A new clamp for patch graft angioplasty of coarctation of the aorta.
A new clamp controls the great vessels in the region of aortic coarctation, allowing unhampered access to the aortotomy in patch graft angioplasty.
Aorta, Thoracic↗
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Stretch-activated channels in heart cells: relevance to cardiac hypertrophy.
Stretch-activated channels have been proposed as the transduction mechanism between load and protein synthesis in cardiac hypertrophy. Under this hypothesis, cardiac deformation is linked to an increased sodium (Na) influx, which, in turn, increases protein synthesis. We have tested whether stretch actually increases Na influx by applying patch-clamp techniques to cultured chick embryo cardiac myocytes and to freshly isolated adult guinea pig cardiomyocytes. Our experiments, in excised and cell-attached patches, revealed the existence of ionic channels that opened, or increased their frequency of opening, upon the application of negative pressures to the lumen of the patch-clamp pipettes. These stretch-sensitive channels allowed the passage of the major monovalent physiological cations, Na and potassium (K), and, to a much lesser extent, the major divalent cations calcium (Ca) and magnesium (Mg). Under normal conditions, the channels had a high open channel noise that prevented the customary, straightforward statistical analysis of single channel data. However, when one of the major monovalent cations was iso-osmotically replaced by sucrose, the open channel noise decreased significantly and permitted a good delineation of the open and closed channel states and, therefore, application of standard patch-clamp, statistical analysis techniques. Under these "sucrose," "monoionic" conditions, the reversal potential was, as one should expect, close to the equilibrium potential for the major monovalent cation present. When high extracellular K solution was used to minimize the cell resting potential, the reversal potential for these stretch-activated currents was estimated to be around -40 mV. Therefore, under normal conditions, stretch should induce an inward, depolarizing current, carried mostly by Na ions.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Role of membrane depolarization and T-type Ca2+ channels in angiotensin II and K+ stimulated aldosterone secretion.
The hypothesis that Ca2+ influx necessary for angiotensin II (AngII) and K+ stimulation of aldosterone secretion is primarily mediated by membrane depolarization and activation of T-type Ca2+ channels was examined in isolated rat adrenal glomerulosa cells. Perforated-patch clamp recordings of membrane potential (Vm) demonstrated that AngII and K+ induce concentration-dependent depolarizations capable of activating T channels and, at high K+ and AngII concentrations, activating L channels and inactivating T channels. K+-induced depolarizations were stable and readily reversible. Vm was proportional to K+ concentration, exhibiting a linear slope of 53.7 mV per 10-fold increase in K+. AngII-induced depolarizations were complex, consisting of a slow maintained component superimposed with small amplitude depolarizing fluctuations. Slow oscillations in Vm were occasionally observed in response to 10(-9) M AngII or greater. The slow, maintained component of depolarization coincided with inhibition of K+ conductance. Neither rapid fluctuations nor slow oscillations in Vm were blocked by mibefradil or other treatments that inhibit voltage-gated Ca2+ channels. Perforated-patch clamp experiments also demonstrated that AngII (10(-8) M) inhibited L channels by 45.6% without affecting T channels. Thus AngII activates T channels by depolarization rather than T channel modulation in rat cells. The concentration dependencies of mibefradil inhibition of T channels and AngII- and K+-induced aldosterone secretion were compared. Under whole-cell patch clamp mibefradil induced a concentration-dependent inhibition of T channels, exhibiting a K(app) of 0.62 microM. Mibefradil inhibition was use-dependent but mibefradil neither acted as an open channel blocker nor significantly affected T channel inactivation or activation. Mibefradil inhibited K+- and AngII-induced secretion at concentrations similar to that for T channel inhibition; at high concentrations (10 microM) mibefradil inhibited AngII-induced secretion by 88% and completely inhibited K+-induced secretion. The IC50 for K+-induced secretion was dependent on K+ concentration, increasing from 0.2 microM for 6 mM K+ to 2.5 microM for 10 mM K+ or greater. Mibefradil exhibited an IC50 of 1.1 microM for inhibition of secretion at all AngII concentrations examined (0.1, 1.0, and 10 nM). Mibefradil also exhibited multiple nonspecific effects, which complicated the assessment of T channel function, including; inhibition of leak and voltage-dependent K+ conductances, inhibition of Ca2+-independent aldosterone secretion, and inhibition of secretion under conditions expected to completely inactivate T channels (10 nM AngII or 20 mM K+). In summary, these results indicate that voltage-gated T channels represent the primary Ca2+ influx pathway activated by physiological concentrations of AngII and K+ but other Ca2+ influx pathways must mediate aldosterone secretion induced by high K+ or AngII concentrations.
