Cell-attached Patch Configuration Research Articles

Modulation by chloramine-T of 4-aminopyridine-sensitive transient outward current in rabbit atrial cells

The effects of an oxidizing agent, chloramine-T, on the 4-aminopyridine-sensitive transient outward current ( I TO) were investigated in rabbit atrial myocytes by using patch–clamp techniques. Extracellular application of chloramine-T at 20 μM irreversibly slowed the time course of inactivation of the whole-cell I TO, and increased the peak by 19.3% ( n=19) at +40 mV. At 100 μM, chloramine-T decreased the peak by 22.5% ( n=9) of the control, and subsequently induced a glibenclamide-sensitive time-independent outward K + current. Under superfusion with dithiothreitol (3 mM), chloramine-T (100 μM) produced no change in I TO. The chloramine-T-induced slowing of I TO inactivation was partially reversed by subsequent application of 3 mM dithiothreitol. In single-channel recordings with the cell-attached patch configuration, chloramine-T (20 μM) increased the open probability of the I TO channel from 0.15 to 0.46 at a potential 100 mV positive to the resting potential, and the mean open lifetime from 5.1 ms to 7.0 ms ( n=5). The unitary current amplitude was not affected. As a result, chloramine-T increased the ensemble current in amplitude and slowed its decay. These results indicated that: (1) inactivation of the native A-type channels of rabbit heart is susceptible to oxidation; and (2) oxidation of I TO channels may contribute to the genesis of arrhythmias.

Mg2+ block and inward rectification of mechanosensitive channels in Xenopus oocytes.

The effects of Mg2+ on single mechanosensitive (MS) channel currents recorded from Xenopus oocytes were studied using cell-attached and inside-out patch configurations. Mg2+ both permeates and blocks MS channels. Under symmetrical ionic conditions, the blocking effects of Mg2+ can be described by a Hill coefficient of 0.9 at +/-100 mV and IC50s of 0.12 mM (-100 mV) and 0.60 mM at (+100 mV). Although block by intracellular Mg2+ may contribute to inward MS channel rectification, significant current rectification is retained even under symmetrical KCl concentrations and in the complete absence of Mg2+. The observed voltage dependencies of the IC50 for Mg2+ block and the Km for K+ current saturation indicate asymmetries in the MS channel pore. In addition, the absence of K+ self block and anomalous mole fraction effects with K+/Tl+ mixtures indicate a single site pore model.

Hypertrophy Decreases Cardiac KATPChannel Responsiveness to Exogenous and Locally Generated (Glycolytic) ATP

This study tests the hypothesis that glycolytic regulation of KATPchannel activity is altered in myocardial hypertrophy. Left ventricular (LV) subendocardial myocytes were isolated from cats with normal or left ventricular hypertrophied hearts (LVH). Saponin-permeabilized open cell-attached patch configurations of normal and LVH cells were exposed to an exogenous ATP consuming system containing hexokinase and 2-deoxyglucose. Phosphoenol pyruvate (PEP, substrate for the last ATP producing step in glycolysis) was applied extracellularly; ADP was present. In both cell types, KATPchannels were activated in the absence of PEP, inhibited when PEP was added and activated again when PEP was removed, indicating the cells retained metabolic integrity and generated ATP in the proximity of their KATPchannels. Single channel conductance in the absence of PEP was similar (70 pS, normal; 66 pS, LVH). However, LVH KATPchannels showed enhanced activity (P0=0.50±0.03); normal (0.41±0.03) in PEP absence (P<0.05). PEP responsiveness was reduced in LVH, with IC50, PEP increased to 23μM; (11μMnormal). Lactate failed to activate KATPchannels in both cell types. The concentration-P0response curves obtained during exposure of open cells to exogenous ATP also revealed reduced responsiveness to ATP of LVH KATPchannels (IC50,ATP=283μMLVH; 93μMnormal). Our data indicate myocardial hypertrophy increases the maximal activity of KATPchannels in the absence of ATP and reduces their responsiveness to ATP, including locally generated glycolytic ATP. These alterations in metabolic regulation of myocardial electrophysiology may contribute to diversity of action potential shortening in hypertrophied hearts during acute ischemia.

