scholarly journals Localization and mobility of the delayed-rectifer K+ channel Kv2.1 in adult cardiomyocytes

2008 ◽  
Vol 294 (1) ◽  
pp. H229-H237 ◽  
Author(s):  
Kristen M. S. O'Connell ◽  
Jennifer D. Whitesell ◽  
Michael M. Tamkun
Keyword(s):  
Hippocampal Neurons ◽  
Intracellular Transport ◽  
Fluorescent Protein ◽  
Ventricular Myocytes ◽  
Recombinant Adenovirus ◽  
Yellow Fluorescent Protein ◽  
K Channel ◽  
Delayed Rectifier ◽  
Transverse Tubules ◽  
Green Fluorescent

The delayed-rectifier voltage-gated K+ channel (Kv) 2.1 underlies the cardiac slow K+ current in the rodent heart and is particularly interesting in that both its function and localization are regulated by many stimuli in neuronal systems. However, standard immunolocalization approaches do not detect cardiac Kv2.1; therefore, little is known regarding its localization in the heart. In the present study, we used recombinant adenovirus to determine the subcellular localization and lateral mobility of green fluorescent protein (GFP)-Kv2.1 and yellow fluorescent protein-Kv1.4 in atrial and ventricular myocytes. In atrial myocytes, Kv2.1 formed large clusters on the cell surface similar to those observed in hippocampal neurons, whereas Kv1.4 was evenly distributed over both the peripheral sarcolemma and the transverse tubules. However, fluorescence recovery after photobleach (FRAP) experiments indicate that atrial Kv2.1 was immobile, whereas Kv1.4 was mobile (τ = 252 ± 42 s). In ventricular myocytes, Kv2.1 did not form clusters and was localized primarily in the transverse-axial tubules and sarcolemma. In contrast, Kv1.4 was found only in transverse tubules and sarcolemma. FRAP studies revealed that Kv2.1 has a higher mobility in ventricular myocytes (τ = 479 ± 178 s), although its mobility is slower than Kv1.4 (τ1 = 18.9 ± 2.3 s; τ2 = 305 ± 55 s). We also observed the movement of small, intracellular transport vesicles containing GFP-Kv2.1 within ventricular myocytes. These data are the first evidence of Kv2.1 localization in living myocytes and indicate that Kv2.1 may have distinct physiological roles in atrial and ventricular myocytes.

scholarly journals Axonal Membrane Proteins Are Transported in Distinct Carriers: A Two-Color Video Microscopy Study in Cultured Hippocampal Neurons

2000 ◽  
Vol 11 (4) ◽  
pp. 1213-1224 ◽  
Author(s):  
Christoph Kaether ◽  
Paul Skehel ◽  
Carlos G. Dotti
Keyword(s):  
Membrane Proteins ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Microscopy Study ◽  
Video Microscopy ◽  
Axonal Membrane ◽  
Color Video ◽  
Green Fluorescent ◽  
Living Neurons

Neurons transport newly synthesized membrane proteins along axons by microtubule-mediated fast axonal transport. Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants. The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 μm/s) and over long distances. In contrast, p38-enhanced GFP-transporting structures were more vesicular and moved four times slower (0.9 μm/s) and over shorter distances only. Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons. Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes. These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier.

GABAC ρ1 Subunits Form Functional Receptors But Not Functional Synapses in Hippocampal Neurons

2001 ◽  
Vol 86 (5) ◽  
pp. 2605-2615 ◽  
Author(s):  
Qing Cheng ◽  
Paul M. Burkat ◽  
John C. Kulli ◽  
Jay Yang
Keyword(s):  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Recombinant Adenovirus ◽  
Short Pulse ◽  
Gaba Release ◽  
Ruthenium Red ◽  
Confocal Imaging ◽  
Imaging Data ◽  
Whole Cell Patch Clamp ◽  
Green Fluorescent

