Cation selective channel in fetal alveolar type II epithelium.

A cation selective channel was identified in the apical membrane of fetal rat (Wistar) alveolar type II epithelium using the patch clamp technique. The single channel conductance was 23 +/- 1.2 pS (n = 16) with symmetrical NaCl (140 mM) solution in the bath and pipette. The channel was highly permeable to Na+ and K+ (PNa/PK = 0.9) but essentially impermeant to chloride and gluconate. Membrane potential did not influence open state probability when measured in a high Ca2+ (1.5 mM) bath. The channel reversibly inactivated when the bath was exchanged with a Ca(2+)-free (less than 10(-9) M) solution. The Na+ channel blocker amiloride (10(-6) M) applied to the extracellular side of the membrane reduced P(open) relative to control patches; P(control) = 0.57 +/- 0.11 (n = 5), P(amiloride) = 0.09 +/- 0.07 (n = 4, p less than 0.01), however, amiloride did not significantly influence channel conductance (g); g(control) 19 +/- 0.9 pS (n = 5), 18 +/- 3.0 pS (n = 4). More than one current level was observed in 42% (16/38) of active patches; multiple current levels (ranging from 2 to 6) were of equal amplitude suggesting the presence of multiple channels or subconductance states. Channel activity was also evident in cell attached patches. Since monolayers of these cells absorb Na+ via an amiloride sensitive transport mechanism we speculate that this amiloride sensitive cation selective channel is a potential apical pathway for electrogenic Na+ transport in the alveolar region of the lung.     

Calcium- and voltage-dependent ion channels in Saccharomyces cerevisiae.

Ion channels in both the tonoplast and the plasma membrane of Saccharomyces cerevisiae have been characterized at the single channel level by patch-clamp techniques. The predominant tonoplast channel is cation selective, has an open-channel conductance of 120 pS in 100 mM KCl, and conducts Na+ or K+ equally well, and Ca2+ to a lesser extent. Its open probability (Po) is voltage-dependent, peaking at about -80 mV (cytoplasm negative), and falling to near zero at +80 mV. Elevated cytoplasmic Ca2+, alkaline cytoplasmic pH, and reducing agents activate the channel. The predominant plasma membrane channel is highly selective for K+ over anions and other cations, and shows strong outward rectification of the time-averaged current-voltage curves in cell-attached experiments. In isolated inside-out patches with micromolar cytoplasmic Ca2+, this channel is activated by positive going membrane voltages: mean Po is zero at negative membrane voltages and near unity at 100 mV. At moderate positive membrane voltages (20-40 mV), elevating cytoplasmic Ca2+ activates the channel to open in bursts of several hundred milliseconds duration. At higher positive membrane voltages, however, elevating cytoplasmic Ca2+ blocks the channel in a voltage-dependent fashion for periods of 2-3 ms. The frequency of these blocking events depends on cytoplasmic Ca2+ and membrane voltage according to second-order kinetics. Alternative cations, such as Mg2+ or Na+, block the yeast plasma-membrane K+ channel in a similar but less pronounced manner.     

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.     

A class of non-selective cation channels in human fibroblasts

Non-selective cation channels were detected in membrane patches of cultured human fibroblasts. The channels had a unitary conductance which ranged from 14 to 25 pS in symmetrical 130 mM NaCl and were permeable to both sodium and potassium ions. Open channel probability was dependent either on the membrane potential and the Ca2+ concentration on the intracellular side of the membrane. High Ca2+ concentrations in the millimolar range were needed to keep the channel active.     

A calcium and ATP sensitive nonselective cation channel in the antiluminal membrane of rat cerebral capillary endothelial cells.

