scholarly journals Electrically induced bacterial membrane potential dynamics correspond to cellular proliferation capacity

2019 ◽  
Author(s):  
James P Stratford ◽  
Conor LA Edwards ◽  
Manjari J Ghanshyam ◽  
Dmitry Malyshev ◽  
Marco A Delise ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Membrane Potential ◽  
Single Cell ◽  
Cellular Response ◽  
Cellular Proliferation ◽  
Model Organisms ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Response Dynamics ◽  
Proliferation Capacity

Membrane-potential dynamics mediate bacterial electrical signaling at both intra- and inter- cellular levels. Membrane potential is also central to cellular proliferation. It is unclear whether the cellular response to external electrical stimuli is influenced by the cell proliferative capacity. A new strategy enabling electrical stimulation of bacteria with simultaneous monitoring of single-cell membrane potential dynamics would allow bridging this knowledge gap and further extend electrophysiological studies into the field of microbiology. Here we report that an identical electrical stimulus can cause opposite polarization dynamics depending on cellular proliferation capacity. This was demonstrated using two model organisms, namely B. subtilis and E. coli, and by developing an apparatus enabling exogenous electrical stimulation and single-cell time-lapse microscopy. Using this bespoke apparatus, we show that a 2.5 sec electrical stimulation causes hyperpolarization in unperturbed cells. Measurements of intracellular K+ and the deletion of the K+ channel suggested that the hyperpolarization response is caused by the K+ efflux through the channel. When cells are pre-exposed to UV-violet light, the same electrical stimulation depolarizes cells instead of causing hyperpolarization. A mathematical model extended from the FitzHugh-Nagumo neuron model suggested that the opposite response dynamics are due to the shift in resting membrane potential. As predicted by the model, electrical stimulation only induced depolarization when cells are treated with antibiotics, protonophore or alcohol. Therefore, electrically induced membrane potential dynamics offer a novel and reliable approach for rapid detection of proliferative bacteria and determination of their sensitivity to antimicrobial agents at the single-cell level.

scholarly journals Electrically induced bacterial membrane-potential dynamics correspond to cellular proliferation capacity

2019 ◽  
Vol 116 (19) ◽  
pp. 9552-9557 ◽  
Author(s):  
James P. Stratford ◽  
Conor L. A. Edwards ◽  
Manjari J. Ghanshyam ◽  
Dmitry Malyshev ◽  
Marco A. Delise ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Membrane Potential ◽  
Single Cell ◽  
Cellular Response ◽  
Cellular Proliferation ◽  
Antimicrobial Agents ◽  
Model Organisms ◽  
Resting Membrane Potential ◽  
Response Dynamics ◽  
Proliferation Capacity

Membrane-potential dynamics mediate bacterial electrical signaling at both intra- and intercellular levels. Membrane potential is also central to cellular proliferation. It is unclear whether the cellular response to external electrical stimuli is influenced by the cellular proliferative capacity. A new strategy enabling electrical stimulation of bacteria with simultaneous monitoring of single-cell membrane-potential dynamics would allow bridging this knowledge gap and further extend electrophysiological studies into the field of microbiology. Here we report that an identical electrical stimulus can cause opposite polarization dynamics depending on cellular proliferation capacity. This was demonstrated using two model organisms, namelyBacillus subtilisandEscherichia coli, and by developing an apparatus enabling exogenous electrical stimulation and single-cell time-lapse microscopy. Using this bespoke apparatus, we show that a 2.5-second electrical stimulation causes hyperpolarization in unperturbed cells. Measurements of intracellular K+and the deletion of the K+channel suggested that the hyperpolarization response is caused by the K+efflux through the channel. When cells are preexposed to 400 ± 8 nm wavelength light, the same electrical stimulation depolarizes cells instead of causing hyperpolarization. A mathematical model extended from the FitzHugh–Nagumo neuron model suggested that the opposite response dynamics are due to the shift in resting membrane potential. As predicted by the model, electrical stimulation only induced depolarization when cells are treated with antibiotics, protonophore, or alcohol. Therefore, electrically induced membrane-potential dynamics offer a reliable approach for rapid detection of proliferative bacteria and determination of their sensitivity to antimicrobial agents at the single-cell level.

scholarly journals Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line.

