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PubMed1987

The distribution of sodium and potassium channels in single demyelinated axons of the frog.

1. Frog sciatic nerves were focally demyelinated by intraneural injection of lysolecithin. Fibres were examined 1-8 days later. At early times axons contained zones just a few micrometers long that were covered by myelin debris. These zones lengthened and the debris was cleared over the next several days. 2. The loose patch clamp technique was used to measure ionic currents in the demyelinated axolemma in both internodal and paranodal regions. 3. Transient inward currents that have been identified as Na+ currents on the basis of voltage dependence and sensitivity to tetrodotoxin were recorded at all stages tested. There was no significant increase in internodal peak Na+ current with time over the first 8 days after injection. 4. Peak inward currents varied relatively little with distance along the internode at 1 day post-injection. However, sharp gradients were detected at demyelinated paranodal and nodal membranes. 5. By combining the patch clamp with external stimulation proximal to the patch site it has been possible to record membrane current responses to propagating signals invading the demyelinated zone. With demyelination only in the distal heminode the extent of Na+ channel activation is similar to that predicted for normal nodes of Ranvier by Frankenhaeuser & Huxley (1964). 6. The ratio of K+ conductance to Na+ conductance is much higher in demyelinated internodes than in normal nodes of Ranvier. 7. It is concluded that Na+ channels are likely to be present in normal internodes. The total number of these internodal channels may be about 40 times the total number at nodes. Steep gradients of Na+ channel density remain at nodal regions at least up to 6 days following demyelination.

Action Potentials↗

PubMed1984

An improved loose patch voltage clamp method using concentric pipettes.

A method for noninvasive voltage-clamp recording from large cells is described. A firepolished pipette having two concentric barrels is pushed against the cell membrane, thereby electrically isolating a circular patch subdivided into an inner and an annular outer region. Both regions are held isopotential, but current is collected from the inner region only. The method electrically simulates a high resistance seal between pipette and cell membrane, allowing accurate and rapid voltage-clamp recording under conditions where the seal resistances actually obtained are low (near 1 M omega). This is useful in applications where one wishes to avoid enzymatic treatment. We provide details of electrode construction and voltage-clamp electronics, and present results obtained from frog skeletal muscle and leech neurons. For sodium channels of frog muscle, extensive data were previously obtained with other methods. There is good agreement between the earlier results and the measurements presented here.

Animals↗

PubMed1989

Low-conductance K channels in apical membrane of rat cortical collecting tubule.

Low-conductance, K-selective channels were identified in the apical membrane of the rat cortical collecting tubule (CCT) by use of the patch-clamp technique. Isolated, split tubules were bathed in K gluconate medium to depolarize the cell while keeping the intracellular K concentration high. With the patch-clamp pipette containing predominantly either Na+ or Li+ but no K, outward currents were observed through channels that had a single-channel conductance (g) of 9 pS and a probability of being open (Po) of greater than 0.9, independent of the voltage (+/- 40 mV) applied to the pipette (Vp). Similarly, only outward currents were observed when the patch was excised into high-K solution, implying a high selectivity of the channel for K+. When 1 mM BaCl2 was added to the pipette, Po decreased to 0.36 at Vp = 0; however, g was not changed but the channels flickered rapidly between open and blocked states; Po decreased as Vp was made positive, and increased as Vp was made negative. With the pipette filled with KCl + 1 mM Ba, the channels conducted K+ in both directions. The inward currents (at positive Vp were larger than the outward currents (at negative Vp) and g near Vp = 0 increased to 25 pS. When the pipette was filled with RbCl + 1 mM Ba the inward and outward currents were similar in magnitude, suggesting that the channels can conduct Rb, although not as well as K. With the tubules bathed in NaCl Ringer solution and the pipette containing KCl, inward currents were observed that could be attributed to the same pathway for K.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Cytosolic Ca2+ spikes evoked by the thiol reagent thimerosal in both intact and internally perfused single pancreatic acinar cells.

