Single acetylcholine-activated channel currents in developing muscle cells

The properties of single acetylcholine-activated ion channels in developing rat myoblasts and myotubes in tissue culture have been investigated using the gigaohm seal patch clamp technique. Two classes of ACh-activated channels were identified. The major class of channels (accounting for >95% of all channel openings) has a conductance of 35 pS and a mean open time of 15 msec (at room temperature and ?80 mV). The minor class of channels has a larger conductance (55 pS) and a briefer mean open time (2–3 msec). Functional ACh-activated channels are present in undifferentiated mononucleated myoblasts 1–2 days in culture, although the channel density on such cells is low. Over the next week in culture, as the myoblasts fuse to form multinucleate myotubes, there is a marked increase in channel density and an increase in the proportion of large conductance channels. No significant change, however, occurs in channel conductance or open time (within a given class of channels) during this period. At high concentrations of ACh, channels desensitize and channel openings occur in groups, similar to what has been previously described in adult muscle. The rate of channel opening within a group of openings increases with increasing agonist concentration while mean open time is independent of agonist concentration, as expected from simple models of drug action. During a group of openings, the channel is open for half the time (i.e., channel opening rate is equal to channel closing rate) at a concentration of approximately 6 μm ACh.     

Rates and equilibria at the acetylcholine receptor of electrophorus electroplaques. A study of neurally evoked postsynaptic currents and of voltage-jump relaxations

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15 degrees C the rate constant, α, equals 1.2 ms(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/τ as the sum of the rate constant α for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate α, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to α-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10(7) to 2x10(8) M(-1)s(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10(2) to 3x10(3)s(-1).     

Roles of G proteins in coupling of receptors to ionic channels and other effector systems

Guanine nucleotide binding (G) proteins are heterotrimers that couple a wide range of receptors to ionic channels. The coupling may be indirect, via cytoplasmic agents, or direct, as has been shown for two K+ channels and two Ca2+ channels. One example of direct G protein gating is the atrial muscarinic K+ channel K+[ACh], an inwardly rectifying K+ channel with a slope conductance of 40 pS in symmetrical isotonic K+ solutions and a mean open lifetime of 1.4 ms at potentials between -40 and -100 mV. Another is the clonal GH3 muscarinic or somatostatin K+ channel, also inwardly rectifying but with a slope conductance of 55 pS. A G protein, Gk, purified from human red blood cells (hRBC) activates K+ [ACh] channels at subpicomolar concentrations; its alpha subunit is equipotent. Except for being irreversible, their effects on gating precisely mimic physiological gating produced by muscarinic agonists. The alpha k effects are general and are similar in atria from adult guinea pig, neonatal rat, and chick embryo. The hydrophilic beta gamma from transducin has no effect while hydrophobic beta gamma from brain, hRBCs, or retina has effects at nanomolar concentrations which in our hands cannot be dissociated from detergent effects. An anti-alpha k monoclonal antibody blocks muscarinic activation, supporting the concept that the physiological mediator is the alpha subunit not the beta gamma dimer. The techniques of molecular biology are now being used to specify G protein gating. A "bacterial" alpha i-3 expressed in Escherichia coli using a pT7 expression system mimics the gating produced by hRBC alpha k.     