1993 ◽  
Vol 102 (4) ◽  
pp. 667-692 ◽  
Author(s):  
E Hamada ◽  
T Nakajima ◽  
S Ota ◽  
A Terano ◽  
M Omata ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Epithelial Cell ◽  
Cell Line ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Induced Response ◽  
Epithelial Cell Line ◽  
Bathing Solution ◽  
Pipette Solution ◽  
K Current

The effects of acetylcholine (ACh) and histamine (His) on the membrane potential and current were examined in JR-1 cells, a mucin-producing epithelial cell line derived from human gastric signet ring cell carcinoma. The tight-seal, whole cell clamp technique was used. The resting membrane potential, the input resistance, and the capacitance of the cells were approximately -12 mV, 1.4 G ohms, and 50 pF, respectively. Under the voltage-clamp condition, no voltage-dependent currents were evoked. ACh or His added to the bathing solution hyperpolarized the membrane by activating a time- and voltage-independent K+ current. The ACh-induced hyperpolarization and K+ current persisted, while the His response desensitized quickly (< 1 min). These effects of ACh and His were mediated predominantly by m3-muscarinic and H1-His receptors, respectively. The K+ current induced by ACh and His was inhibited by charybdotoxin, suggesting that it is a Ca(2+)-activated K+ channel current (IK.Ca). The measurement of intracellular Ca2+ ([Ca2+]i) using Indo-1 revealed that both agents increased [Ca2+]i with similar time courses as they increased IK.Ca. When EGTA in the pipette solution was increased from 0.15 to 10 mM, the induction of IK.Ca by ACh and His was abolished. Thus, both ACh and His activate IK.Ca by increasing [Ca2+]i in JR-1 cells. In the Ca(2+)-free bathing solution (0.15 mM EGTA in the pipette), ACh evoked IK.Ca transiently. Addition of Ca2+ (1.8 mM) to the bath immediately restored the sustained IK.Ca. These results suggest that the ACh response is due to at least two different mechanisms; i.e., the Ca2+ release-related initial transient activation and the Ca2+ influx-related sustained activation of IK.Ca. Probably because of desensitization, the Ca2+ influx-related component of the His response could not be identified. Intracellularly applied inositol 1,4,5-trisphosphate (IP3), with and without inositol 1,3,4,5-tetrakisphosphate (IP4), mimicked the ACh response. IP4 alone did not affect the membrane current. Under the steady effect of IP3 or IP3 plus IP4, neither ACh nor His further evoked IK.Ca. Intracellular application of heparin or of the monoclonal antibody against the IP3 receptor, mAb18A10, inhibited the ACh and His responses in a concentration-dependent fashion. Neomycin, a phospholipase C (PLC) inhibitor, also inhibited the agonist-induced response in a concentration-dependent fashion. Although neither pertussis toxin (PTX) nor N-ethylmaleimide affected the ACh or His activation of IK,Ca, GDP beta S attenuated and GTP gamma S enhanced the agonist response.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of external ions on 45Ca efflux from cat intestinal muscle

1980 ◽  
Vol 238 (6) ◽  
pp. G520-G525
Author(s):  
B. A. Curtis ◽  
D. Kreulen ◽  
C. L. Prosser
Keyword(s):  
Smooth Muscle ◽  
Small Intestine ◽  
Electrical Stimulation ◽  
Membrane Potential ◽  
Ethylene Glycol ◽  
Resting Membrane Potential ◽  
Krebs Solution ◽  
Tetraacetic Acid ◽  
Circular Smooth Muscle ◽  
45Ca Efflux

The surface-bound Ca of isolated circular smooth muscle of cat small intestine can be removed by substitution of LiCl for NaCl in Krebs solution. This substitution removed surface-bound Ca (45Ca) and allowed us to study transmembrane 45Ca efflux. Neither the resting membrane potential nor contractility changed when Li was substituted for Na. Li removed the same extracellular 45Ca store as did ethylene glycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid. The resting transmembrane 45Ca efflux was inhibited by La3+ and was unchanged in Li, tris(hydroxymethyl)aminomethane, arginine, and sucrose Krebs solution. The extra 45Ca efflux observed upon electrical stimulation was no greater in Na-Krebs than Li-Krebs, but during response to acetylcholine the extra 45Ca efflux was greater in Na-Krebs than Li-Krebs. We conclude that the surface-bound Ca is sensitive to external Na and that the transmembrane Ca efflux is not completely dependent on external Na.

scholarly journals Contribution of KCNQ and TREK Channels to the Resting Membrane Potential in Sympathetic Neurons at Physiological Temperature