Aldosterone↗
Long-term induction of a unique C1- current by endothelin-1 in an epithelial cell line from rat lung: evidence for regulation of cytoplasmic calcium.
1. Using conventional microelectrodes, the perforated patch clamp technique and fluorescence microscopy with fura-2, we investigated the relationship between the cell membrane potential, whole-cell currents and the free cytoplasmic Ca2+ concentration ([Ca2+]i) in response to 10 nM endothelin-1 (ET) in a rat respiratory epithelial cell line (L2). 2. Microelectrode experiments revealed that ET caused an immediate depolarization of the cell membrane potential (Vm) by 25 mV, which was unaffected by Na+ replacement with N-methyl-D-glucamine+ (NMDG+) or by omission of bath Ca2+. In contrast, ET depolarized the cells by 61 mV in the presence of low C1- (6 mM), resulting in a complete breakdown of Vm. 3. In perforated patch clamp experiments, the ET-induced whole-cell current (IET) exhibited a slight outward rectification with a reversal potential (Vrev) of -22.7 mV. IET was reduced by 85 % in low C1- (6 mM), but was unaffected by Ca2+ removal, Na+ replacement with NMDG+, pipette K+ replacement with Cs+ or 1 mM Ni2+ in the bath. 4. IET was unaffected by (+)-isradipine (100 nM), a specific L-type Ca2+ channel (L-VDCC) blocker. Transient inward Sr2+ currents through L-VDCCs were blocked by ET. 5. ET induced a biphasic Ca2+ signal, consisting of a 'peak' and a 'plateau' elevation of [Ca2+]i. Simultaneous patch clamp and fura-2 measurements revealed that IET coincided with intracellular Ca2+ release but clearly outlasted the elevation of [Ca2+]i. When the rise of [Ca2+]i was prevented by pretreatment with thapsigargin in a Ca2+-free bath, both activation time and amplitude of IET were reduced. Under these conditions, ET caused a decrease of [Ca2+]i. 6. The C1- channel blocker mefenamic acid (MFA) had a dual, concentration-dependent effect on both IET and the ET-induced 'plateau' elevation of [Ca2+]i: an increase at 10 microM, but an almost complete block at 100 microM. The effect of MFA on IET preceded the effect on [Ca2+]i. 7. The ET-induced 'plateau' [Ca2+]i fell below control values in a low-C1- (6 mM) solution. 8. These data indicate an amplifying function of intracellular Ca2+ release on an otherwise Ca2+-independent, unique C1- current by ET. Moreover, this C1- current appears to be functionally coupled with dihydropyridine (DHP)-insensitive Ca2+ entry, suggesting a modulatory role for long-lasting effects of ET.
Animals↗
Ca2+ dependence of the Ca2+-selective TRPV6 channel.
Microfluorimetry and patch-clamp experiments were performed on TRPV6-expressing HEK cells to determine whether this Ca(2+)-sensing Ca(2+) channel is constitutively active. Intact cells loaded with fura-2 had an elevated intracellular free Ca(2+) concentration ([Ca(2+)](i)), which decreased to the same level such as in non-transfected cells if external Ca(2+) was chelated by EGTA. Whole cell recordings from non-transfected HEK cells and cells expressing human TRPV6 revealed the presence of a basal inward current in both types of cells when the internal solution contained 0.1 mm EGTA and 100 nm [Ca(2+)](i) or if the cytosolic Ca(2+) buffering remained undisturbed in perforated patch-clamp experiments. If recombinantly expressed TRPV6 forms open channels, one would expect Ca(2+)-induced current inhibition, because TRPV6 is negatively regulated by internal Ca(2+). However, dialyzing solutions with high [Ca(2+)] such as 1 microm into TRPV6-expressing cells did not block the basal inward current, which was not different from the recordings from non-transfected cells. In contrast, dialyzing 0.5 mm EGTA into TRPV6-expressing cells readily activated Ca(2+) inward currents, which were undetectable in non-transfected cells. Interestingly, monovalent cations permeated the TRPV6 channels under conditions where no Ca(2+) permeation was detectable, indicating that divalent cations block TRPV6 channels from the extracellular side. Like human TRPV6, the truncated human TRPV6(Delta695-725), which lacks the C-terminal domain required for Ca(2+)-calmodulin binding, does not form constitutive active channels, whereas the human TRPV6(D542A), carrying a point mutation in the presumed pore region, does not function as a channel. In summary, no constitutive open TRPV6 channels were detected in patch-clamp experiments from transfected HEK cells. However, channel activity is highly regulated by intracellular and extracellular divalent cations.