Two Types of Stretch-Activated Channels Coexist in the Rabbit Corneal Epithelial Cell

Ion channels contribute to the regulation of cellular function through control of the membrane potential and intracellular concentration of various ions. We examined stretch-activated channels in the corneal epithelial cell. Patch clamping was applied to enzymatically dissociated corneal epithelial cells to characterize their stretch-activated ion channels. The plasma membrane was stretched by applying suction to the patch pipette in cell-attached or inside-out patch configuration. The ion selectivity, voltage-dependence, and stretch-dependence were examined. Two kinds of stretch-activated channel events were observed; the previously-reported large conductance (L) channel and a novel small conductance (S) channel. The probability of recording L vs. S channels in the cell-attached configuration was about 2:1. The L channel was potassium selective with single channel conductance (γ) of about 160 pS under the symmetrical (150 mmK+) solution. The S channel was permeable to Na+and K+with γof about 20 pS under the same conditions. Both L and S channels showed little activity in the absence of suction applied to the recording pipette. Channel activity was evoked by suction (negative pressure) stronger than -20 mmHg in both channels. The open probability (Po) and the mean current increased in proportion to further applied stretch and did not saturate for applied suction as strong as -80 mmHg, the pressure at which the gigaseal started to break. Thus, two types of stretch-activated channels coexist in corneal epithelial cells; a potassium-selective L channel and non-selective S channel. The contribution of these channels to the membrane potential is discussed.

Two parallel signaling pathways couple 5HT1A receptors to N- and L-type calcium channels in C-like rat dorsal root ganglion cells.

The coupling of serotonin receptors to Ca2+ channels was studied in a subpopulation of acutely isolated rat dorsal root ganglion (DRG) cell bodies (type 1 DRG cells), which have membrane properties similar to C-type nociceptive sensory neurons. In these cells, serotonin (5HT) inhibited high-threshold Ca2+ channel current and decreased action potential duration. The inhibitory effects of 5HT and the 5HT1A agonist 8-OH-DPAT were shown to be antagonized by the 5HT1A antagonists spiperone and pindolol, respectively, indicating involvement of a 5HT1A receptor. Several observations suggest that 5HT1A receptors couple to N- and L-type Ca2+ channels by two different signaling pathways in type 1 DRG cells. The inhibition of Ca2+ channel currents produced by 10 microM 5HT occurred in two phases, an initial slowing of current activation rate (kinetic slowing), which was complete within 10 s, and a simultaneous reduction in steady state current amplitude (steady state inhibition), which peaked in approximately 1 min. The kinetic slowing, but not steady state inhibition, was reversed by a positive prepulse to +70 mV (prepulse). Blockade of N-type Ca2+ channels selectively reduced the kinetic slowing and its reversal by prepulses. Chelation of intracellular Ca2+ or blockade of L-type Ca2+ channels selectively reduced the steady state inhibition. Recordings using the cell-attached patch configuration suggest that steady state inhibition required a component that was diffusible in the cytosol, while kinetic slowing occurred via a membrane delimited pathway. The application of 5HT to the cell body outside the patch pipette reduced macroscopic Ca2+ channel currents in 33% of small-diameter DRG cells tested, indicating the participation of a cytosolic diffusible component. Application of 5HT (a membrane impermeant compound) outside the patch pipette produced steady state inhibition only, whereas similar application of membrane permeant 5HT1A agonists, 8-OH-DPAT or 5-methoxy-N,N-dimethyl-tryptamine, produced kinetic slowing and steady state inhibition. Together these data suggest that 5HT1A receptors couple negatively to Ca2+ channels via two pathways: a membrane-delimited pathway that couples to N-channels and actuates voltage-sensitive kinetic slowing and a pathway dependent on a cytosolic diffusible component and free intracellular Ca2+, which couples to L channels and actuates steady state inhibition.

