-evoked [Ca2+]i increases and delayed the onset of store-operated Ca2+ influx. Megakaryocyte development was normal in Kv1.3−/− mice, but the platelet count was increased, consistent with a role of Kv1.3 in apoptosis or decreased platelet activation. We conclude that Kv1.3 forms the Kv channel of the platelet and megakaryocyte, which sets the resting membrane potential, regulates agonist-evoked Ca2+ increases and influences circulating platelet numbers.

Role of membrane potential in the regulation of cell proliferation and differentiation.

Biophysical signaling, an integral regulator of long-term cell behavior in both excitable and non-excitable cell types, offers enormous potential for modulation of important cell functions. Of particular interest to current regenerative medicine efforts, we review several examples that support the functional role of transmembrane potential (V(mem)) in the regulation of proliferation and differentiation. Interestingly, distinct V(mem) controls are found in many cancer cell and precursor cell systems, which are known for their proliferative and differentiation capacities, respectively. Collectively, the data demonstrate that bioelectric properties can serve as markers for cell characterization and can control cell mitotic activity, cell cycle progression, and differentiation. The ability to control cell functions by modulating bioelectric properties such as V(mem) would be an invaluable tool for directing stem cell behavior toward therapeutic goals. Biophysical properties of stem cells have only recently begun to be studied and are thus in need of further characterization. Understanding the molecular and mechanistic basis of biophysical regulation will point the way toward novel ways to rationally direct cell functions, allowing us to capitalize upon the potential of biophysical signaling for regenerative medicine and tissue engineering.

Role of membrane potential on artificial transformation of E. coli with plasmid DNA

The standard method of transformation of Escherichea coli with plasmid DNA involves two important steps: cells are first suspended in 100mM CaCl(2) at 0 degrees C (in which DNA is added), followed by the administration of a heat-pulse from 0 to 42 degrees C for 90s [Cohen, S., Chang, A., Hsu, L., 1972. Nonchromosomal antibiotic resistance in bacteria. Proc. Natl. Acad. Sci. U.S.A., 69, 2110-2114]. The first step makes the cells competent for uptake of DNA and the second step is believed to facilitate the DNA entry into the cells by an unknown mechanism. In this study, the measure of membrane potential of the intact competent cells, at different steps of transformation process, either by the method of spectrofluorimetry or that of flow cytometry, indicates that the heat-pulse step (0-->42 degrees C) heavily decreases the membrane potential. A subsequent cold shock (42-->0 degrees C) raises the potential further to its original value. Moreover, the efficiency of transformation of E. coli XL1 Blue cells with plasmid pUC19 DNA remains unaltered when the heat-pulse step is replaced by the incubation of the DNA-adsorbed competent cells with 10 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 90s at 0 degrees C. Since the CCCP, a well-known protonophore, reduces membrane potential by dissipating the proton-motive-force (PMF) across E. coli plasma membrane, our experimental results suggest that the heat-pulse step of the standard transformation procedure facilitates DNA entry into the cells by lowering the membrane potential.

Role of Membrane Potential in Endothelium-Dependent Relaxation of Isolated Mouse Main Pulmonary Artery

The physiology of smooth muscle and endothelial cells of a particular vascular bed and from different species differs from each other. Acetylcholine causes an endothelium-dependent relaxation of preconstricted pulmonary arteries from the rat. This relaxation is mediated by nitric oxide (NO) plus a yet-unidentified endothelium-derived hyperpolarizing factor, which relaxes the smooth muscles by hyperpolarizing them. Our aim is to test whether these observations could be generalized to the smooth muscle cells from the mouse pulmonary artery. Smooth muscle or endothelial cell membrane potential of strips of murine pulmonary artery were measured simultaneously with the force developed by the strip. Acetylcholine hyperpolarized the endothelial cells. However, acetylcholine did not induce an endothelium-dependent hyperpolarization of the smooth muscle, while it relaxed the strip in an endothelium-dependent manner. This relaxation was abolished by an inhibitor of NO synthesis, nitro-L-arginine. Moreover, nitroglycerin relaxed the strips without changing the membrane potential of the smooth muscle cells. Injection of Lucifer yellow into the endothelial cells and the smooth muscle cells did not show heterocellular dye coupling. Furthermore, electron microscopy did not show gap junction plate at the myoendothelial junctions. We conclude that in the mouse main pulmonary artery, NO alone is responsible for the acetylcholine-induced endothelium-dependent vasodilatation, whereas the phenomenon called endothelium-derived hyperpolizing factor is not present. Therefore, caution should be taken when comparing different animal models to study pulmonary circulation and its reactivity.