Animals↗
Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.
The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.
Animals↗
Single K+ channels in membrane evaginations of smooth muscle cells.
Attempts have been made to apply the patch-clamp technique to enzymatically dispersed smooth muscle cells of frog and toad stomach. The rate of successful gigaseal formation has been extremely low, but better results can be obtained when patches are taken from membrane evaginations which develop on single cells after mechanical agitation and incubation in Ca2+-containing solutions at 25 degrees C. Also ball-shaped single cells formed by the confluence of membrane evaginations were found to be equally useful for patch-clamp studies. Giga-seal formation was obtained in more than 80% of all attempts. Electron micrographs indicate that the myofilaments in membrane evaginations and in ball-shaped cells are separated from the cell membrane. Channel activity in membrane patches of such "myoballs" or evaginations is similar to the channel activity as found in intact cells. Two types of K+ channels (100 and 200 pS) have been observed that can be blocked by tetraethylammonium. Channels with the conductance of 200 pS are activated by intracellular Ca2+. The formation of evaginations has also been observed in other cells and may help to apply the patch-clamp technique to cells contaminated with surface coats.
Animals↗
Microchip technology in ion-channel research.
The electrical activity of living cells can be monitored in various ways, but for the study of ion channels and the drugs that affect them, the patch-clamp techniques are the most sensitive. Recent developments in microfabricated patch-clamp electrodes are reviewed, and technical challenges for the future are discussed.
Action Potentials↗
Developmental regulation of a novel outwardly rectifying mechanosensitive anion channel in Caenorhabditis elegans.
The nematode Caenorhabditis elegans offers unique experimental advantages for defining the molecular basis of anion channel function and regulation. However, the relative inaccessibility of somatic cells in adult animals greatly limits direct electrophysiological studies of channel activity. We developed methods to routinely isolate and patch clamp C. elegans embryo cells and oocytes and to culture and patch clamp neurons and muscle cells. Dissociated embryonic cells express a robust outwardly rectifying anion current that is activated by membrane stretch and depolarization. This current, termed I(Cl,mec), is inhibited by anion and mechanosensitive channel inhibitors. I(Cl,mec) has broad anion selectivity and the channel has a unitary conductance of 5-7 picosiemens. I(Cl,mec) is not detectable in whole-cell or isolated patch recordings from oocytes, cultured muscle cells, and cultured neurons but is expressed in single cell and later embryos. Channel density is high, and the current is observed in >80% of membrane patches. Macroscopic currents of 40-120 pA at +100 mV are typically observed in inside-out membrane patches formed using low resistance patch pipettes. Isolated membrane patches of early embryonic cells therefore contain 60-200 I(Cl,mec) channels. The apparent activation of I(Cl,mec) shortly after fertilization and its down-regulation in terminally differentiated cells suggests that the channel may play important roles in embryogenesis and/or cytokinesis.
Animals↗
Characterization of voltage-gated, whole-cell ionic currents from conjunctival epithelial cells.
PURPOSE: These studies were performed to characterize the voltage-gated, whole-cell ionic currents in rabbit bulbar conjunctival epithelial and goblet cells. METHODS: New Zealand White rabbits were killed, and the bulbar conjunctiva was isolated. Conjunctival cells were dissociated for patch clamp analysis of whole-cell currents. The amphotericin, perforated-patch, whole-cell technique was used. RESULTS: Conjunctival epithelial cells had a mean capacitance of 6.72 pF (SE = 0.49; n = 25). The primary currents found were an inwardly rectifying K+ current, a saturating K+ current, and an outwardly rectifying nonselective cation current. A second nonselective cation current also appeared to be present. The inward current was observed in a KCl Ringer's bath and was almost nonexistent in a NaCl bath. The current was Ba(2+)- and Cs(+)-sensitive. The second K+ current became saturated at depolarized voltages and was Ba(2+)- and quinidine-sensitive. The first outward nonselective cation current was typically less than 100 pA in amplitude and activated at voltages positive to 0 mV. Tail current experiments showed that the current was cation selective. The current was blocked by Gd3+ but not by the Cl- current blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid. The second nonselective cation current was larger and Gd(3+)-insensitive. The primary current observed in goblet cells was a large outward K+ current. CONCLUSIONS: The primary currents observed during whole-cell patch clamping of bulbar conjunctival epithelium are a Ba(2+)- and Cs(+)-sensitive, inwardly rectifying K+ current, a saturating K+ current, and two outwardly rectifying nonselective cation currents. Goblet cells contain a large outward K+ current.
