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Results for “patch-clamp”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1996

Laser microsurgery of higher plant cell walls permits patch-clamp access

Plasma membranes of guard cells in epidermal peels of Vicia faba and Commelina communis can be made accessible to a patch-clamp pipet by removing a small portion (1-3 micrometers in diameter) of the guard cell wall using a microbeam of ultraviolet light generated by a nitrogen laser. Using this laser microsurgical technique, we have measured channel activity across plasma membranes of V. faba guard cells in both cell-attached and isolated patch configurations. Measurements made in the inside-out patch configuration revealed two distinct K(+)-selective channels. Major advantages of the laser microsurgical technique include the avoidance of enzymatic protoplast isolation, the ability to study cell types that have been difficult to isolate as protoplasts or for which enzymatic isolation protocols result in protoplasts not amenable to patch-clamp studies, the maintenance of positional information in single-channel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling through studies of cells remaining situated within tissue.

Non-NASA Center↗
NASA NTRS1996

Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells

By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.

NASA Discipline Musculoskeletal↗
NASA NTRS2000

Extracellular calcium-sensing-receptor (CaR)-mediated opening of an outward K(+) channel in murine MC3T3-E1 osteoblastic cells: evidence for expression of a functional CaR

The existence in osteoblasts of the G-protein-coupled extracellular calcium (Ca(o)(2+))-sensing receptor (CaR) that was originally cloned from parathyroid and kidney remains controversial. In our recent studies, we utilized multiple detection methods to demonstrate the expression of CaR transcripts and protein in several osteoblastic cell lines, including murine MC3T3-E1 cells. Although we and others have shown that high Ca(o)(2+) and other polycationic CaR agonists modulate the function of MC3T3-E1 cells, none of these actions has been unequivocally shown to be mediated by the CaR. Previous investigations using neurons and lens epithelial cells have shown that activation of the CaR stimulates Ca(2+)-activated K(+) channels. Because osteoblastic cells express a similar type of channel, we have examined the effects of specific "calcimimetic" CaR activators on the activity of a Ca(2+)-activated K(+) channel in MC3T3-E1 cells as a way of showing that the CaR is not only expressed in those cells but is functionally active. Patch-clamp analysis in the cell-attached mode showed that raising Ca(o)(2+) from 0.75 to 2.75 mmol/L elicited about a fourfold increase in the open state probability (P(o)) of an outward K(+) channel with a conductance of approximately 92 pS. The selective calcimimetic CaR activator, NPS R-467 (0.5 micromol/L), evoked a similar activation of the channel, while its less active stereoisomer, NPSS-467 (0.5 micromol/L), did not. Thus, the CaR is not only expressed in MC3T3-E1 cells, but is also functionally coupled to the activity of a Ca(2+)-activated K(+) channel. This receptor, therefore, could transduce local or systemic changes in Ca(o)(2+) into changes in the activity of this ion channel and related physiological processes in these and perhaps other osteoblastic cells.

NASA Discipline Musculoskeletal↗
NASA NTRS1994

Chronic, intermittent loading alters mechanosensitive channel characteristics in osteoblast-like cells

The effects of chronic, intermittent strain on the mechanosensitive cation (SA-cat) channels in UMR-106.01 osteoblast-like osteosarcoma cells were studied using patch-clamp techniques. Chronically strained cells demonstrated significantly larger increases in whole cell conductance when subjected to additional mechanical strain than nonstrained controls (69.0 +/- 15.1 vs. 14.1 +/- 3.1%; P < 0.001). This increase could be blocked by the SA-cat channel inhibitor, gadolinium, and corresponded to a three- to fivefold increase in SA-cat channel activity. Chronic strain increased the number of open channels in response to stretch and induced spontaneous SA-cat channel activity in 33% of the patches of strained cells. Graded increases in negative patch pressure demonstrated that SA-cat channels in chronically strained cells were activated at significantly lower levels of mechanical perturbation than nonstrained controls. These data suggest that chronic, cyclic strain reduces the activation threshold of the SA-cat channel and further strengthen our hypothesis that this channel may act as a mechanotransducer for the activation of bone remodeling by physical strain.

NASA Discipline Musculoskeletal↗
NASA NTRS1999

A laser microsurgical method of cell wall removal allows detection of large-conductance ion channels in the guard cell plasma membrane

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.

Non-NASA Center↗
NASA NTRS1993

Aluminium and hydrogen ions inhibit a mechanosensory calcium-selective cation channel

The tension-dependent activity of mechanosensory calcium-selective cation channels in excised plasmalemmal patches from onion bulb scale epidermis is modulated by pH in the physiologically meaningful range between 4.5 and 7.2. It is rapidly lowered by lowering pH and rapidly raised by raising pH. Channel activity is effectively inhibited by low levels of aluminium ions and activity can be partially restored by washing for a few minutes. We suggest that under normal conditions the sensitivity of the mechanosensory channels to pH of the wall free space plays important roles in regulation of plant activities such as growth. We further suggest that, when levels of acid and aluminium ions in the soil solution are high, they might inhibit similar sensory channels in cells of the root tip, thus contributing critically to the acid soil syndrome.