Some properties of acetylcholine receptors in human cultured myotubes

The distribution and single channel properties of acetylcholine (ACh) receptors in human myotubes grown in tissue culture have been examined. Radioautography of myotubes labelled with [125I]alpha-bungarotoxin showed that ACh receptors are distributed uniformly over the myotube surface at a density of 3.9 +/- 0.5 receptors per square micrometre. Accumulations of ACh receptors (hot spots) were found rarely. The conductance and kinetics of ACh-activated channels were investigated with the patch-clamp technique. Cell-attached membrane patches were used in all experiments. A single channel conductance in the range 40-45 pS was calculated. No sublevels of conductance (substates) of the activated channel were observed. The distribution of channel open-times varied with ACh concentration. With 100 nM ACh, the distribution was best fitted by the sum of two exponentials, whereas with 1 microM ACh a single exponential could be fitted. The mean channel open-time at the myotube resting potential (ca. -70 mV, 22 degrees C) was 8.2 ms. The distribution of channel closed-times was complex at all concentrations of ACh studied (100 nM to 10 microM). With desensitizing doses of ACh (10 microM), channel openings occurred in obvious bursts; each burst usually appeared as part of a 'cluster' of bursts. Both burst duration and mean interval between bursts increased with membrane hyperpolarization. Individual channel open-times and burst durations showed similar voltage dependence (e-fold increase per 80 mV hyperpolarization), whereas both the channel closed-times within a burst and the number of openings per burst were independent of membrane potential.     

The frog interosseal muscle fiber as a new model for patch clamp studies of chemosensitive- and voltage-sensitive ion channels: actions of acetylcholine and batrachotoxin

The patch clamp technique was used to record the currents flowing through single ion channels in isolated frog muscle fibers. The majority of the acetylcholine (ACh)-activated channels had a conductance of 32 pS, although 20 pS channels were also occasionally observed. Lifetimes of ACh-activated channels increased with the transmembrane potential in the range from - 30 mV to - 105 mV. In these same fibers we also observed channels which were activated by low concentrations of batrachotoxin (BTX; 10 nM). These channels, presumed to be Na channels, had a conductance of 19 pS and opened at potentials at which Na channels would not normally open. A notable feature of these BTX-activated channels was that they opened and closed repeatedly. Therefore, it appears that the toxin, in addition to activating Na channels, also blocks the inactivation process. The physiological properties of these channels reveal significant differences between the ion channels of tissue-cultured and mature tissues.     

Opening rate of acetylcholine receptor channels.

The nicotinic acetylcholine (ACh) receptor is responsible for rapid conversion of chemical signals to electrical signals at the neuromuscular junction. Because the receptor and its ion channel are components of a single transmembrane protein, the time between ACh binding and channel opening can be minimized. To determine just how quickly the channel opens, we made rapid (100-400 microseconds) applications of 0.1-10 mM ACh to outside-out, multichannel membrane patches from BC3H-1 cells, while measuring the onset of current flow through the channels at 11 degrees C. Onset time is steeply dependent upon ACh concentration when channel activation is limited by binding of ACh (0.1-1 mM). At +50 mV, the 20-80% onset time reaches a plateau near 110 microseconds above 5 mM ACh as channel opening becomes rate limiting. Thus, we calculate the opening rate, beta = 12/ms, without reference to specific channel activation schemes. At -50 mV, the combination of a rapid, voltage-dependent block of channels by ACh with a finite solution exchange time distorts onset. To determine opening rate at -50 mV, we determine the kinetic parameters of block from "steady-state" current and noise analyses, assume a sequential model of channel activation/block, and numerically simulate current responses to rapid perfusion of ACh. Using this approach, we find beta = 15/ms. In contrast to the channel closing rate, the opening rate is relatively insensitive to voltage.     

Properties of membrane ion conductances evoked by hormonal stimulation of guinea-pig and rabbit isolated hepatocytes