The ability to control the physiological and pharmacological properties of synaptic receptors is a powerful tool for studying neuronal function and may be of therapeutic utility. We designed a recombinant adenovirus to deliver either a GABAC receptor ρ1 subunit or a mutant GABAA receptor β2 subunit lacking picrotoxin sensitivity [β2(mut)] to hippocampal neurons. A green fluorescent protein (GFP) reporter molecule was simultaneously expressed. Whole cell patch-clamp recordings demonstrated somatic expression of both bicuculline-resistant GABAC receptor-mediated and picrotoxin-resistant GABAA receptor-mediated GABA-evoked currents in ρ1- and β2(mut)-transduced hippocampal neurons, respectively. GABAergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in the presence of 6-cyano-7-nitroquinoxalene-2,3-dione, Mg2+, and TTX revealed synaptic events with monoexponential activation and biexponential decay phases. Despite the robust expression of somatic GABAC receptors in ρ1-neurons, no bicuculline-resistant mIPSCs were observed. This suggested either a kinetic mismatch between the relatively brief presynaptic GABA release and slow-activating ρ1 receptors or failure of the ρ1 subunit to target properly to the subsynaptic membrane. Addition of ruthenium red, a presynaptic release enhancer, failed to unmask GABACreceptor-mediated mIPSCs. Short pulse (2 ms) application of 1 mM GABA to excised outside-out patches from ρ1 neurons proved that a brief GABA transient is sufficient to activate ρ1 receptors. The simulated-IPSC experiment strongly suggests that if postsynaptic GABACreceptors were present, bicuculline-resistant mIPSCs would have been observed. In contrast, in β2(mut)-transduced neurons, picrotoxin-resistant mIPSCs were observed; they exhibited a smaller peak amplitude and faster decay compared with control. Confocal imaging of transduced neurons revealed ρ1immunofluorescence restricted to the soma, whereas punctate β2(mut) immunofluorescence was seen throughout the neuron, including the dendrites. Together, the electrophysiological and imaging data show that despite robust somatic expression of the ρ1 subunit, the GABACreceptor fails to be delivered to the subsynaptic target. On the other hand, the successful incorporation of β2(mut) subunits into subsynaptic GABAA receptors demonstrates that viral transduction is a powerful method for altering the physiological properties of synapses.

A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family

2011 ◽  
Vol 441 (1) ◽  
pp. 209-217 ◽  
Author(s):  
Iraia García-Santisteban ◽  
Sonia Bañuelos ◽  
Jose A. Rodríguez
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
The Novel ◽  
Sequence Motifs ◽  
Leptomycin B ◽  
Specific Amino Acid ◽  
Green Fluorescent ◽  
Nuclear Export Receptor

The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export receptor CRM1 binds to specific amino acid motifs termed NESs (nuclear export sequences). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These results allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified in the present study reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor LMB (leptomycin B) on the localization of YFP (yellow fluorescent protein)- or GFP (green fluorescent protein)-tagged versions of six NES-bearing deubiquitinases [USP (ubiquitin-specific peptidase) 1, USP3, USP7, USP21, CYLD (cylindromatosis) and OTUD7B (OTU-domain-containing 7B)]. YFP–USP21 and, to a lesser extent, GFP–OTUD7B relocated from the cytoplasm to the nucleus in the presence of LMB, revealing their nucleocytoplasmic shuttling capability. Two sequence motifs in USP21 had been identified during our survey as active NESs in the export assay. Using site-directed mutagenesis, we show that one of these motifs mediates USP21 nuclear export, whereas the second motif is not functional in the context of full-length USP21.

Characterization of an early afterhyperpolarization after a brief train of action potentials in rat hippocampal neurons in vitro

1990 ◽  
Vol 63 (1) ◽  
pp. 72-81 ◽  
Author(s):  
A. Williamson ◽  
B. E. Alger
Keyword(s):  
Action Potentials ◽  
Hippocampal Neurons ◽  
Pyramidal Cells ◽  
Chloride Ions ◽  
Membrane Potentials ◽  
Resting Potential ◽  
K Channel ◽  
Delayed Rectifier ◽  
Extracellular Potassium