Seal-promoting solutions and pipette perfusion for patch clamping plant cells.

Patch-clamp technology has greatly increased our knowledge of plant membrane transport. However, the success of patch clamping crucially relies on establishing a high resistance (G omega) seal between the membrane and the patch-clamp pipette. This can prove problematic in many plant-cell preparations. It is therefore of great importance to develop protocols for protoplast isolation, maintenance and seal formation that improve seal rate. This study investigated whether the pH and the K+ and the Cl(-)concentration of the pipette solution had an effect on the seal formation. High pH and absence of K+ significantly promoted membrane sealing, whereas the concentration of Cl- had no effect. To reap the benefit of seal-promoting pipette solutions and yet retain the option to adjust this solution to experimental requirements, a pipette perfusion apparatus was implemented. The perfusion system was successfully applied in cell-attached patch, excised-patch and whole-cell configurations, using plasma membrane and tonoplast of three different species. The system enables complete solution exchange within minutes and is potentially of great benefit in the study of channel selectivity, the application of (cytoplasmic) channel blockers and the study of primary and secondary transport.

The Novel Class III Antiarrhythmic Agent MS-551 Blocks the Cardiac Inward Rectifier With Greater Potency Than Sotalol or E-4031: Possible Relevance to Reverse Use Dependence.

BACKGROUND: The tendency for the electrophysiologic effect of class III antiarrhythmic agents (action potential prolongation) to be diminished at faster heart rates represents a major drawback of this class of drug and is usually referred to as "reverse use dependence." A novel class III agent, MS-551, has recently been reported to exhibit less reverse use dependence than E-4031. We set out to investigate whether this observation may be due to differential blockade of the inward rectifier current (i(K1)) by these drugs. METHODS AND RESULTS: We recorded i(K1) using single channel methods and cell attached patch configurations, with standard patch clamp technology. Neither E-4031 nor racemic sotalol in concentrations up to 100 µM had any significant effect on the open probability or kinetics of i(K1) without altering the single-channel conductance. Openings to subconductance levels were abolished in three of six patches in which they had been frequently present in the absence of drug. MS-551 had no effect on mean channel open time but increased the slower component of the closed time. CONCLUSIONS: MS-551, unlike E-4031 and sotalol, appears to produce significant blockade of the inwardly rectifying potassium channel at clinically relevant concentrations. We propose that this might provide a partial explanation for the observed differences in their response to rate changes.

Competition between Mg2+ and spermine for a cloned IRK2 channel expressed in a human cell line.

1. A cloned inwardly rectifying K+ channel, IRK2, was expressed in a human cell line, human embryonic kidney (HEK) 293T. Its electrophysiological properties were examined using the patch clamp technique in the whole-cell, cell-attached and inside-out patch configurations. 2. The cells transfected with IRK2 cDNA exhibited a K+ current which showed classical properties of inwardly rectifying K+ channels at both whole-cell and single-channel levels. 3. In the inside-out patch configuration, intracellular Mg2+ (Mg2+i blocked the outward currents in a voltage-dependent and virtually time-independent manner. Mg2+i (1-100 microM) caused a decrease in the unitary current amplitude of the IRK2 channel by inducing subconducting levels. 4. In the absence of Mg2+i, intracellular spermine blocked the outwardly flowing IRK2 currents in a voltage- and time-dependent manner. Spermine (1-100 nM) did not affect the unitary channel current amplitude but reduced the channel open probability. The spermine block showed a slower time and steeper voltage dependence than the Mg2+i++ block. 5. When both these blockers were present, Mg2+i apparently attenuated the inhibitory effect of spermine on the outwardly flowing IRK2 currents. This interaction was voltage and time dependent, and could be well explained by a model in which Mg2+i and spermine competitively bind to the channel with their individual first-order kinetics. This competition would induce time-dependent transits of the channel between the Mg2+i -and spermine-blocked states via a single open state, thereby preserving a certain size of persistent outward currents at depolarized potentials.