Membrane conductance changes evoked in isolated guinea-pig or rabbit hepatocytes by hormonal stimulation were studied with the whole-cell patch clamp technique. In Cl-containing solutions, noradrenaline (NA), ATP or angiotensin II (AII) evoked an increase of conductance to both K (GK) and Cl (GCl) ions. Activation of GK occurred after a delay of several seconds and was sustained in the presence of hormone. Activation of GCl was transient, lasting several seconds, and arose either at the same time or shortly after the increase in GK. Conductances showed an initial rapid rise and slow oscillatory changes during maintained hormone application. The NA-induced current reversed at -19 mV in Cl solutions, between the equilibrium potentials for chloride (ECl = 0 mV) and potassium ions (EK = -85 mV), and at -75 mV, near EK, in Cl-free solution. In both conditions whole-cell current-voltage curves were linear in the range -100 mV to +40 mV. The conductance increase produced by NA to Cl- ions was about 50 nS, that to K+ ions was 6 nS. The potassium conductance increase was abolished by the polypeptide toxin apamin (50 nM). An increase in membrane current noise was associated with NA-evoked outward K+ current and blocked by apamin. Spectral analysis gave estimates of the elementary K channel conductance of 1.7 pS. Power spectra were fitted by two Lorentzian components, with average half-power frequencies of 2 and 190 Hz. These results are discussed in relation to the single-channel properties and indicate that the open probability of K channels during the NA response is high. In Cl solutions, with apamin to block the K conductance, no increase in current noise was detected during the large Cl conductance evoked by NA. This suggests either that Cl channels are of very low unitary conductance (less than 1 pS) or that Cl transport is due to a membrane carrier. The complex time-course of hormonally evoked conductances is not due to the properties of ion conductances per se but probably to underlying changes of intracellular second-messenger concentration.     

Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.     

Inward rectifier K+-channel kinetics from analysis of the complex conductance of Aplysia neuronal membrane.

Conduction in inward rectifier, K+-channels in Aplysia neuron and Ba++ blockade of these channels were studied by rapid measurement of the membrane complex admittance in the frequency range 0.05 to 200 Hz during voltage clamps to membrane potentials in the range -90 to -40 mV. Complex ionic conductances of K+ and Cl- rectifiers were extracted from complex admittances of other membrane conduction processes and capacitance by vector subtraction of the membrane complex admittance during suppressed inward K+ current (near zero-mean current and in zero [K+]0) from complex admittances determined at other [K+]0 and membrane potentials. The contribution of the K+ rectifier to the admittance is distinguishable in the frequency domain above 1 Hz from the contribution of the Cl- rectifier, which is only apparent at frequencies less than 0.1 Hz. The voltage dependence (-90 to -40 mV) of the chord conductance (0.2 to 0.05 microS) and the relaxation time (4-8 ms) of K+ rectifier channels at [K+]0 = 40 mM were determined by curve fits of admittance data by a membrane admittance model based on the linearized Hodgkin-Huxley equations. The conductance of inward rectifier, K+ channels at a membrane potential of -80 mV had a square-root dependence on external K+ concentration, and the relaxation time increased from 2 to 7.5 ms for [K+]0 = 20 and 100 mM, respectively. The complex conductance of the inward K+ rectifier, affected by Ba++, was obtained by complex vector subtraction of the membrane admittance during blockage of inward rectifier, K+ channels (at -35 mV and [Ba++]0 = 5 mM) from admittances determined at -80 mV and at other Ba++ concentrations. The relaxation time of the blockade process decreased with increases in Ba++ concentration. An open-closed channel state model produces the inductive-like kinetic behavior in the complex conductance of inward rectifier, K+ channels and the addition of a blocked channel state accounts for the capacitive-like kinetic behavior of the Ba++ blockade process.     

Characterization of inositol-1,4,5-trisphosphate-gated channels in the plasma membrane of rat olfactory neurons