Animals↗
Activation of volume-regulated Cl(-) channels by ACh and ATP in Xenopus follicles.
Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two-electrode voltage clamp and single-channel activity was measured using the patch-clamp technique. In follicles held at -60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume-regulated current elicited by external hyposmolarity (ICl,swell), and the second was a slow and smooth current (Sin) generated by ACh or ATP application. In follicles, the permeability ratios for different anions with respect to Cl- were similar for both ICl,swell and Sin, with a sequence of: SCN- > I- > Br- >= NO3- >= Cl- > gluconate >= cyclamate > acetate > SO42-. Extracellular ATP blocked the outward component of Sin. Also, extracellular pH modulated the inactivation kinetics of Sin elicited by ACh; e.g. inactivation at +80 mV was approximately 100 % slower at pH 8.0 compared with that at pH 6.0. Lanthanides inhibited ICl, swell and Sin. La3+ completely inhibited ICl,swell with a half-maximal inhibitory concentration (IC50) of 17 +/- 1.9 microM, while Sin was blocked up to 55 % with an apparent IC50 of 36 +/- 2.6 microM. Patch-clamp recordings in follicular cells showed that hyposmotic challenge opened inward single-channel currents. The single channel conductance (4.7 +/- 0.4 pS) had a linear current-voltage relationship with a reversal membrane potential close to -20 mV. This single-channel activity was increased by application of ACh or ATP. The ICl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named Fin. Thus, ICl,swell was not generated via neurotransmitters released during cellular swelling. All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single-channel activity, strongly suggest that ICl,swell and Sin currents depend on the opening of the same type or a closely related class of volume-regulated chloride channels.
Acetylcholine↗
Free radicals enhance Na+/Ca2+ exchange in ventricular myocytes.
Oxygen-derived free radicals (OFR) have been implicated in the pathogenesis of intracellular Ca2+ overload and the arrhythmias that characterize cardiac reperfusion. These arrhythmias may in large part be due to activation of the pathological transient inward current (ITI). However, the identity of the ITI generated by OFR is uncertain. We previously found that H2O2, an OFR-generating compound, markedly stimulated the ITI elicited by brief caffeine pulses in patch-clamped guinea pig ventricular myocytes. In the present study, using patch-clamped rabbit ventricular myocytes loaded with the Ca(2+)-sensitive indicator fura 2, we have further characterized this ITI and have identified its major component to be Na+/Ca2+ exchange based on its dependence on extracellular Na+ and sarcoplasmic reticulum Ca2+ release, its sensitivity to Ni2+, and the effects of its inhibition on relaxation. The effect on ITI was not unique to H2O2, because another free radical-generating system, xanthine + xanthine oxidase, produced a similar response. We hypothesize that enhancement of Na+/Ca2+ exchange by OFR during reperfusion, when intracellular Na+ is elevated, may promote intracellular Ca2+ overload and triggered arrhythmias.
Animals↗
Enhancement by arginine vasopressin of the L-type Ca2+ current in guinea pig ventricular myocytes.
Effects of arginine-vasopressin (AVP) on the cardiac L-type Ca2+ current (ICa,L) were investigated in single ventricular cells of guinea pig hearts by the conventional and nystatin-perforated patch clamp methods. Using the conventional whole-cell clamp method, AVP (0.01-1 micromol/l) appeared to have little effect on ICa,L, but the same concentrations of AVP consistently increased ICa,L using the nystatin-perforated patch clamp mode. The stimulatory effect was blocked by either OPC-21268 (8 micromol/l), a selective V1 receptor antagonist, or staurosporine (10 nmol/l), an inhibitor of protein kinases. AVP further increased the amplitude of ICa,L previously augmented maximally by isoprenaline (1 micromol/l). When myocytes were pretreated with ryanodine (2 micromol/l) or cyclopiazonic acid (3 micromol/l), the increase in ICa,L by AVP was partially reduced. The present results indicate that protein kinase C might be involved in the AVP-induced increase of ICa,L. The AVP-induced increase in ICa,L may require intracellular constituents, which might be washed out during the use of the conventional whole-cell patch clamp method.
Animals↗
Effect of calcium on membrane potential behavior in a rat pituitary cell line (GH3).