NASA Discipline Plant Biology↗
NASA NTRS2000

Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

NASA Discipline Plant Biology↗
NASA NTRS1999

Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was DeltaE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (DeltaA), the free energy, TDeltaA, would correspond to DeltaA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest DeltaA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to DeltaA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.

Non-NASA Center↗
NASA NTRS1999

Characterization of ionic currents of cells of the subfornical organ that project to the supraoptic nuclei

The subfornical organ (SFO) is a forebrain structure that converts peripheral blood-borne signals reflecting the hydrational state of the body to neural signals and then through efferent fibers conveys this information to several central nervous system structures. One of the forebrain areas receiving input from the SFO is the supraoptic nucleus (SON), a source of vasopressin synthesis and control of release from the posterior pituitary. Little is known of the transduction and transmission processes by which this conversion of systemic information to brain input occurs. As a step in elucidating these mechanisms, the present study characterized the ionic currents of dissociated cells of the SFO that were identified as neurons that send efferents to the SON. A retrograde tracer was injected into the SON area in eleven-day-old rats. After three days for retrograde transport of the label, the SFOs of these animals were dissociated and plated for tissue culture. The retrograde tracer was used to identify the soma of SFO cells projecting to the SON so that voltage-dependent ionic currents using whole-cell voltage clamp methods could be studied. The three types of currents in labeled SFO neurons were characterized as a 1) rapid, transient inward current that can be blocked by tetrodotoxin (TTX) characteristic of a sodium current; 2) slow-onset sustained outward current that can be blocked by tetraethylammonium (TEA) characteristic of a delayed rectifier potassium current; and 3) remaining outward current that has a rapid-onset and transient characteristic of a potassium A-type current. Copyright 1999 Elsevier Science B.V.

Non-NASA Center↗
NASA NTRS1998

Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

NASA Discipline Regulatory Physiology↗
NASA NTRS1997

Laser-assisted patch clamping: a methodology

Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

Non-NASA Center↗
NASA NTRS1996

An anion channel in Arabidopsis hypocotyls activated by blue light

A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition.

NASA Discipline Plant Biology↗
NASA NTRS1996

Electrical filtering in gerbil isolated type I semicircular canal hair cells

1. Membrane potential responses of dissociated gerbil type I semicircular canal hair cells to current injections in whole cell current-clamp have been measured. The input resistance of type I cells was 21.4 +/- 14.3 (SD) M omega, (n = 25). Around the zero-current potential (Vz = -66.6 +/- 9.3 mV, n = 25), pulsed current injections (from approximately -200 to 750 pA) produced only small-amplitude, pulse-like changes in membrane potential. 2. Injecting constant current to hyperpolarize the membrane to around -100 mV resulted in a approximately 10-fold increase in membrane resistance. Current pulses superimposed on this constant hyperpolarization produced larger and more complex membrane potential changes. Depolarizing currents > or = 200 pA caused a rapid transient peak voltage before a plateau. 3. Membrane voltage was able to faithfully follow sine-wave current injections around Vz over the range 1-1,000 Hz with < 25% attenuation at 1 kHz. A previously described K conductance, IKI, which is active at Vz, produces the low input resistance and frequency response. This was confirmed by pharmacologically blocking IKI. This conductance, present in type I cells but not type II hair cells, would appear to confer on type I cells a lower gain, but a much broader bandwidth at Vz, than seen in type II cells.

Non-NASA Center↗
NASA NTRS1995

Regulation of cyclic nucleotide-gated channels and membrane excitability in olfactory receptor cells by carbon monoxide

1. The effect of the putative neural messenger carbon monoxide (CO) and the role of the cGMP second-messenger system for olfactory signal generation was examined in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. With the use of whole cell voltage-clamp recordings in combination with a series of ionic and pharmological tests, it is demonstrated that exogenously applied CO is a potent activator (K1/2 = 2.9 microM) of cyclic nucleotide-gated (CNG) channels previously described to mediate odor transduction. 3. Several lines of evidence suggest that CO mediates its effect through stimulation of a soluble guanylyl cyclase (sGC) leading to formation of the second-messenger cGMP. This conclusion is based on the findings that CO responses show an absolute requirement for guanosine 5'-triphosphate (GTP) in the internal solution, that no direct effect of CO on CNG currents in the absence of GTP is detectable, and that a blocker of sGC activation, LY85383 (10 microM), completely inhibits the CO response. 4. The dose-response curve for cGMP at CNG channels is used as a calibration to provide a quantitative estimate of the CO-stimulated cGMP formation. This analysis implies that CO is a potent activator of olfactory sGC. 5. Perforated patch recordings using amphotericin B demonstrate that low micromolar doses of CO effectively depolarize the membrane potential of ORNs through tonic activation of CNG channels. This effect in turn regulates excitable and adaptive properties of ORNs and modulates neuronal responsiveness. 6. These data argue for an important role of the cGMP pathway in olfactory signaling and support the idea that CO may function as a diffusible messenger in the olfactory system.

NASA Discipline Neuroscience↗