It is generally accepted that inositol-1,4,5-trisphosphate (InsP3) plays a role in olfactory transduction. However, the precise mode of action of InsP3 remains controversial. We have characterized the conductances activated by the addition of 10 microM InsP3 to excised patches of soma plasma membrane from rat olfactory neurons. InsP3 induced current fluctuations in 25 of 121 inside-out patches. These conductances could be classified into two groups according to the polarity of the current at a holding potential of +40 to +60 mV (with Ringer's in the pipette and pseudointracellular solution in the bath). Conductances mediating outward currents could be further divided into large- (64 +/- 4 pS, n = 4) and small- (16 +/- 1.7 pS, n = 11) conductance channels. Both small- and large-conductance channels were nonspecific cation channels. The large-conductance channel displayed bursting behavior at +40 mV, with flickering increasing at negative holding potentials to the point where single-channel currents were no longer discernible. The small-conductance channel did not display flickering behavior. The conductance mediating inward currents at +40 to +60 mV reversed at +73 +/- 4 mV (n = 4). The current traces displayed considerable fluctuations, and single-channel currents could not be discerned. The current fluctuations returned to baseline after removal of InsP3. The power density spectrum for the excess noise generated by InsP3 followed a 1/f dependence consistent with conductance fluctuations in the channel mediating this current, although other mechanisms are not excluded. These experiments demonstrate the presence of plasma membrane InsP3-gated channels of different ionic specificity in olfactory receptor cells.     

Two types of Ca2+ currents with different sensitivities to organic Ca2+ channel antagonists in guinea pig pancreatic alpha 2 cells

The possibility that guinea pig pancreatic alpha 2 cells are equipped with more than one type of Ca2+ channel was explored using the patch-electrode voltage-clamp technique. At a holding potential of -100 mV, a slowly developing (tau m approximately 5 ms at -40 mV assuming m2 kinetics) Ca2+ current appeared. This conductance first became detectable at potentials of about -60 mV and reached a maximum amplitude of 50-100 pA between -30 and -20 mV. During long depolarizations, it inactivated completely (tau h approximately 100 ms at -40 mV). Half-maximal steady state inactivation was observed at about -60 mV. A second, more rapidly developing (tau m approximately 2 ms at 0 mV) Ca2+ current was observed during pulses to -40 mV and above. It had a peak amplitude of 150-200 pA between 0 and 10 mV, was less dependent on the holding potential, and inactivated very little, even during long pulses. Both conductances were blocked by Co2+ but were unaffected by tetrodotoxin. The rapidly developing current differed from the slowly developing one in being sensitive to the antagonists D-600 and nifedipine, conducting Ba2+ better than Ca2+, increasing upon exposure to forskolin, and showing time-dependent decay (rundown). These findings indicate that the alpha 2 cells are equipped with two kinds of Ca2+ channels.     

Overexpressed Ca(v)beta3 inhibits N-type (Cav2.2) calcium channel currents through a hyperpolarizing shift of ultra-slow and closed-state inactivation

It has been shown that beta auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of beta3 subunit on macroscopic Ba(2+) currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at "physiological" holding potentials (HPs) of -60 and -80 mV. At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca(v)2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced current suppression was reversed at a HP of -120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba(2+) (40 mM) as a charge carrier also largely diminished the effect of beta3 at -80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and beta3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel beta3 effect. Steady-state inactivation curves revealed that N-type channels exhibited "closed-state" inactivation without beta3, and that beta3 caused an approximately 40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of beta3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV. Similar "ultra-slow" inactivation was observed for N-type channels without beta3. Thus, beta3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting "ultra-slow" and "closed-state" inactivation properties.     

Intrinsic gating of inward rectifier in bovine pulmonary artery endothelial cells in the presence or absence of internal Mg2+

Inward rectifier (IR) currents were studied in bovine pulmonary artery endothelial cells in the whole-cell configuration of the patch-clamp technique with extracellular K+ concentrations, [K+]o, ranging from 4.5 to 160 mM. Whether the concentration of free Mg2+ in the intracellular solution, [Mg2+]i, was 1.9 mM or nominally 0, the IR exhibited voltage- and time-dependent gating. The IR conductance was activated by hyperpolarization and deactivated by depolarization. Small steady-state outward IR currents were present up to approximately 40 mV more positive than the K+ reversal potential, EK, regardless of [Mg2+]i. Modeled as a first-order C in equilibrium O gating process, both the opening rate, alpha, and the closing rate, beta, were exponentially dependent on voltage, with beta more steeply voltage dependent, changing e-fold for 9 mV compared with 18 mV for an e-fold change in alpha. Over all [K+]o studied, the voltage dependence of alpha and beta shifted along with EK, as is characteristic of IR channels in other cells. The steady-state voltage dependence of the gating process was well described by a Boltzmann function. The half-activation potential was on average approximately 7 mV negative to the observed reversal potential in all [K+]o regardless of [Mg2+]i. The activation curve was somewhat steeper when Mg-free pipette solutions were used (slope factor, 4.3 mV) than when pipettes contained 1.9 mM Mg2+ (5.2 mV). The simplest interpretation of these data is that IR channels in bovine pulmonary artery endothelial cells have an intrinsic gating mechanism that is not due to Mg block.     