1. In rat hippocampal pyramidal cells in vitro, a brief train of action potentials elicited by direct depolarizing current pulses injected through an intracellular recording electrode is followed by a medium-duration afterhyperpolarization (mAHP) and a longer, slow AHP. We studied the mAHP with the use of current-clamp techniques in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) to block the slow AHP and isolate the mAHP. 2. The mAHP evoked at hyperpolarized membrane potentials was complicated by a potential generated by the anomalous rectifier current, IQ. The mAHP is insensitive to chloride ions (Cl-), whereas it is sensitive to the extracellular potassium concentration ([K+]o). 3. At slightly depolarized levels, the mAHP is partially Ca2+ dependent, being enhanced by increased [Ca2+]o and BAY K 8644 and depressed by decreased [Ca2+]o, nifedipine, and Cd2+. The Ca2(+)-dependent component of the mAHP was also reduced by 100 microM tetraethylammonium (TEA) and charybdotoxin (CTX), suggesting it is mediated by the voltage- and Ca2(+)-dependent K+ current, IC. 4. Most of the Ca2(+)-independent mAHP was blocked by carbachol, implying that IM plays a major role. In a few cells, a small Ca2(+)- and carbachol-insensitive mAHP component was detectable, and this component was blocked by 10 mM TEA, suggesting it was mediated by the delayed rectifier current, IK. The K+ channel antagonist 4-aminopyridine (4-AP, 500 microM) did not reduce the mAHP. 5. We infer that the mAHP is a complex potential due either to IQ or to the combined effects of IM and IC. The contributions of each current depend on the recording conditions, with IC playing a role when the cells are activated from depolarized potentials and IM dominating at the usual resting potential. IQ is principally responsible for the mAHP recorded at hyperpolarized membrane potentials.

Properties of voltage-gated Ca2+ channels in rabbit ventricular myocytes expressing Ca2+ channel α1E cDNA

2001 ◽  
Vol 280 (1) ◽  
pp. C175-C182 ◽  
Author(s):  
Michihiro Tateyama ◽  
Shuqin Zong ◽  
Tsutomu Tanabe ◽  
Rikuo Ochi
Keyword(s):  
Cardiac Myocytes ◽  
Fluorescent Protein ◽  
Ventricular Myocytes ◽  
Foreign Gene ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Green Fluorescent ◽  
Ca2 Channels

Using the whole-cell patch-clamp technique, we have studied the properties of α1ECa2+ channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current ( I Ca,L). Expression of green fluorescent protein significantly decreased the I Ca,L. By contrast, expression of α1E with β2b and α2/δ significantly increased the total Ca2+ current, and in these cells a Ca2+ antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni2+and was little affected by changing the charge carrier from Ca2+ to Ba2+ or by β-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of α1E did not generate T-like current in cardiac myocytes. On the other hand, expression of α1E decreased I Ca,L and slowed the I Ca,L inactivation. This inactivation slowing was attenuated by the β2b coexpression, suggesting that the α1E may slow the inactivation of I Ca,L by scrambling with α1C for intrinsic auxiliary β.

High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells

2003 ◽  
Vol 284 (5) ◽  
pp. H1647-H1654 ◽  
Author(s):  
Jean-Philippe Fortin ◽  
Johanne Bouthillier ◽  
François Marceau
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Brefeldin A ◽  
Metabolic Inhibitors ◽  
Maximal Effect ◽  
Hek 293 ◽  
B1 Receptors ◽  
Green Fluorescent

We hypothesized that the inducible kinin B1 receptor (B1R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B1Rs and related B2 receptors (B2Rs) was investigated. Endocytosis of the B1R-yellow fluorescent protein (YFP) conjugate was more intense than that of B2R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B1R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B2R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B1R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B2R-GFP remained constant. Wild-type B1Rs were also cleared faster than B2Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B1Rs are more rapidly eliminated than B2Rs (decreased maximal effect of agonist over 2 h). The highly regulated B1R is rapidly degraded, relative to the constitutive B2R.

NRSF Causes cAMP-Sensitive Suppression of Sodium Current in Cultured Hippocampal Neurons

2002 ◽  
Vol 88 (1) ◽  
pp. 409-421 ◽  
Author(s):  
H. Nadeau ◽  
H. A. Lester
Keyword(s):  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Sodium Current ◽  
Striking Similarity ◽  
Secondary Effects ◽  
Functional Effect ◽  
Green Fluorescent ◽  
Altered Sensitivity ◽  
Cultured Hippocampal Neurons

The neuron restrictive silencer factor (NRSF/REST) has been shown to bind to the promoters of many neuron-specific genes and is able to suppress transcription of Na+channels in PC12 cells, although its functional effect in terminally differentiated neurons is unknown. We constructed lentiviral vectors to express NRSF as a bicistronic message with green fluorescent protein (GFP) and followed infected hippocampal neurons in culture over a period of 1–2 wk. NRSF-expressing neurons showed a time-dependent suppression of Na+channel function as measured by whole cell electrophysiology. Suppression was reversed or prevented by the addition of membrane-permeable cAMP analogues and enhanced by cAMP antagonists but not affected by increasing protein expression with a viral enhancer. Secondary effects, including altered sensitivity to glutamate and GABA and reduced outward K+currents, were duplicated by culturing GFP-infected control neurons in TTX. The striking similarity of the phenotypes makes NRSF potentially useful as a genetic “silencer” and also suggests avenues of further exploration that may elucidate the transcription factor's in vivo role in neuronal plasticity.