Cytosolic calcium signals evoked by the sulphydryl-group-oxidising agent, thimerosal, have been investigated in acutely isolated pancreatic acinar cells. Two techniques were employed for the assessment of the cytosolic free-calcium concentration ([Ca2+]i): measurement of calcium-dependent chloride and non-specific cation currents (whole-cell patch-clamp recording) and microfluorimetry (fura-2). Thimerosal (0.5-100 microM) evoked repetitive spikes in both chloride and cation currents as seen by patch-clamp recording, and in [Ca2+]i as seen by microfluorimetry, with a latency of 1-3 min. The response increased in magnitude over time and was not reversed on removal of thimerosal. The thimerosal-induced spikes were reversibly blocked by 2 mM dithiothreitol and by 20 mM caffeine. Inclusion of heparin (200 micrograms/ml) in the pipette solution blocked the thimerosal-induced spikes. The calcium spikes continued after the removal of extracellular calcium; however, low concentrations of thimerosal (0.5-5 microM) were unable to initiate a current response in the absence of external calcium. High concentrations of thimerosal (50-100 microM) could initiate spikes without extracellular calcium. Thimerosal, at concentrations that failed to produce an independent effect, potentiated the acetylcholine-evoked oscillations in [Ca2+]i. We conclude that thimerosal is able to mobilise calcium from an intracellular store; the blockade by heparin may indicate that thimerosal exerts an action on the inositol trisphosphate pathway. The dependence on extracellular calcium for initiation, but not for continuation of the thimerosal-induced calcium spikes suggests that thimerosal may have the additional effect of inhibiting the plasma membrane calcium ATPase.

Action Potentials↗

PubMed2006

Photometric recording of transmembrane potential in outer hair cells.

Cochlear outer hair cells (OHCs) are polarized epithelial cells that have mechanoelectrical transduction channels within their apical stereocilia and produce electromotile force along their lateral wall. Phase shifts, or time delays, in the transmembrane voltage occurring at different axial locations along the cell may contribute to our understanding of how these cells operate at auditory frequencies. We developed a method to optically measure the phase of the OHC transmembrane potential using the voltage-sensitive dye (VSD) di-8-ANEPPS. The exit aperture of a fibre-optic light source was driven in two dimensions so that a 24 microm spot of excitation light could be positioned along the length of the OHC. We used the whole-cell patch-clamp technique in the current-clamp mode to stimulate the OHC at the base. The photometric response and the voltage response were monitored with a photodetector and patch-clamp amplifier, respectively. The photometric response was used to measure the regional changes in the membrane potential in response to maintained (dc) and sinusoidal (ac) current stimuli applied at the base of the cell. We used a neutral density filter to lower the excitation light intensity and reduce phototoxicity. A sensitive detector and lock-in amplifier were used to measure the small ac VSD signal. This permitted measurements of the ac photometric response below the noise floor of the static fluorescence. The amplitude and phase components of the photometric response were recorded for stimuli up to 800 Hz. VSD data at 400-800 Hz show the presence of a small phase delay between the stimulus voltage at the base of the cell and the local membrane potential measured along the lateral wall. Results are consistent with the hypothesis that OHCs exhibit inhomogeneous membrane potentials that vary with position in analogy with the voltage in nerve axons.

Animals↗

PubMed1998

Valproate selectively reduces the persistent fraction of Na+ current in neocortical neurons.

The effect of valproate (VPA) on Na+ currents (INa), was studied by means of voltage clamp recordings using whole-cell patch clamp configuration in 21 acutely dissociated neocortical neurons. Concentrations of VPA up to 200 microM failed to induce any detectable decrease in fast INa (I(Naf)), but the persistent fraction (I(NaP)) was significantly reduced by low VPA concentrations (10-30 microM), corresponding to the lower values of the 'therapeutic' range in epileptic patients. Since it is known that I(NaP) critically regulates the firing properties of pyramidal neurons, it is suggested that the anticonvulsant effectiveness of VPA is mainly due to its effect on I(NaP).