High Extracellular Ca2+ Hyperpolarizes Human Parathyroid Cells via Ca2+-activated K+ Channels

Membrane potential has a major influence on stimulus-secretion coupling in various excitable cells. The role of membrane potential in the regulation of parathyroid hormone secretion is not known. High K+-induced depolarization increases secretion from parathyroid cells. The paradox is that increased extracellular Ca2+, which inhibits secretion, has also been postulated to have a depolarizing effect. In this study, human parathyroid cells from parathyroid adenomas were used in patch clamp studies of K+ channels and membrane potential. Detailed characterization revealed two K+ channels that were strictly dependent of intracellular Ca2+ concentration. At high extracellular Ca2+, a large K+ current was seen, and the cells were hyperpolarized (-50.4 +/- 13.4 mV), whereas lowering of extracellular Ca2+ resulted in a dramatic decrease in K+ current and depolarization of the cells (-0.1 +/- 8.8 mV, p < 0.001). Changes in extracellular Ca2+ did not alter K+ currents when intracellular Ca2+ was clamped, indicating that K+ channels are activated by intracellular Ca2+. The results were concordant in cell-attached, perforated patch, whole-cell and excised membrane patch configurations. These results suggest that [Ca2+]o regulates membrane potential of human parathyroid cells via Ca2+-activated K+ channels and that the membrane potential may be of greater importance for the stimulus-secretion coupling than recognized previously.

Role of membrane potential and expression of endothelial factors in restenosis after angioplasty in SHR.

We examined the roles played by impaired K+ channels, diminished nitric oxide (NO) production, endothelin release, and smooth muscle membrane potential in the increased restenosis observed in spontaneously hypertensive rat (SHR) carotid arteries after angioplasty. The SHR carotid was found to be less polarized than that of normotensive Wistar rats (NWR), and it was further depolarized by the alpha2 agonist UK 14,304. This response was blocked by iberiotoxin, indicating that calcium-dependent K+ channels operate normally in the SHR carotid. Acetylcholine caused a hyperpolarization that was significantly smaller in SHR than in NWR carotids, indicating a deficient release of NO in the SHR. After angioplasty, SHR and NWR vessels were depolarized, returning to baseline after 10 days. In the SHR but not in the NWR the contralateral carotid was also depolarized, and this was prevented by the endothelin A/B receptor antagonist bosentan. After angioplasty, endothelin-1 plasma levels increased in both SHR and NWR, but the increase was significantly more prolonged in SHR. We found that the more pronounced restenosis observed in the SHR carotid after angioplasty is not due to impairment of calcium-dependent K+ channels but is related to the relatively depolarized vascular smooth muscles, involving endothelin release caused by reduced NO levels in that strain.

Role of membrane potential in vasomotion of isolated pressurized rat arteries

Vasomotion, the phenomenon of vessel diameter oscillation, regulates blood flow and resistance. The main parameters implicated in vasomotion are particularly the membrane potential and the cytosolic free calcium in smooth muscle cells. In this study, these parameters were measured in rat perfused-pressurized mesenteric artery segments. The application of norepinephrine (NE) caused rhythmic diameter contractions and membrane potential oscillations (amplitude; 5.3 ± 0.3 mV, frequency; 0.09 ± 0.01 Hz). Verapamil (1 μM) abolished this vasomotion. During vasomotion, 10 −5 M ouabain (Na +–K + ATPase inhibitor) decreased the amplitude of the electrical oscillations but not their frequency (amplitude; 3.7 ± 0.3 mV, frequency; 0.08 ± 0.002 Hz). Although a high concentration of ouabain (10 −3 M) (which exhibits non-specific effects) abolished both electrical membrane potential oscillations and vasomotion, we conclude that the Na+–K+ ATPase could not be implicated in the generation of the membrane potential oscillations. We conclude that in rat perfused-pressurized mesenteric artery, the slow wave membrane type of potential oscillation by rhythmically gating voltage-dependent calcium channels, is responsible for the oscillation of intracellular calcium and thus vasomotion.

Cardiac excitation-contraction coupling: role of membrane potential in regulation of contraction.