ROMK1 (Kir1.1) Causes Apoptosis and Chronic Silencing of Hippocampal Neurons

2000 ◽  
Vol 84 (2) ◽  
pp. 1062-1075 ◽  
Author(s):  
H. Nadeau ◽  
S. McKinney ◽  
D. J. Anderson ◽  
H. A. Lester
Keyword(s):  
Cell Death ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Input Resistance ◽  
Firing Frequency ◽  
Compensatory Mechanisms ◽  
Spike Shape ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Potential Threshold

Lentiviral vectors were constructed to express the weakly rectifying kidney K+ channel ROMK1 (Kir1.1), either fused to enhanced green fluorescent protein (EGFP) or as a bicistronic message (ROMK1-CITE-EGFP). The channel was stably expressed in cultured rat hippocampal neurons. Infected cells were maintained for 2–4 wk without decrease in expression level or evidence of viral toxicity, although 15.4 mM external KCl was required to prevent apoptosis of neurons expressing functional ROMK1. No other trophic agents tested could prevent cell death, which was probably caused by K+loss. This cell death did not occur in glia, which were able to support ROMK1 expression indefinitely. Functional ROMK1, quantified as the nonnative inward current at −144 mV in 5.4 mM external K+blockable by 500 μM Ba2+, ranged from 1 to 40 pA/pF. Infected neurons exhibited a Ba2+-induced depolarization of 7 ± 2 mV relative to matched EGFP-infected controls, as well as a 30% decrease in input resistance and a shift in action potential threshold of 2.6 ± 0.5 mV. This led to a shift in the relation between injected current and firing frequency, without changes in spike shape, size, or timing. This shift, which quantifies silencing as a function of ROMK1 expression, was predicted from Hodgkin-Huxley models. No cellular compensatory mechanisms in response to expression of ROMK1 were identified, making ROMK1 potentially useful for transgenic studies of silencing and neurodegeneration, although its lethality in normal K+ has implications for the use of K+ channels in gene therapy.

scholarly journals pH of the Cytoplasm and Periplasm of Escherichia coli: Rapid Measurement by Green Fluorescent Protein Fluorimetry

2007 ◽  
Vol 189 (15) ◽  
pp. 5601-5607 ◽  
Author(s):  
Jessica C. Wilks ◽  
Joan L. Slonczewski
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Time Resolution ◽  
Fluorescent Protein ◽  
Osmotic Shock ◽  
Yellow Fluorescent Protein ◽  
Fluorescence Signal ◽  
Cytoplasmic Ph ◽  
Green Fluorescent ◽  
External Ph

ABSTRACT Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the external pH was lowered from pH 7.5 to 5.5, the cytoplasmic pH fell within 10 to 20 s to pH 5.6 to 6.5. Rapid recovery occurred until about 30 s after HCl addition and was followed by slower recovery over the next 5 min. As a control, KCl addition had no effect on fluorescence. In the presence of 5 to 10 mM acetate or benzoate, recovery from external acidification was diminished. Addition of benzoate at pH 7.0 resulted in cytoplasmic acidification with only slow recovery. Periplasmic pH was observed using TorA-GFPmut3* exported to the periplasm through the Tat system. The periplasmic location of the fusion protein was confirmed by the observation that osmotic shock greatly decreased the periplasmic fluorescence signal by loss of the protein but had no effect on the fluorescence of the cytoplasmic protein. Based on GFPmut3* fluorescence, the pH of the periplasm equaled the external pH under all conditions tested, including rapid acid shift. Benzoate addition had no effect on periplasmic pH. The cytoplasmic pH of E. coli was measured with 4-s time resolution using a method that can be applied to any strain construct, and the periplasmic pH was measured directly for the first time.

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