Animals↗

PubMed1992

Cell swelling activates K+ and Cl- channels as well as nonselective, stretch-activated cation channels in Ehrlich ascites tumor cells.

Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba2+ and hence presumably to Ca2+. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca2+ permeability, as part of the volume-activated signal system. In isolated inside-out patches a Ca(2+)-dependent, inwardly rectifying K+ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mM K+ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K+ channel takes place after an increase in Ca2+ from 10(-7) to 10(-6) M which is in the physiological range. Patch-clamp studies in cell-attached mode show K+ channels with spontaneous activity and with characteristics similar to those of the K+ channel seen in excised patches. The single-channel conductance for outward current at 5 mM external K+ is estimated at about 7 pS. A K+ channel with similar properties can be activated in the cell-attached mode by addition of Ca2+ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K+ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K+ channels following addition of Ca2+ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K+ channels in the presence of 3 mM Ca2+ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca2+ and could not be activated by membrane stretch (suction). In cell-attached mode single-channel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca2+ plus ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1998

Ca2+-permeable non-NMDA glutamate receptors in rat magnocellular basal forebrain neurones.

1. Ionotropic glutamate receptor-mediated responses were recorded from rat magnocellular basal forebrain neurones under voltage clamp from a somatically located patch-clamp pipette. Currents were recorded from both acutely dissociated neurones and neurones maintained in culture for up to 6 weeks. 2. Non-NMDA and NMDA receptor-mediated events could be distinguished pharmacologically using the selective agonists (S)-alpha-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA), and antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphonopentanoic acid (AP5). 3. Responses to rapid application of AMPA displayed pronounced and rapid desensitization. Responses to kainate showed no desensitization. Steady-state EC50 values for AMPA and kainate were 2.7 +/- 0.4 microM (n = 5) and 138 +/- 25 microM (n = 10), respectively. Cyclothiazide markedly increased current amplitude of responses to both agonists, whereas concanavalin A had no clear effect on either response. The selective AMPA receptor antagonist GYKI 53655 inhibited responses to kainate with an IC50 of 1.2 +/- 0.08 microM (n = 5) at -70 mV. These data strongly suggest that AMPA receptors are the predominant non-NMDA receptors expressed by basal forebrain neurones. 4. At -70 mV, approximately 6 % of control current amplitude remained, at a maximally effective concentration of GYKI 53655. This residual response displayed desensitization, was insensitive to cyclothiazide and was potentiated by concanavalin A, suggesting that it was mediated by a kainate receptor. 5. Current-voltage relationships for non-NMDA receptor-mediated currents were obtained from both nucleated patches pulled from neurones in culture and from acutely dissociated neurones. With 30 microM spermine in the recording pipette, currents frequently displayed double-rectification characteristic of non-NMDA receptors with high Ca2+ permeabilities. Ca2+ permeability, relative to Na+ and Cs+, was investigated using constant field theory. The measured Ca2+ to Na+ permeability coefficient ratio was 0.26-3.6; median, 1.27 (n = 15). 6. Current flow through non-NMDA receptors was inhibited by Ca2+, Cd2+ and Co2+ ions. At a holding potential of -70 mV, a maximally effective concentration of Cd2+ (> 30 mM) reduced current amplitude by approximately 90 %, with an IC50 of 44 microM. In six out of seven cells tested, block by Cd2+ was voltage sensitive. 7. Ca2+ permeability of many of the non-NMDA receptors expressed by magnocellular basal forebrain neurones may underlie the unusual sensitivity of cholinergic basal forebrain neurones to non-NMDA receptor-mediated excitotoxicity.

Animals↗

PubMed1996

Development of firing patterns and electrical properties in neurons of the rat ventrobasal thalamus.