2020 ◽  
Vol 21 (16) ◽  
pp. 5796
Author(s):  
Paula Rivas-Ramírez ◽  
Antonio Reboreda ◽  
Lola Rueda-Ruzafa ◽  
Salvador Herrera-Pérez ◽  
Jose Antonio Lamas
Keyword(s):  
Membrane Potential ◽  
Room Temperature ◽  
Sympathetic Neurons ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Physiological Temperature ◽  
M Current ◽  
Key Players ◽  
Cervical Ganglion ◽  
Ionic Mechanisms

The ionic mechanisms controlling the resting membrane potential (RMP) in superior cervical ganglion (SCG) neurons have been widely studied and the M-current (IM, KCNQ) is one of the key players. Recently, with the discovery of the presence of functional TREK-2 (TWIK-related K+ channel 2) channels in SCG neurons, another potential main contributor for setting the value of the resting membrane potential has appeared. In the present work, we quantified the contribution of TREK-2 channels to the resting membrane potential at physiological temperature and studied its role in excitability using patch-clamp techniques. In the process we have discovered that TREK-2 channels are sensitive to the classic M-current blockers linopirdine and XE991 (IC50 = 0.310 ± 0.06 µM and 0.044 ± 0.013 µM, respectively). An increase from room temperature (23 °C) to physiological temperature (37 °C) enhanced both IM and TREK-2 currents. Likewise, inhibition of IM by tetraethylammonium (TEA) and TREK-2 current by XE991 depolarized the RMP at room and physiological temperatures. Temperature rise also enhanced adaptation in SCG neurons which was reduced due to TREK-2 and IM inhibition by XE991 application. In summary, TREK-2 and M currents contribute to the resting membrane potential and excitability at room and physiological temperature in the primary culture of mouse SCG neurons.

scholarly journals Spatiotemporal mapping of bacterial membrane potential responses to extracellular electron transfer

2020 ◽  
Vol 117 (33) ◽  
pp. 20171-20179 ◽  
Author(s):  
Sahand Pirbadian ◽  
Marko S. Chavez ◽  
Mohamed Y. El-Naggar
Keyword(s):  
Electron Transfer ◽  
Membrane Potential ◽  
Single Cell ◽  
Electrochemical Techniques ◽  
Model Organisms ◽  
Extracellular Electron Transfer ◽  
Single Cell Level ◽  
Cell Level ◽  
Membrane Hyperpolarization ◽  
Wide Range

Extracellular electron transfer (EET) allows microorganisms to gain energy by linking intracellular reactions to external surfaces ranging from natural minerals to the electrodes of bioelectrochemical renewable energy technologies. In the past two decades, electrochemical techniques have been used to investigate EET in a wide range of microbes, with emphasis on dissimilatory metal-reducing bacteria, such asShewanella oneidensisMR-1, as model organisms. However, due to the typically bulk nature of these techniques, they are unable to reveal the subpopulation variation in EET or link the observed electrochemical currents to energy gain by individual cells, thus overlooking the potentially complex spatial patterns of activity in bioelectrochemical systems. Here, to address these limitations, we use the cell membrane potential as a bioenergetic indicator of EET byS. oneidensisMR-1 cells. Using a fluorescent membrane potential indicator during in vivo single-cell-level fluorescence microscopy in a bioelectrochemical reactor, we demonstrate that membrane potential strongly correlates with EET. Increasing electrode potential and associated EET current leads to more negative membrane potential. This EET-induced membrane hyperpolarization is spatially limited to cells in contact with the electrode and within a near-electrode zone (<30 μm) where the hyperpolarization decays with increasing cell-electrode distance. The high spatial and temporal resolution of the reported technique can be used to study the single-cell-level dynamics of EET not only on electrode surfaces, but also during respiration of other solid-phase electron acceptors.

Inward rectifier K+ channel Kir2.3 is localized at the postsynaptic membrane of excitatory synapses

2002 ◽  
Vol 282 (6) ◽  
pp. C1396-C1403 ◽  
Author(s):  
Atsushi Inanobe ◽  
Akikazu Fujita ◽  
Minoru Ito ◽  
Hitonobu Tomoike ◽  
Kiyoshi Inageda ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Granule Cells ◽  
Pdz Domain ◽  
Postsynaptic Membrane ◽  
Equilibrium Potential ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Kir Channel ◽  
Anchoring Proteins