Evidence for two concentration-dependent processes for beta-subunit effects on alpha1B calcium channels

beta-Subunits of voltage-dependent Ca(2+) channels regulate both their expression and biophysical properties. We have injected a range of concentrations of beta3-cDNA into Xenopus oocytes, with a fixed concentration of alpha1B (Ca(V)2.2) cDNA, and have quantified the corresponding linear increase of beta3 protein. The concentration dependence of a number of beta3-dependent processes has been studied. First, the dependence of the a1B maximum conductance on beta3-protein occurs with a midpoint around the endogenous concentration of beta3 (approximately 17 nM). This may represent the interaction of the beta-subunit, responsible for trafficking, with the I-II linker of the nascent channel. Second, the effect of beta3-subunits on the voltage dependence of steady-state inactivation provides evidence for two channel populations, interpreted as representing alpha1B without or with a beta3-subunit, bound with a lower affinity of 120 nM. Third, the effect of beta3 on the facilitation rate of G-protein-modulated alpha1B currents during a depolarizing prepulse to +100 mV provides evidence for the same two populations, with the rapid facilitation rate being attributed to Gbetagamma dissociation from the beta-subunit-bound alpha1B channels. The data are discussed in terms of two hypotheses, either binding of two beta-subunits to the alpha1B channel or a state-dependent alteration in affinity of the channel for the beta-subunit.     

Two types of single inward rectifying potassium channels in rat myocardial cells

The patch-clamp method was used to examine inward rectifying potassium channels in the membrane of rat ventricular myocytes. Two types of inward rectifying channels strongly selective for K+ ions and with different conductance and kinetics coexist in rat myocardial cells. When the concentration of K+ was 140 mmol/l on the extracellular side of the patch, the conductance was 38.9 pS for type I channels and 25.7 pS for the type II. The type II channels had a detectable conductance (4 pS) at potentials positive than the potassium equilibrium potential. The mean open time was 18 ms at -60 mV patch membrane potential and 12 ms at -100 mV for type I channels, and 1.3 s at -60 mV and 0.94 s at -105 mV for type II channels, respectively. The opening probability of type II channels decreased with hyperpolarization. The type II channels can adopt several (about 10 or more) conductance states, which can occur either within one opening or as individual events.     

Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts. Single channel current kinetics reveal distinct agonist binding affinities.