Functional interaction between K(ATP) channels and the Na(+)-K(+) pump in metabolically inhibited heart cells of the guinea-pig.

1. Transmembrane current through ATP-regulated K(+) channels (IK(ATP)) was measured in ventricular heart cells of the guinea-pig in the whole-cell and cell-attached patch configurations under conditions of metabolic poisoning with the mitochondrial uncoupler 2,4-dinitrophenol (DNP). 2. Maintained exposure of the cells to DNP resulted in a transient appearance of whole-cell IK(ATP) When IK(ATP) had reached several nanoamps, blocking the forward-running Na(+)-K(+) pump with 0.5 mM strophanthidin decreased IK(ATP) after a delay. The time course of this decrease could be described by a single exponential function, which yielded a time constant(T)of 4.51+/- 1.89 s (n=8). 3. Hyperpolarization from 0 mV to -100 or -150 mV for 2 s caused IK(ATP) (measured at 0 mV) to decrease by 34.2 +/- 14.1 % (n = 8) and 37.6 +/- 9.4% (n = 8), respectively. After the hyperpolarizing pulse, IK(ATP) returned to its higher initial level within a couple of seconds. 4. Driving the pump backwards by removing the extracellular K(+) ions caused the permanent disappearance of DNP-induced IK(ATP). 5. Application of 0.5 mM strophanthidin in the absence of external K(+) ions induced a transient increase in IK(ATP), as did washing out the glycoside (n = 5). 6. When pump action was inhibited by using Na(+), K(+)-free Tyrode solution (see Methods) in the bath, strophanthidin did not have a comparable direct effect on IK(ATP). 7. In cell-attached patches, strophanthidin applied via the bath caused a reduction in IK(ATP) with a similar time course to that in whole-cell experiments. This suggests that the interaction between the pump molecules and the K(ATP) channels is not restricted to closely neighbouring molecules. 8. The data support the hypothesis that [ATP] at the cytosolic face of the membrane may drop to practically zero, thereby passing an 'ATP window' in which the channels first open and then close, and that the submembrane [ATP] is readily controlled by the cytosolic [ATP].

Activation of Maxi-K Channels by Parathyroid Hormone and Prostaglandin E 2 in Human Osteoblast Bone Cells

Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuration (CAP) no channel activity was observed in 2 mM Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon addition of 50 nM parathyroid hormone (PTH), 5 nM prostaglandin E2 (PGE2) or 0.1 mM dibutyryl cAMP + 1 microM forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that in symmetrical solutions (140 mM K) the channel has a conductance of 246 +/- 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (PK/PNa) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability (Po) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the Po vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mM ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE2 or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (45Ca2+) by MG-63 cells was stimulated in the presence of PTH and PGE2 an effect inhibited by Nitrendipine (10 microM). Second, whereas PGE2 stimulated the calcium-activated maxi-K channel in 2 mM Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE2 did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mM Ca Ringer and 10 microM Nitrendipine in CAP configuration, PGE2 (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of this channel, it is concluded that activation of this channel by PTH, PGE2 or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx.

Endotoxin-Induced L-Arginine Pathway Produces Nitric Oxide and Modulates the Ca2+-Activated K+ Channel in Cultured Human Dermal Papilla Cells