Classical inwardly rectifying K+ channels (Kir2.0) are responsible for maintaining the resting membrane potential near the K+ equilibrium potential in various cells, including neurons. Although Kir2.3 is known to be expressed abundantly in the forebrain, its precise localization has not been identified. Using an antibody specific to Kir2.3, we examined the subcellular localization of Kir2.3 in mouse brain. Kir2.3 immunoreactivity was detected in a granular pattern in restricted areas of the brain, including the olfactory bulb (OB). Immunoelectron microscopy of the OB revealed that Kir2.3 immunoreactivity was specifically clustered on the postsynaptic membrane of asymmetric synapses between granule cells and mitral/tufted cells. The immunoprecipitants for Kir2.3 obtained from brain contained PSD-95 and chapsyn-110, PDZ domain-containing anchoring proteins. In vitro binding assay further revealed that the COOH-terminal end of Kir2.3 is responsible for the association with these anchoring proteins. Therefore, the Kir channel may be involved in formation of the resting membrane potential of the spines and, thus, would affect the response of N-methyl-d-aspartic acid receptor channels at the excitatory postsynaptic membrane.

Evidence for a Ca-activated inwardly rectifying K channel in human macrophages

1989 ◽  
Vol 257 (1) ◽  
pp. C77-C85 ◽  
Author(s):  
E. K. Gallin
Keyword(s):  
Membrane Potential ◽  
Single Channel ◽  
Reversal Potential ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Base Line ◽  
Channel Activity ◽  
Human Macrophages ◽  
Excised Patches ◽  
Single Channel Currents

Cell-attached patch studies of cultured human macrophages demonstrate that exposure to ionomycin induces inward-rectifying single-channel currents that differ from the voltage-dependent 28 pS inward-rectifying K currents previously described in these cells (J. Membr. Biol. 103: 55-66, 1988). With 150 mM KCl in the electrode and NaCl Hanks' solution in the bath, the ionomycin-induced single-channel conductance for inward currents was 37 pS, and the reversal potential was 57 mV. Channel activity was often associated with a shift in the base-line current level indicating that the cell membrane potential hyperpolarized. The ability of ionomycin to induce channel activity depended on extracellular [Ca] supporting the view that the channels were gated by calcium. Ionomycin-induced channels were permeable to K, relatively impermeable to Cl or Na, exhibited bursting kinetics, and had no apparent voltage dependence. Barium (3 mM in the patch electrode) did not significantly block the ionomycin-induced channel at rest but blocked channel activity when the patch was hyperpolarized beyond the resting membrane potential. Exposure of macrophages to platelet-activating factor, which is known to increase intracellular [Ca] [( Ca]i) (J. Cell Biol. 103: 439-450, 1986), also transiently induced channel activity. In excised patches with 3 microM [Ca]i bursting inward-rectifying channels with a 41 pS conductance were noted that probably correspond to the ionomycin-induced channels present in cell-attached patches. Increasing [Ca]i from 10(-8) to 3 x 10(-6) M induced inward-rectifying channel activity in previously quiescent excised patches.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Single-Cell Electrophysiological Measurements Reveal Bacterial Membrane Potential Dynamics during Extracellular Electron Transfer

2020 ◽  
Author(s):  
Sahand Pirbadian ◽  
Marko S. Chavez ◽  
Mohamed Y. El-Naggar
Keyword(s):  
Electron Transfer ◽  
Membrane Potential ◽  
Single Cell ◽  
Solid Phase ◽  
Electrochemical Techniques ◽  
Model Organisms ◽  
Extracellular Electron Transfer ◽  
Single Cell Level ◽  
Cell Level ◽  
Wide Range

AbstractExtracellular electron transfer (EET) allows microorganisms to gain energy by linking intracellular reactions to external surfaces ranging from natural minerals to the electrodes of bioelectrochemical renewable energy technologies. In the past two decades, electrochemical techniques have been used to investigate EET in a wide range of microbes, with emphasis on dissimilatory metal-reducing bacteria, such as Shewanella oneidensis MR-1, as model organisms. However, due to the typically bulk nature of these techniques, they are unable to reveal the subpopulation variation in EET or link the observed electrochemical currents to energy gain by individual cells, thus overlooking the potentially complex spatial patterns of activity in bioelectrochemical systems. Here, to address these limitations, we use the cell membrane potential as a bioenergetic indicator of EET by S. oneidensis MR-1 cells. Using a fluorescent membrane potential indicator during in vivo single-cell level fluorescence microscopy in a bioelectrochemical reactor, we demonstrate that membrane potential strongly correlates with the electrode potential, produced current, and position of cells relative to the electrodes. The high spatial and temporal resolution of the reported technique can be used to study the single-cell level dynamics of EET not only on electrode surfaces, but also during respiration of other solid-phase electron acceptors.