The electrophysiological properties of thalamic neurons of the rat ventrobasal complex (VB) in vitro were studied during early postnatal development. Current clamp recordings using the whole cell patch clamp method revealed that immature thalamic neurons had less negative membrane potential and higher input resistance than mature neurons. One of the most remarkable differences was the absence of spike bursts riding on the low threshold calcium spike (LTS) in VB neurons before postnatal day 12 (P12). Action potentials recorded from immature neurons had longer duration than those of mature cells and were followed by a longer afterhyperpolarization (spike-ahp). The spike-ahp became shorter as maturation progressed, reaching mature characteristics around P12, coinciding with the appearance of spike bursting on the LTS. The calcium activated potassium conductance, IC, played a prominent role in the spike-ahp in immature neurons. In conclusion, the major differences in intrinsic membrane properties of VB neurons occur during the first 12 postnatal days. The appearance of spike bursting riding on the LTS at P12 is consistent with the emergence of synchronized thalamocortical oscillations in rats around that age.

Action Potentials↗

PubMed1997

Epidermal growth factor reduces L-type voltage-activated calcium current density in GH4C1 rat pituitary cells.

Long-term treatment of rat pituitary tumor cells with epidermal growth factor (EGF) inhibits 45Ca2+ uptake, intracellular calcium levels and subsequent prolactin secretion in response to membrane depolarization. In the present study we have used whole-cell voltage-clamp and single-channel patch-clamp recording to determine directly the effects of EGF (10 nM for 48 h) on L-type calcium current density, the current-voltage relationship, single-channel amplitude, and opening and closing dwell times in rat GH4C1 pituitary tumor cells. Sustained, nimodipine-sensitive inward currents (barium as the carrier) with an activation threshold of approximately -30 mV were elicited in both control and EGF-treated GH4C1 cells by depolarization. Mean current density normalized to membrane capacitance was reduced to 45% of control after EGF treatment. There was no difference in the voltage-dependent activation of L-type channels between control and EGF-treated cells. Analysis of single-channel current recordings showed that EGF treatment had no effect on unitary current amplitude or channel open and close durations. These results suggest that EGF reduces the number of voltage-gated calcium channels in GH4C1 cell membranes, which likely contributes to the decreased calcium uptake.

Animals↗

PubMed2003

The effects of H2O2 on electromechanical activity of the ileum longitudinal muscle.

The effects of H2O2 on electrical and mechanical activity of the longitudinal layer from the guinea-pig ileum were studied using sucrose-gap technique and the influence of H2O2 on ionic current was investigated in single smooth muscle cells by the patch-clamp method. In most of the preparations tested, the spontaneous activity observed was composed of slow waves with superimposed action potentials (APs). Both were resistant to tetrodotoxin and atropine. H2O2 (1 mmol/l) evoked sustained 3-5 mV membrane depolarisation, doubled the amplitude of the slow waves and increased their frequency, augmented the APs and reduced their splitting. These changes were accompanied with significant contraction, which had an amplitude comparable to that of the tonic component of 50 mmol/l K+-induced contraction. Calcium-free solution caused membrane depolarisation, reduction of the slow wave amplitude and frequency, disappearance of APs and decreased the mechanical tension of the preparations. Application of H2O2 (1 mmol/l) into the zero-calcium bath solution recovered the APs, which was accompanied by a low amplitude contraction. H2O2 (up to 1 mmol/l) increased the L-type calcium current (I(Ca)) both under conventional whole-cell patch-clamp configuration and under amphotericin-perforated patches by 16 +/- 3%. These data demonstrated that contractile response of the ileum longitudinal smooth muscle preparation evoked by H2O2 was mainly due to the enhanced electrical activity.

Action Potentials↗

PubMed1997

The Ca2+-inactivated Cl- channel at work: selectivity, blocker kinetics and transport visualization.