The effects of intracellular calcium buffering and increasing bath Ca2+ on spontaneous membrane depolarizations expressed by the clonal rat pituitary cell line GH3 were examined by use of the whole-cell patch-clamp technique. Increasing intracellular calcium buffering capacity caused the duration of spontaneous depolarizations to increase without altering other parameters of membrane potential activity. Increasing bath Ca2+ caused a decrease in duration. These results suggest that the duration of spontaneous membrane depolarization in GH3 cells is regulated by the accumulation of free intracellular Ca2+. The behavior of spontaneous depolarizations measured with the perforated-patch variation of whole-cell patch-clamp techniques closely resembled that obtained in standard whole-cell patch-clamp measurements with an intracellular calcium buffer of 200 microM EGTA with free Ca2+ adjusted to 100 nM.
Animals↗
Characterization of GABAA receptor function in human temporal cortical neurons.
1. Surgically resected tissue from the tip of the human temporal lobe of seven patients undergoing temporal lobectomy was employed to study functional properties of GABAergic inhibition mediated through activation of GABAA receptors, using patch-clamp recording techniques in acutely isolated neurons and in slices of human temporal cortex. 2. Human temporal cortical pyramidal neurons from surgically resected tissue could be acutely isolated with the use of conventional methods. These neurons appeared normal in morphology, in their intrinsic membrane properties, and in their response to application of exogenous gamma-aminobutyric acid (GABA). 3. Application of GABA to acutely isolated human temporal cortical neurons elicited a large current with an average reversal potential of -65 mV, presumably mediated through a GABAA-activated chloride conductance. Application of varying concentrations of GABA generated a concentration/response relationship that could be well-fitted by a conventional sigmoidal curve, with an EC50 of 25.5 microM and a Hill coefficient of 1.0 4. Coapplication of the benzodiazepine clonazepam and 10 microM GABA augmented the amplitude of the GABA response. The concentration dependence of this benzodiazepine augmentation could be best-fitted by an equation assuming that the benzodiazepine interacted with two distinct binding sites, with differing potencies. The high-potency site had an EC50 of 0.06 nM and maximally contributed 38.5% augmentation to the total effect of clonazepam. The lower potency site had an EC50 of 16.4 nM, and contributed 66.1% maximal augmentation to the overall effect of clonazepam. These data derived from adult human temporal cortical neurons were very similar to our findings in adult rat sensory cortical neurons. 5. The effects of equimolar concentrations (100 nM) of clonazepam, a BZ1 and BZ2 agonist, and zolpidem, a selective BZ1 agonist, on acutely isolated human temporal cortical neurons were also investigated. Zolpidem and clonazepam were equally effective (71.5 vs. 65.0%, respectively) in potentiating GABA responses elicited by application of 10 microM GABA. This suggests that many of the functional benzodiazepine receptors in these neurons were of the BZ1 variety. 6. GABAergic synaptic inhibition was also studied with the use of patch-clamp recordings in slices of human temporal cortex. Extracellular stimulation at the white matter/gray matter border elicited compound synaptic events in layer II-V cortical neurons. These events usually consisted of an early excitatory postsynaptic potential (EPSP) and a late multiphasic inhibitory postsynaptic potential (IPSP). Application of either clonazepam or zolpidem (both at 100 nM) to the slice during extracellular stimulation reversibly augmented the late compound IPSP. 7. Spontaneous IPSPs were also recorded in approximately 50% of human temporal cortical neurons. These events did not have a preceding EPSP and were usually monopolar, with a single exponential rise and decay. This supported the idea that these events were triggered by spontaneous activity of GABAergic interneurons. Bath application of either clonazepam or zolpidem (both at 100nM) to the slice during ongoing spontaneous IPSP activity increased the amplitude and lengthened the time constant of decay of these events. 8. To our knowledge, this is one of the first detailed characterizations of the functional properties of GABAA-mediated inhibition in human cortical neurons using patch-clamp recordings in both isolated cells and slices of resected temporal cortex. Isolated pyramidal neurons exhibited GABAA-mediated currents that were comparable in many aspects with GABA currents recorded from adult rat cortical neurons, including similar GABA concentration/response curves, and similar two differing potency site effects for clonazepam augmentation of GABA currents. In addition, evoked and spontaneous IPSPs recorded in human cortical neurons appeared similar to IPSPs in rat cortical
Animals↗
Caffeine rapidly decreases potassium conductance of dissociated outer hair cells of guinea-pig cochlea.