The patch-clamp technique was applied to the antiluminal membrane of freshly isolated capillaries of rat brain (blood-brain barrier). With 1.3 mM Ca2+ in the bath, excision of membrane patches evoked ion channels, which could not be observed in cell-attached mode. The channel was about equally permeable to Na+ and K+ ions, but not measurable permeable to Cl- and the divalent ions Ca2+ and Ba2+. The current-voltage curve was linear in the investigated voltage range (-80 mV to +80 mV), and the single-channel conductance was 31 +/- 2 pS (n = 22). The channel open probability was not dependent on the applied potential. Lowering of Ca2+ to 1 microM or below on the cytosolic side inactivated the channels, whereas addition of cytosolic ATP (1 mM) inhibited channel activity completely and reversibly. The channel was blocked by the inhibitor of nonselective cation channels in rat exocrine pancreas 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10 microM) and by the antiinflammatory drugs flufenamic acid (greater than 10 microM) and tenidap (100 microM), as well as by gadolinium (10 microM). Thus, these nonselective cation channels have many properties in common with similar channels observed in fluid secreting epithelia. The channel could be involved in the transport of K+ ions from brain to blood side.     

Spontaneous and GABA-induced single channel currents in cultured murine spinal cord neurons

Intracellular and patch clamp recordings were made from embryonic mouse spinal cord neurons growing in primary cell culture. Outside-out membrane patches obtained from these cells usually showed spontaneous single channel currents when studied at the resting potential (-56 +/- 1.5 mV). In 18 out of 30 patches tested, spontaneous single channel activity was abolished by making Tris+ the major cation on both sides of the membrane. The remaining patches continued to display spontaneous single channel currents under these conditions. These events reversed polarity at a patch potential of 0 mV and displayed a mean single channel conductance of 24 +/- 1.2 pS. Application of the putative inhibitory transmitter gamma-aminobutyric acid (0.5-10 microM) to outside-out patches of spinal cord cell membrane induced single channel currents in 10 out of 15 patches tested. These channels had a primary conductance of 29 +/- 2.8 pS in symmetrical 145 mM Cl- solutions. Frequency distributions for the open times of these channels were well fit by the sum of a fast exponential term ("of") with a time constant tau of = 4 +/- 1.3 ms and a slow exponential term ("os") with a time constant tau os = 24 +/- 8.1 ms. Frequency distributions for channel closed times were also well fit by a double exponential equation, with time constants tau cf = 2 +/- 0.2 ms and tau cs = 62 +/- 20.9 ms.     

K+-selective channel from sarcoplasmic reticulum of split lobster muscle fibers

The patch clamp technique has been used to study channels in a membrane inside a cell. A single muscle fiber is skinned in relaxing saline (high K+, low Ca2+ with EGTA and ATP), leaving the native sarcoplasmic reticulum (SR) membrane exposed for patching. Fibers are dissected from the second antenna remotor muscles of the American lobster, Homarus americanus. Transmission and scanning electron microscopy confirm the large volume fraction of SR (approximately 70%) and absence of sarcolemma in this unusual skinned preparation. The resting potential of the SR was measured after the resistance of the patch of membrane was broken down. It is near 0 mV (-0.4 +/- 0.6 mV). The average input resistance of the SR is 842 +/- 295 M omega. Some 25% of patches contain a K+-selective channel with a mean open time of seconds and the channel displays at least two conducting states. The open probability is weakly voltage dependent, large at zero and positive potentials (cytoplasm minus SR lumen), and decreasing at negative potentials. The maximal conductance of this channel is 200 +/- 1 pS and the substate conductance is 170 +/- 3 pS in symmetrical 480 mM K+ solution. The current-voltage relation of the open channel is linear over a range of +/- 100 mV. The selectivity is similar to the SR K+ channel of vertebrates: PK/PNa is 3.77 +/- 0.03, determined from reversal potential measurements, whereas gamma K/gamma Na is 3.28 +/- 0.06, determined from open-channel conductance measurements in symmetrical 480 mM solutions. Voltage-dependent block in the lobster SR K+ channel is similar to, but distinct from, that reported for the vertebrate channels. It occurs asymmetrically when hexamethonium is added to both sides of the membrane. The block is more effective from the cytoplasmic side of the channel.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

Apical membrane ionic channels in the rabbit cortical thick ascending limb in primary culture.