Removal of extracellular divalent cations activated a Cl- channel in the plasma membrane of Xenopus laevis oocytes. This so-called Ca2+-inactivated Cl- channel (CaIC) was present in every oocyte and was investigated using two-electrode whole-cell voltage clamp and single-channel patch-clamp techniques. Beside other Cl- channel inhibitors, anthracene-9-carboxylic acid (9-AC) and 3' azido-3'deoxythymidine (AZT), a nucleoside analog we used as an antiviral drug, blocked at least partly the CalC-mediated currents. Using the Cl--sensitive dye 6-methoxy-N-(sulfopropyl)quinolinium (SPQ) we could visualize the transport of Cl- from the oocyte cytoplasm to the surrounding medium after activation of the CaIC by Ca2+ removal. In the absence of external Cl- and Ca2+, the emission intensity of SPQ declined continuously, indicating a quenching of fluorescence by the efflux of Cl- in the millimolar range. In the presence of external Ca2+, no emission changes could be observed during the same time period. Chelating external Ca2+ in absence of Cl- immediately activated Ca2+-inactivated Cl- channels leading to subsequent emission decrease of SPQ. Investigations on the selectivity of the CaIC revealed only poor discrimination between different anions. With single-channel measurements, we found an anion selectivity sequence I- > Br- > Cl- >> gluconate as it is also typical for maxi Cl- channels. Contrary to the majority of all other transport systems of the Xenopus oocyte, which show reduced activity due to membrane depolarization or endocytotic removal of the transport protein from the plasma membrane during oocyte maturation, the CaIC remained active in maturated oocytes. Single-channel measurements on maturated oocytes, also known as eggs, showed the presence of Ca2+-inactivated Cl- channels. However, this egg CaIC revealed an altered sensitivity to external Ca2+ concentrations. All these data confirm and extend our previous observations on the CaIC and give clear evidence that this channel is peculiar among all Cl- channels described up to now.

Animals↗

PubMed2000

Regulation of basal intracellular calcium concentration by the sarcoplasmic reticulum in myocytes from the rat gastric antrum.

The intracellular calcium concentration ([Ca2+]i) was monitored in fura-2-loaded myocytes isolated from the rat gastric antrum and voltage clamped at -60 1r1rqmV1qusing the perforated patch clamp technique. The rate of quench of fura-2 fluorescence by Mn2+ was used as a measure of capacitative Ca2+ entry. Cyclopiazonic acid (5 microM) did not affect the holding current but produced a sustained elevation in steady-state [Ca2+]i that was dependent on the presence of external calcium. Cyclopiazonic acid increased Mn2+ influx with physiological external [Ca2+], but not in Ca2+-free conditions. Cyclopiazonic acid increased the rate of [Ca2+]i rise following a rapid switch from Ca2+-free to physiological [Ca2+] solution. Sustained application of carbachol (10 microM) produced an elevation in steady-state [Ca2+]i that was associated with an increased rate of Mn2+ influx. Application of cyclopiazonic acid in the presence of carbachol further elevated steady-state [Ca2+]i without changing Mn2+ influx. Ryanodine (10 microM) elevated steady-state [Ca2+]i either on its own or following a brief application of caffeine (10 9i1s1sqmMc1q). Cyclopiazonic acid had no further effect when added to cells pre-treated with ryanodine. Neither caffeine nor ryanodine increased the rate of Mn2+ influx. When brief applications of ionomycin (25 microM) in Ca2+-free solution were used to release stored Ca2+, ryanodine reduced the amplitude of the resulting [Ca2+]i transients by approximately 30 %, indicating that intracellular stores were partially depleted. These findings suggest that continual uptake of Ca2+ by the sarcoplasmic reticulum Ca2+-ATPase into a ryanodine-sensitive store limits the bulk cytoplasmic [Ca2+]i under resting conditions. This pathway can be short circuited by 10 microM ryanodine, presumably by opening Ca2+ channels in the sarcoplasmic reticulum. Depletion of stores with cyclopiazonic acid or carbachol also activates capacitative Ca2+ entry.

Animals↗

PubMed1999

Positive and negative coupling of the endothelin ETA receptor to Ca2+-permeable channels in rabbit cerebral cortex arterioles.