The effects of caffeine on the outer hair cells (OHCs) freshly dissociated from guinea-pig cochlea were investigated with the whole-cell patch-clamp technique, in both the conventional and the nystatin perforated patch-clamp configurations under voltage-clamp condition. Application of caffeine (> 1 mM for 10-30 s) induced an inward current (Icaffeine) with decrease of conductance in a dose-dependent manner at a holding potential (VH) of -60 mV. The reversal potential of Icaffeine (Ecaffeine) was close to the K+ equilibrium potential. The Icaffeine was not affected by Ca(2+)-free external solution. The internal perfusion of the Ca2+ chelator BAPTA had no effect on Icaffeine. The Icaffeine was not modulated by the external application of H-8 or staurosporine and by the internal perfusion of GDP-beta S. The amplitude of Icaffeine was the largest at the basal region of OHCs when caffeine was locally applied by the 'puffer' method. These results suggest that caffeine induces a decrease in membrane potassium conductance of the OHCs mainly at the basal region without mediating the intracellular signaling pathway.
Animals↗
Inward-rectifying potassium channels in retinal glial (Müller) cells.
The voltage- and K(+)-dependent properties of Müller cell currents and channels were characterized in freshly dissociated salamander Müller cells. In whole-cell voltage-clamp experiments, cells with endfeet intact and cells missing endfeet both displayed strong inward rectification. The rectification was similar in shape in both groups of cells but currents were 9.2 times larger in cells with endfeet. Ba2+ at 100 microM reduced the inward current to 6.8% of control amplitude. Decreasing external K+ concentration shifted the cell current-voltage (I-V) relation in a hyperpolarizing direction and reduced current magnitude. In multichannel, cell-attached patch-clamp experiments, patches from both endfoot and soma membrane displayed strong inward rectification. Currents were 38 times larger in endfoot patches. In single-channel, cell-attached patch-clamp experiments, inward-rectifying K+ channels were, in almost all cases, the only channels present in patches of endfoot, proximal process, and soma membrane. Channel conductance was 27.8 pS in 98 mM external K+. Reducing external K+ shifted the channel reversal potential in a hyperpolarizing direction and reduced channel conductance. Channel open probability varied as a function of voltage, being reduced at more negative potentials. Together, these observations demonstrate that the principal ion channel in all Müller cell regions is an inward-rectifying K+ channel. Channel density is far higher on the cell endfoot than in other cell regions. Whole-cell I-V plots of cells bathed in 12, 7, 4, and 2.5 mM K+ were fit by an equation including Boltzmann relation terms representing channel rectification and channel open probability. This equation was incorporated into a model of K+ dynamics in the retina to evaluate the significance of inward-rectifying channels to the spatial buffering/K+ siphoning mechanism of K+ regulation. Compared with ohmic channels, inward-rectifying channels increased the rate of K+ clearance from the retina by 23% for a 1 mM K+ increase and by 137% for a 9.5 mM K+ increase, demonstrating that Müller cell inward-rectifying channels enhance K+ regulation in the retina.
Ambystoma↗
Passive current flow and morphology in the terminal arborizations of the posterior pituitary.
1. Patch-clamp techniques were used to study the morphology and electrotonic properties of the terminal arborizations of the posterior pituitary. 2. Neurobiotin-labeling experiments revealed axons and swellings connected to the structure that was patch clamped. The large swellings were en passant and situated along axons in a topological arrangement identical to that of the small varicosities. Axons had many varicosities and few branches, reflecting a predominant architectural motif of beads on a string rather than berries on a bush. 3. Cable theory was used to analyze passive current transients produced by voltage steps under whole-cell clamp. Most charging transients were not consistent with an equivalent cylinder representation as posited by the Rall model for a motoneuron. A few charging transients were consistent with the Rall model and provided estimates for basic membrane and cable properties. 4. Some of the charging transients that violated predictions of the Rall model were consistent with an alternative model, in which the patch-clamped swelling was assumed to be coupled to another swelling by a segment of axon. This model was called the Dumbbell model, and it, together with the neurobiotin-labeling experiments, indicated that a significant number of large swellings were less than one length constant away from another large swelling. 5. Large swellings can have diameters approximately 30 times larger than the diameters of the connecting axons. These swellings lie along the axon such that action potentials must propagate through them to spread excitation through the entire terminal arborization. These large swellings could be sites where action-potential propagation is more likely to fail. 6. The information presented here about neurohypophysial nerve terminals should be useful in further investigations of how terminal arborization geometry and membrane properties influence neurosecretion and synaptic transmission.
Animals↗