Endotoxin includes an enzyme that synthesizes nitric oxide (NO) from l-arginine (NO synthase) in vascular smooth muscles cells, macrophages, and fibroblasts, leading to the release of NO. We evaluated the release of NO and its intracellular action on the Ca2+-activated K+ channel (KCa channel) in cultured human dermal papilla cells by use of the electron paramagnetic response (EPR) spin trapping method and the patch clamp technique. In dermal papilla cells pretreated for 24 h with endotoxin (1 microgram/microliter), application of 1 microM L-arginine generated NO, although no measurable release of NO was observed in cells without endotoxin pretreatment, as determined by the EPR spin trapping method. With the patch clamp technique, we found that the KCa channel of dermal papilla cells had high conductance and was voltage dependent. In addition, after endotoxin pretreatment, the extracellular application of 100 microM l-arginine modulated the KCa channel in the cell-attached patch configurations. In inside-out patch configuration, however, NO produced by L-arginine itself did not modulate the Kca channel. The modulation of the KCa channel was suppressed by pretreatment with 100 microM N omega-nitro-L arginine methyl ester, and inhibitor of inducible and constitutive NO synthases. Methylene blue, a blocker of guanylate cyclase, inhibited the L-arginine-induced activation of the Kca channel. Theses results indicate that the endotoxin-induced L-arginine pathway cell generates No, which consequently modulated the KCa channel in cultured human dermal papilla cells by increasing of cyclic GMP-dependent phosphorylation.

Delayed rectifier channels in human ventricular myocytes.

Previous studies have shown that in heart there are two kinetically distinct components of delayed rectifier current: a rapidly activating component (IKr) and a more slowly activating component (IKs). The presence of IKr and/or IKs appears to be species dependent. We studied the nature of the delayed rectifier current in human ventricle in whole-cell and single-channel experiments. Ventricular myocytes were obtained from hearts of patients with ischemic or dilated cardiomyopathy. Single-channel currents and whole-cell tail currents were recorded at negative potentials directly after return from a depolarizing step. Single-channel currents were measured in the cell-attached patch configuration with 140 mmol/L K+ in the pipette. In the present study, we identified a voltage-dependent channel with a single-channel conductance of 12.9 +/- 0.8 pS (mean +/- SEM, n = 5) and a reversal potential near to the K+ equilibrium potential, suggesting that the channel is selective to K+ ions. Channel activity was observed only after a depolarizing step and increased with the duration and amplitude of the depolarization, indicating time- and voltage-dependent activation. Activation at +30 mV was complete within 300 milliseconds, and the time constant of activation, determined in the whole-cell configuration, was 101 +/- 25 milliseconds (mean +/- SEM, n = 4). The voltage dependence of activation could be described by a Boltzmann equation with a half-activation potential of -29.9 mV and a slope factor of 9.5 mV. The addition of the class III antiarrhythmic drug E-4031 completely blocked channel activity in one patch. No indications for the presence of IKs were found in these experiments. The conformity between the properties of IKr and those of the K+ channel in the present study strongly suggests that IKr is present in human ventricle.

Pentobarbitone modulation of NMDA receptors in neurones isolated from the rat olfactory brain

1. The action of pentobarbitone on the N-methyl-D-aspartate (NMDA) receptors of neurones freshly dissociated from the olfactory bulb and olfactory tubercle has been studied using patch-clamp techniques. 2. Pentobarbitone produced a concentration-dependent depression of the currents evoked by NMDA with an IC50 value of c. 250 microM. 3. Analysis of the NMDA-evoked noise produced power spectra that could be fitted by the sum of two Lorentzians with corner frequencies of 17 and 82 Hz. Pentobarbitone increased the corner frequency of the high frequency component but did not alter the apparent single channel conductance estimated from the noise. 4. Single channel recordings in either the cell-attached or outside-out patch configurations revealed that NMDA (20 or 50 microM) opened channels with a main conductance level around 55 pS and a principal subconductance around 44 pS. The uncorrected mean open time of the channels was 3.4 ms and mean burst length was 6.0 ms. Mean cluster length was about 12 ms. 5. Pentobarbitone produced a concentration-dependent reduction in both mean open time and burst length. Mean cluster length was much less affected. Pentobarbitone did not decrease unitary current amplitude or bias the open-state current amplitude distribution in favour of a particular substate. 6. From these data it appears that pentobarbitone depresses the inward current evoked by NMDA by reducing the probability of channel opening and this results from a shortening of the lifetime of the channel open state and by decreasing burst length.