The steps that couple depolarization of the cardiac cell membrane to initiation of contraction remain controversial. Depolarization triggers a rise in intracellular free Ca(2+) which activates contractile myofilaments. Most of this Ca(2+) is released from the sarcoplasmic reticulum (SR). Two fundamentally different mechanisms have been proposed for SR Ca(2+) release: Ca(2+)-induced Ca(2+) release (CICR) and a voltage-sensitive release mechanism (VSRM). Both mechanisms operate in the same cell and may contribute to contraction. CICR couples the release of SR Ca(2+) closely to the magnitude of the L-type Ca(2+) current. In contrast, the VSRM is graded by membrane potential rather than Ca(2+) current. The electrophysiological and pharmacological characteristics of the VSRM are strikingly different from CICR. Furthermore, the VSRM is strongly modulated by phosphorylation and provides a new regulatory mechanism for cardiac contraction. The VSRM is depressed in heart failure and may play an important role in contractile dysfunction. This review explores the operation and characteristics of the VSRM and CICR and discusses the impact of the VSRM on our understanding of cardiac excitation-contraction coupling.

Mitochondrial status of caspase inhibitor-saved neurons: Role of membrane potential in commitment(s) to die

Mitochondrial status of caspase inhibitor-saved neurons: Role of membrane potential in commitment(s) to die

A novel role for membrane potential in the modulation of intracellular Ca2+ oscillations in rat megakaryocytes.

1. The effect of membrane potential (Vm) on ADP-evoked [Ca2+]i oscillations was investigated in rat megakaryocytes, a non-excitable cell type recently shown to exhibit depolarisation-evoked Ca2+ release from intracellular stores during metabotropic purinoceptor stimulation. 2. Hyperpolarising voltage steps caused a transient fall in [Ca2+]i and either abolished Ca2+ oscillations or reduced the oscillation amplitude. These effects were observed in both the presence and absence of extracellular Ca2+ and also in Na+-free saline solutions, suggesting that hyperpolarisation leads to a reduction in the level of ADP-dependent Ca2+ release without a requirement for altered transmembrane Ca2+ fluxes. 3. In the presence of Ca2+ oscillations, depolarising voltage steps transiently enhanced the amplitude of Ca2+ oscillations. Following run-down of Ca2+ oscillations, depolarisation briefly restimulated oscillations. 4. Simultaneous [Ca2+]i and current-clamp recordings showed that Ca2+ and Vm oscillate in synchrony, with an average fluctuation of approximately 30-40 mV, due to activation and inactivation of Ca2+-dependent K+ channels. Application of a physiological oscillating Vm waveform to non-oscillating cells under voltage clamp stimulated [Ca2+]i oscillations. 5. Analysis of the relationship between [Ca2+]i and Vm showed a threshold for activation of hyperpolarisation at about 250-300 nM. The implications of this threshold in the interaction between Vm and Ca2+ release during oscillations are discussed. 6. We conclude that the ability of voltage to control release of endosomal Ca2+ in ADP-stimulated megakaryocytes is bipolar in nature. Our data suggest that Vm changes are active components of the feedback/feedforward mechanisms contributing to the generation of Ca2+ oscillations.

Effect of acidosis on tension and [Ca2+]i in rat cerebral arteries: is there a role for membrane potential?

The cellular mechanism responsible for the reduction of tension in cerebral small arteries to acidosis is not known. In this study the role of smooth muscle intracellular Ca2+ concentration ([Ca2+]i) and membrane potential for the relaxation to acidosis was investigated in isolated rat cerebral small arteries. Isometric force was measured simultaneously with [Ca2+]i (fura 2) or with membrane potential (intracellular microelectrodes), and acidosis was induced by increasing PCO2 or reducing HCO3- of the bathing solution. Both hypercapnic and normocapnic acidosis were associated with a reduction of intracellular pH [measured with 2',7'-bis-(carboxyethyl)-5 (and -6)-carboxyfluorescein], caused relaxation, and reduced [Ca2+]i. However, whereas hypercapnic acidosis caused hyperpolarization, normocapnic acidosis was associated with depolarization. It is concluded that a reduction of [Ca2+]i is in part responsible for the direct effect of the acidosis on the vascular smooth muscle both during normo- and hypercapnia. The mechanism responsible for the reduction of [Ca2+]i differs between the hypercapnic and normocapnic acidosis, being partly explained by hyperpolarization during hypercapnic acidosis, whereas it is seen despite depolarization during normocapnic acidosis.