1. Arteriolar segments were isolated from pial membrane and studied within 10 h. Current-clamp and voltage-clamp measurements were made by patch-clamp recording from smooth muscle cells within arterioles. [Ca2+]i was measured from the smooth muscle cell layer by digital imaging of emission from fura-PE3 which was loaded into arterioles by pre-incubation with the acetoxymethyl ester derivative. The external diameter of arterioles was measured using a video-dimension analyser. 2. Endothelin-1 (ET1) was a potent constrictor of isolated arterioles and induced a sustained depolarization up to -27 mV and reduced membrane resistance (EC50 140-170 pm). At a constant holding potential of -60 mV ET-1 induced a transient followed by a sustained inward current. ET1 inhibited L-type voltage-dependent Ca2+ current. 3. ET1 induced a transient followed by sustained elevation of [Ca2+]i. The sustained effect was dependent on extracellular Ca2+. It occurred at a constant holding potential of -60 mV and was not inhibited by the Ca2+ antagonists nicardipine (1 microM) or D600 (10 microM). Thapsigargin (1 microM) completely depleted Ca2+ from caffeine- and ET1-sensitive sarcoplasmic reticulum but did not inhibit the ET1-induced sustained elevation of [Ca2+]i. ET1 effects on [Ca2+]i were prevented by the ETA receptor antagonist BQ123 (cyclo-D-Asp-Pro-D-Val-Leu-D-Trp). 4. The data suggest that ETA receptors are negatively coupled to L-type Ca2+ channels and positively coupled to receptor-operated Ca2+-permeable channels. Inhibition of L-type Ca2+ channel activity may suppress autoregulation, and Ca2+ influx through receptor-operated channels may have a major functional role in the potent long-lasting constrictor effect of endothelin-1 in the cerebral microcirculation.

Animals↗

PubMed2000

VIP and PACAP potentiation of nicotinic ACh-evoked currents in rat parasympathetic neurons is mediated by G-protein activation.

The effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP27 and PACAP38) on isolated parasympathetic neurons of rat intracardiac and submandibular ganglia were examined under voltage clamp using whole-cell patch-clamp recording techniques. VIP and PACAP (</= 10 nM) selectively and reversibly increased the affinity of nicotinic acetylcholine receptor channels (nAChRs) for their agonists resulting in a potentiation of acetylcholine (ACh)-evoked whole-cell currents at low agonist concentrations. VIP-induced potentiation was observed with either ACh or nicotine as the cholinergic agonist. The VIP- but not the PACAP-induced potentiation of ACh-evoked currents was inhibited by [Ac-Tyr1, D-Phe2]-GRF 1-29, amide (100 nM), a selective antagonist of VPAC1 and VPAC2 receptors; whereas the PACAP38- but not the VIP-induced potentiation was inhibited by 100 nM PACAP6-38, a PAC1 and VPAC2 receptor antagonist. The signal transduction pathway mediating VIP- and PACAP-induced potentiation of nicotinic ACh-evoked currents involves a pertussis toxin (PTX)-sensitive G-protein. Intracellular application of 200 microM GTPgammaS or GDPbetaS inhibited VIP-induced potentiation of ACh-evoked whole-cell currents. GTPgammaS alone potentiated ACh- and nicotine-evoked currents and the magnitude of these currents was not further increased by VIP or PACAP. The G-protein subtype modulating the neuronal nAChRs was examined by intracellular dialysis with antibodies directed against alphao, alphai-1,2, alphai-3 or beta G-protein subunits. Only the anti-Galphao and anti-Gbeta antibodies significantly inhibited the effect of VIP and PACAP on ACh-evoked currents. The potentiation of ACh-evoked currents by VIP and PACAP may be mediated by a membrane-delimited signal transduction cascade involving the PTX-sensitive Go protein.

Acetylcholine↗

PubMed1994

Pump and K+ inward rectifiers in the plasmalemma of wheat root protoplasts.