Voltage-activated and spontaneous Cl −-channel activity in cultured human melanoma cell-line A2058

Voltage-activated and spontaneous chloride-channel activity was studied in melanoma cell-line A2058 by patch-clamp technique. Whole-cell and inside-out patch recordings carried out with leak subtraction show voltage-activated chloride-conductance. In addition, a large leak-type conductance typical of epithelial cells was found in whole-cell experiments. This current was carried mostly by chloride-ions but also a leak-type potassium conductance was found showing KCl fluxes to be possible. Cell-attached and inside-out patch recordings showed at least two types of spontaneous chloride-channel activity. Bursting, flickering-type channels were found only in cell-attached recordings. That led to the conclusion that some intracellular factors are needed for that kind of activity. A second spontaneous, 30 pS chloride-channel with slow kinetics was found both in cell-attached and inside-out patch configuration. A voltage-activated chloride-channel found had a conductance of approximately 25 pS. In our experiments these channels did not need external calcium for activation. Voltage ramp recordings in cell-attached configuration gives the intracellular chloride concentration of 163 mM on the basis of chloride reversal potential (extracellular 146 mM in our experiments). Intracellular chloride concentration thus seems to be maintained slightly higher than the extracellular one.

Potassium currents in human freshly isolated bronchial smooth muscle cells.

1. K+ currents were studied in smooth muscle cells enzymatically dissociated from human bronchi, by use of the patch-clamp technique. 2. In whole-cell recordings a depolarization-induced, 4-aminopyridine (4-AP)-sensitive current was observed in only 26 of 155 cells, and in 20 of these 26 cells its amplitude at a test potential of 0 mV was less than 100 pA. 3. In the majority of cells depolarization to -40 mV or more positive potentials induced a noisy outward current which activated within milliseconds and showed almost no inactivation even during a 5 s depolarizing voltage step. This current was insensitive to 4-AP (up to 5 mM) but was strongly inhibited in the presence of tetraethylammonium (TEA, 1 mM), charybdotoxin (ChTX, 100 nM) or iberiotoxin (IbTX, 50 nM) in the bath. The same current was also recorded by the nystatin-perforated patch technique. 4. Single channels with a conductance of about 210 pS were recorded in cell-attached patch, inside-out patch, outside-out patch and whole-cell recording configurations. Channel open state probability in inside-out patches was 0.5 at a membrane potential of 4 +/- 14 mV (mean +/- s.d., n = 13) mV even with a free Ca2+ concentration on the cytosolic side of the patch of less than 0.1 nM. Open state probability increased with depolarization and internal Ca2+ concentration. Single channels could be reversibly blocked by externally applied TEA, ChTX and IbTX. 5. In current-clamp recordings with 100 nM free Ca2+ in the intracellular solution both TEA and ChTX caused substantial concentration-dependent depolarization. 6. These results suggest that in human bronchial smooth muscle cells, in marked contrast to other species, the majority of the outward current induced by depolarization is not due to a delayed rectifier,but to the activity of a large conductance, ChTX-sensitive K+ channel. The Ca2+- and voltage-dependency of this channel may well allow a sufficiently high open state probability for it to play a partin the regulation of the resting membrane potential.

MCI-154 activates the Ca 2+-activated K + channel of vascular smooth muscle cells

The aim of this study was to examine the effects of MCI-154, a new positive inotropic agent with vasodilating properties, on the Ca 2+-activated K + channel (K Ca channel) of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 μM MCI-154 activated the K Ca channel in intact cell-attached patch configurations. In excised inside-out patch configurations, application of 100μM MCI-154 to the cytosolic side activated the K Ca channel directly, suggesting that the Ca 2+ sensitivity of the K Ca channel itself is modulated. Though extracellular application of 100 μM amrinone, a phosphodiesterase inhibitor, activated the K Ca channel in the cell-attached patch configurations, application of 100 μm amrinone to the cytosolic side could not activate the K Ca channel in inside-out patch configurations. These results indicate that different from amrinone, MCI-154 can modulate Ca 2+ sensitivity of the K Ca channel in vascular smooth muscle cells.