The experiments described examine single channel currents recorded through Torpedo acetylcholine receptor channels stably expressed by a mouse fibroblast cell line. Closed-duration histograms were constructed from currents elicited by 0.5-300 microM acetylcholine (ACh). The concentration dependence of closed durations is well described by a four-state linear scheme with the addition of open-channel block by ACh. Analysis of closed durations measured at low concentrations gives estimates of the rate of opening of doubly liganded receptors, beta, the rate of dissociation of ACh from doubly liganded receptors, k-2, and the rate of channel closing, alpha. The rate of ACh dissociation from singly liganded receptors, k-1, is then deduced from closed-duration histograms obtained at intermediate ACh concentrations. With k-1, k-2 and beta determined, the rates of ACh association, k+1 and k+2, are estimated from fitting closed-duration histograms obtained over a range of high ACh concentrations. A complete set of rate constants is presented for three experimental conditions: (a) Ca2(+)-free extracellular solution containing 1 mM free Mg2+ at 22 degrees C, (b) Ca2(+)-free solution at 12 degrees C, and (c) extracellular Ca2+ and Mg2+, both at 0.5 mM, at 22 degrees C. For all three conditions the dissociation constant for the first agonist binding site is approximately 100-fold lower than that for the second site. The different affinities are due primarily to different dissociation rates. Both the association and dissociation rates depend strongly on temperature. At 22 degrees C ACh associates at diffusion-limited rates, whereas at 12 degrees C association is 30- to 60-fold slower. Also slowed at 12 degrees C are beta (4-fold), k-2 (3-fold), k-1 (25-fold), and alpha (15-fold). In contrast to the activation rate constants, those for ACh-induced block decrease only twofold between 22 and 12 degrees C. Changing from a Ca2(+)-free to a Ca2(+)-containing extracellular solution does not affect k+1 and k+2, but increases beta (twofold) and decreases k-2, k-1, and alpha (all twofold). Spectral analysis of single channel currents supports the parameter estimates obtained from fitting the open- and closed-duration histograms, and improves resolution of brief channel blockages produced by ACh.     

Alteration of the conductance of Na+ channels in the nodal membrane of frog nerve by holding potential and tetrodotoxin

(1) Na+ currents and Na+-current fluctuations were measured in myelinated frog nerve fibres at 15 degrees C during 7.7 ms depolarizations to V = 40, 60 and 80 mV. (2) The conductance gamma of a single Na+ channel and the number No of channels per node were calculated from ensemble average values of the mean Na+ current and the variance of Na+-current fluctuations. (3) For a hyperpolarizing holding potential of VH = -28 mV the mean values of the channel conductance and number were gamma = 9.8 pS and No = 74000. (4) After changing the holding potential to the resting potential (VH = 0) the conductance gamma increased by a factor of 1.37 whereas the number No decreased by a factor of 0.60. (5) Addition of 8 nM tetrodotoxin at a holding potential of VH = -28 mV increased gamma by a factor of 1.55 and reduced No by a factor of 0.25. (6) The increase of the channel conductance at reduced channel numbers suggests negative cooperativity between Na+ channels in the nodal membrane.     

Single channel K+ currents from HeLa cells

The extracellular patch-clamp technique was used in order to investigate the presence of ionic channels in HeLa cells, a well-known cultured cell type obtained from an epidermoid carcinoma of the cervix. Under Gigohm-seal conditions, discrete current jumps could be observed with patch electrodes containing KCl. These channels were found to be mainly permeable to K+ and showed multiple levels of conductance. From single-channel I-V curve measurements, a strong rectification effect, characterized by a large inward and no detectable outward current, was observed. For negative membrane potentials (0 to -90 mV), the measured current-voltage relationship was found to be mostly linear, corresponding to a single-channel conductance of 40 pS. An analysis of some selected time records has revealed in addition that the probability of the channel to be in the open state was a function of the KCl concentration in the patch pipette.     

Calcium- and voltage-activated potassium channels in human macrophages.

Single calcium-activated potassium channel currents were recorded in intact and excised membrane patches from cultured human macrophages. Channel conductance was 240 pS in symmetrical 145 mM K+ and 130 pS in 5 mM external K+. Lower conductance current fluctuations (40% of the larger channels) with the same reversal potential as the higher conductance channels were noted in some patches. Ion substitution experiments indicated that the channel is permeable to potassium and relatively impermeable to sodium. The frequency of channel opening increased with depolarization and intracellular calcium concentration. At 10(-7) M (Ca++)i, channel activity was evident only at potentials of +40 mV or more depolarized, while at 10(-5) M, channels were open at all voltages tested (-40 to +60 mV). In intact patches, channels were seen at depolarized patch potentials of +50 mV or greater, indicating that the ionized calcium concentration in the macrophage is probably less than 10(-7) M.