Cortical thick ascending limbs of Henle's loop (cTAL) were microdissected from rabbit kidneys and cultured in a hormonally-defined medium. The cultured cells grew as a monolayer and retained the morphological and biochemical characteristics of the original tubule. Cyclic AMP production of the cultured cells was increased by human calcitonin (x13) and parathyroid hormone (x2). The cultured epithelial developed a transepithelial potential of 4.1 +/- 1.3 mV that was orientated positively towards the apical compartment. The basolateral membrane of the cells exhibited a chloride conductance sensitive to diphenylamine 2-carboxylate (DPC) and the apical membrane a barium-sensitive K+ permeability. Patch clamp analysis conducted on the apical membrane of the cells revealed the presence of three types of ionic channel. The first is a large conductance Ca(2+)-activated K+ channel (95 pS). The second K+ channel has a much smaller conductance (18.3 pS) and is insensitive to Ca2+. It may represent the conductive pathway for K+ recycling into the lumen in the original tubule. The last channel is cation selective, does not discriminate between Na+ and K+ and was found to have a conductance of 20.5 pS. Channel activity required a high cytoplasmic calcium concentration (1 mM), and was blocked by ATP (10 microM) applied on its cytoplasmic face.     

Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells

Ba2+ currents through L-type Ca2+ channels were recorded from cell- attached patches on mouse pancreatic beta cells. In 10 mM Ba2+, single- channel currents were recorded at -70 mV, the beta cell resting membrane potential. This suggests that Ca2+ influx at negative membrane potentials may contribute to the resting intracellular Ca2+ concentration and thus to basal insulin release. Increasing external Ba2+ increased the single-channel current amplitude and shifted the current-voltage relation to more positive potentials. This voltage shift could be modeled by assuming that divalent cations both screen and bind to surface charges located at the channel mouth. The single- channel conductance was related to the bulk Ba2+ concentration by a Langmuir isotherm with a dissociation constant (Kd(gamma)) of 5.5 mM and a maximum single-channel conductance (gamma max) of 22 pS. A closer fit to the data was obtained when the barium concentration at the membrane surface was used (Kd(gamma) = 200 mM and gamma max = 47 pS), which suggests that saturation of the concentration-conductance curve may be due to saturation of the surface Ba2+ concentration. Increasing external Ba2+ also shifted the voltage dependence of ensemble currents to positive potentials, consistent with Ba2+ screening and binding to membrane surface charge associated with gating. Ensemble currents recorded with 10 mM Ca2+ activated at more positive potentials than in 10 mM Ba2+, suggesting that external Ca2+ binds more tightly to membrane surface charge associated with gating. The perforated-patch technique was used to record whole-cell currents flowing through L-type Ca2+ channels. Inward currents in 10 mM Ba2+ had a similar voltage dependence to those recorded at a physiological Ca2+ concentration (2.6 mM). BAY-K 8644 (1 microM) increased the amplitude of the ensemble and whole-cell currents but did not alter their voltage dependence. Our results suggest that the high divalent cation solutions usually used to record single L-type Ca2+ channel activity produce a positive shift in the voltage dependence of activation (approximately 32 mV in 100 mM Ba2+).     

Ion channels in mammalian proximal renal tubules.