Developmental differences in Ca2+-activated K+ channel activity in ovine basilar artery

2003 ◽  
Vol 285 (2) ◽  
pp. H701-H709 ◽  
Author(s):  
Mike T. Lin ◽  
David A. Hessinger ◽  
William J. Pearce ◽  
Lawrence D. Longo
Keyword(s):  
Membrane Potential ◽  
Cerebral Arteries ◽  
Outward Current ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Channel Activity ◽  
Open Probability ◽  
Fetal Sheep ◽  
Voltage Gated ◽  
Maximal Activation

A primary determinant of vascular smooth muscle (VSM) tone and contractility is the resting membrane potential, which, in turn, is influenced heavily by K+ channel activity. Previous studies from our laboratory and others have demonstrated differences in the contractility of cerebral arteries from near-term fetal and adult animals. To test the hypothesis that these contractility differences result from maturational changes in voltage-gated K+ channel function, we compared this function in VSM myocytes from adult and fetal sheep cerebral arteries. The primary current-carrying, voltage-gated K+ channels in VSM myocytes are the large conductance Ca2+-activated K+ channels (BKCa) and voltage-activated K+ (KV) channels. We observed that at voltage-clamped membrane potentials of +60 mV in perforated whole cell studies, the normalized outward current densities in fetal myocytes were >30% higher than in those of the adult ( P < 0.05) and that these were predominately due to iberiotoxin-sensitive currents from BKCa channels. Excised, insideout membrane patches revealed nearly identical unitary conductances and Hill coefficients for BKCa channels. The plot of log intracellular [Ca2+] ([Ca2+]i) versus voltage for half-maximal activation ( V½) yielded linear and parallel relationships, and the change in V½ for a 10-fold change in [Ca2+] was also similar. Channel activity increased e-fold for a 19 ± 2-mV depolarization for adult myocytes and for an 18 ± 1-mV depolarization for fetal myocytes ( P > 0.05). However, the relationship between BKCa open probability and membrane potential had a relative leftward shift for the fetal compared with adult myocytes at different [Ca2+]i. The [Ca2+] for half-maximal activation (i.e., the calcium set points) at 0 mV were 8.8 and 4.7 μM for adult and fetal myocytes, respectively. Thus the increased BKCa current density in fetal myocytes appears to result from a lower calcium set point.

An intermediate-conductance Ca2+-activated K+ channel is important for secretion in pancreatic duct cells

2012 ◽  
Vol 303 (2) ◽  
pp. C151-C159 ◽  
Author(s):  
Mikio Hayashi ◽  
Jing Wang ◽  
Susanne E. Hede ◽  
Ivana Novak
Keyword(s):  
Membrane Potential ◽  
Single Channel ◽  
Short Circuit ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Pcr Analysis ◽  
Pancreatic Ducts ◽  
Anion Secretion ◽  
Duct Cells ◽  
Ik Channel

Potassium channels play a vital role in maintaining the membrane potential and the driving force for anion secretion in epithelia. In pancreatic ducts, which secrete bicarbonate-rich fluid, the identity of K+ channels has not been extensively investigated. In this study, we investigated the molecular basis of functional K+ channels in rodent and human pancreatic ducts (Capan-1, PANC-1, and CFPAC-1) using molecular and electrophysiological techniques. RT-PCR analysis revealed mRNAs for KCNQ1, KCNH2, KCNH5, KCNT1, and KCNT2, as well as KCNN4 coding for the following channels: KVLQT1; HERG; EAG2; Slack; Slick; and an intermediate-conductance Ca2+-activated K+ (IK) channel (KCa3.1). The following functional studies were focused on the IK channel. 5,6-Dichloro-1-ethyl-1,3-dihydro-2 H-benzimidazole-2-one (DC-EBIO), an activator of IK channel, increased equivalent short-circuit current ( Isc) in Capan-1 monolayer, consistent with a secretory response. Clotrimazole, a blocker of IK channel, inhibited Isc. IK channel blockers depolarized the membrane potential of cells in microperfused ducts dissected from rodent pancreas. Cell-attached patch-clamp single-channel recordings revealed IK channels with an average conductance of 80 pS in freshly isolated rodent duct cells. These results indicated that the IK channels may, at least in part, be involved in setting the resting membrane potential. Furthermore, the IK channels are involved in anion and potassium transport in stimulated pancreatic ducts.

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