An electrogenic pump, a slowly activating K+ inward rectifier and an intermittent, "spiky," K+ inward rectifier, have been identified in the plasmalemma of whole protoplasts from root cortical cells of wheat (Triticum) by the use of patch clamping techniques. Even with high external concentrations of K+ of 100 mM, the pump can maintain the membrane potential difference (PD) down to -180mV, more negative than the electrochemical equilibrium potentials of the various ions in the system. The slowly activating K+ inward rectifier, apparent in about 23% of protoplasts, allows inward current flow when the membrane PD becomes more negative than the electrochemical equilibrium potential for K+ by about 50 mV. The current usually consists of two exponentially rising components, the time constant of one about 10 times greater than the other. The longer time constant is voltage dependent, while the smaller time constant shows little voltage dependence. The rectifier deactivates, on return of the PD to less negative levels, with a single exponential time course, whose time constant is strongly voltage dependent. The spiky K+ inward rectifier, present in about 68% of protoplasts, allows intermittent current, of considerable magnitude, through the plasmalemma at PDs usually more negative than about -140mV. Patch clamp experiments on detached outside-out patches show that a possibly multi-state K+ channel, with maximum conductance greater than 400 pS, may constitute this rectifier. The paper also considers the role of the pump and the K+ inward rectifiers in physiological processes in the cell.

Biological Transport↗

PubMed1997

Ca(2+)-activated K+ channels in human smooth muscle cells of coronary atherosclerotic plaques and coronary media segments.

The behavior of Ca(2+)-activated K+ channels of large conductance (BKCa) in smooth muscle cells, which were obtained from atherosclerotic plaque material (SMCP) and from media segments (SMCM) of human coronary arteries, were compared using the patch-clamp technique. Voltage-clamp protocols in cell-attached patches revealed the characteristic voltage-dependent activation of BKCa in both cell groups. Single-channel conduction as 216.4 +/- 16.7 pS (n = 6) in SMCP and 199.9 +/- 6.7 pS (n = 6) in SMCM in symmetrical 140 mM K+ solutions. Using outside-out patches, external perfusion with 500 microM tetraethylammonium ions caused a typical "flickery block" of the unitary current. The selective BKCa channel inhibitor iberiotoxin (50 nM) effectively blocked BKCa, channel activity. Comparing BKCa open-state probabilities (P0) at +80 mV in cell-attached patches, a highly significant difference between SMCP (P0 = 0.1438 +/- 0.1301; n = 15) and SMCM (P0 = 0.0093 +/- 0.0044; n = 15; Kruskal-Wallis test, p < 0.001) was found. In contrast to this finding, there was no significant difference in the open-state probability of BKCa, between SMCP (P0 = 0.542 +/- 0.0237; n = 9) and SMCM (P0 = 0.0472 +/- 0.0218; n = 10; p = n.s.) using inside-out patches. The results show an interesting difference in the behavior of large conductance Ca(2+)-activated K+ channel in SMCP compared to SMCM with a significantly higher channel activity in human smooth muscle cells obtained from coronary atherosclerotic plaque material. This finding may indicate an important functional role of BKCa channels in the development of atherosclerosis.

Calcium↗

PubMed1995

Stable and functional expression of the CIC-3 chloride channel in somatic cell lines.

The CIC family is the superfamily of voltage-gated Cl- channels. Although the CIC channels expressed in Xenopus oocytes have been characterized, their channel properties are still poorly understood. We recently cloned a unique member of the CIC family, CIC-3, that is expressed abundantly in neurons. Its channel activity was regulated by phorbol esters. Now, we have established a stably transfected somatic cell line expressing functional CIC-3 channels and examined the CIC-3 single-channel current by patch-clamp techniques. In inside-out patches from the stably transfected cells, a rise of bath Ca2+ concentration in the physiological range of intracellular Ca2+ concentrations inhibited the CIC-3 single-channel currents. This inhibition by Ca2+ was independent of phosphorylation and ATP. Thus, the CIC-3 channel is a Ca(2+)-sensitive Cl- channel localized in neuronal cells, and its Ca2+ sensitivity implies a physiological role in neuronal functions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