Calcitonin gene-related peptide activates the K+ channels of vascular smooth muscle cells via adenylate cyclase.

The aim of the present study was to examine the effects of calcitonin gene-related peptide (CGRP) on the K+ channels of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 nM CGRP activated two types of K+ channels, the Ca(2+)-activated K+ channel (KCa channel) and the ATP-sensitive K+ channel (KATP channel) in cell-attached patch configurations. In cells pretreated with Rp-cAMPS, a membrane-permeable inhibitor of cAMP-dependent protein kinase (PKA), extracellular application of 100 nM CGRP could not activate the KCa or KATP channel, indicating that the activation of the K+ channels by CGRP occurs in connection with PKA. In the cell-attached patch configurations, extracellular application of 1 mM dibutyryl cAMP, a membrane permeable cAMP, activated KCa and KATP channels. In inside-out patch configurations, application of PKA to the cytosolic side activated both the KCa and KATP channels. These results indicate that CGRP modulates the K+ channels of vascular smooth muscle cells via adenylate cyclase, i.e., cAMP-PKA pathway, and contributes to control of vascular tone.

An analysis of cardiac sodium channel properties using digital signal processing techniques

The properties of single sodium channels in membrane patches from isolated rat ventricular myocytes were analyzed. Both cell-attached and inside-out patch configurations were examined. A digital signal processing method was used which allowed accurate determination of single-channel conductance and mean open time, even in situations where many channels coexisted in the patch. The results show that the cardiac sodium channel has a conductance of 5.3 pS at room temperature, and shows no rectification or saturation in the physiological ranges of ion concentration and membrane potential. The active channels contained in the membrane patch opened and closed independently of each other. A transition probability matrix, which describes transitions between all current levels, can also be generated, analysis of which permits an estimate of individual mean channel open time as well as the degree of coupling between channels.

Hg2+ increases the open probability of carbachol-activated Cl- channels in Aplysia neurons.

Carbachol and acetylcholine (ACh)-activated whole-cell chloride currents were recorded in identified neurons of Aplysia californica. Application of 0.1-10 microM HgCl2 potentiated the chloride response and reduced the rate of desensitization. To investigate the underlying mechanism we recorded single chloride channels in a cell-attached patch configuration. Recordings were performed successively on the same neuron with 1 microM carbachol and with 1 microM carbachol + 1 microM HgCl2, respectively. The slope conductance did not change significantly, but there was a manyfold increase in the open probability, Po. This may underlie the Hg(2+)-induced enhancement of carbachol- and ACh-evoked chloride currents in Aplysia neurons and may serve as a model for the neurotoxic effect of Hg2+ on the mammalian ACh receptor.

Patch clamp analysis of the dominant plasma membrane K+ channel in root cell protoplasts of Plantago media L. Its significance for the P and K state

Ion channels in the plasma membrane of root cell protoplasts of Plantago media L. were studied with the patch clamp technique in the cell-attached patch and outside-out patch configuration. An outward rectifying potassium channel was dominantly present in the plasma membrane. It appears responsible for the diffusional part, dominated by the K+ diffusion potential, of the cell membrane potential, in vivo. This channel is activated at potentials near to and more positive than the K+ diffusion potential. The dependence of this ion channel on K+ activity and voltage has been characterized. The current-voltage relationships of the open channel at various K+ concentrations are described by a four-state model. The membrane potential of intact protoplasts appears either dominated by the K+ diffusion potential, the protoplast is then said to be in the K state, or by the pump potential generated by the plasma membrane-bound proton pump/H+ ATPase, the P state. An experimental procedure is described to determine in cell-attached patch mode the state of the protoplast, either K or P state.