In the plasma membranes of mammalian proximal renal tubules single ion channels were investigated mainly in isolated tubules perfused on one side, in isolated nonperfused (collapsed) tubules and in primary cell cultures. With these techniques, the following results were obtained: in the luminal membrane of isolated one-sided perfused tubules of rabbit and mouse S3 segments, K(+)-selective channels with single-channel conductance (g) of 33 pS and 63 pS, respectively, were recorded. In primary cultures of rabbit S1 segments, a small-conductance (42 pS) as well as a large-conductance (200 pS) K+ channel were observed. The latter was Ca2(+)- and voltage-sensitive. In cultured cells a Ca2(+)-activated, nonselective cation channel with g = 25 pS was also recorded. On the other hand, an amiloride-sensitive channel with g = 12 pS, which was highly selective for Na+ over K+, was observed in the isolated perfused S3 segment. In the basolateral membrane of isolated perfused S3 segments, two types of K+ channels with g = 46 pS and 36 pS, respectively, were observed. The latter channel was not dependent on cytosolic Ca2+ in cell-excised patches. A K+ channel with g = 54 pS was recorded in isolated nonperfused S1 segments. This channel showed inward rectification and was more active at depolarizing potentials. In isolated perfused S3 segments, in addition to the K+ channels also a nonselective cation channel with g = 28 pS was observed. This channel was highly dependent on cytosolic Ca2+ in cell-free patches. It can be concluded that the K+ channels both in the luminal and contraluminal cell membrane are involved in the generation of the cell potential. Na+ channels in the luminal membrane may participate in Na+ reabsorption, whereas the function of a basolateral cation channel remains unclear. Recently, single anion-selective channels were recorded in membranes of endocytotic vesicles, isolated from rat proximal tubules. Vesicles were enlarged by the dehydration/rehydration method and investigated with the patch clamp technique. The Cl- channel had a conductance of 73 pS, the current-voltage curve was linear and the channel inactivated at high negative clamp potentials. It is suggested that this channel is responsible for charge neutrality during active H+ uptake into the endosomes.     

Single K+ channels in endocrine cells dispersed from the cricket (Gryllus bimaculatus) corpora allata

Single channel currents were recorded from cell-attached patches of endocrine cells of the adult male cricket corpora allata. Three distinct types of K+ channels were identified; a weak inward rectifier (Type 1), a strong inward rectifier (Type 2) and a weak outward rectifier (Type 3). The type 1 channel had a slope conductance of 191 +/- 9 pS (n = 4) at negative membrane potentials (Vm) and 101 +/- 6 pS (n = 6) at positive Vm. In addition, the channel showed fast open-closed kinetics at negative Vm and slow open-closed kinetics at positive Vm. The open probability (Po) of this channel was strongly voltage-dependent at positive Vm, but less voltage-dependent at negative Vm. The reversal potential was not modified significantly by the substitution of gluconate for external Cl- but was modified after N-methyl-D-glucamine (NMDG+) was substituted for external K+, according to the Nernst equation for a K+-selective channel. The type 2 channel had a slope conductance of 44 +/- 2 pS (n = 5) at negative Vm, but no detectable outward current was observed at positive Vm. This channel showed very slow open-closed kinetics at negative Vm and its Po was not voltage-dependent. The type 3 channel had a limit conductance of 55 +/- 12 pS (n = 3) at negative Vm and 88 +/- 10 pS (n = 3) at positive Vm. This channel showed slow open-closed kinetics at negative Vm and fast open-closed kinetics at positive Vm. The Po for the channel was voltage-dependent at positive Vm but was voltage-independent at negative Vm. These three types of K+ channels may be important for the control of the resting membrane potential, and may thus participate in the regulation of Ca2+ influx and juvenile hormone secretion in corpora allata cells.     

Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation.

The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel.     

Characterization of ion channels in the plasma membrane of epidermal cells of expanding pea (Pisum sativum arg) leaves

Ion channels in isolated patches of the plasma membrane of pea (Pisum sativum arg) epidermal cells were studied with the patch-clamp technique. One anion and one cation channel were dominantly present in most trials. The anion channel conducts nitrate, halides and malate, with a conductance in symmetrical 100 mm Cl^– of 300 pS and can be blocked by SITS when applied to the cytoplasmic side of the membrane. The cation channel poorly discriminates between potassium, sodium and lithium, is not blocked by either TEA or Ba²⁺, and has a conductance of 35 pS in symmetrical 100 mm K⁺. The open probability of the cation channel increases with increase of the Ca²⁺ concentration on the cytoplasmic side of the membrane from 0.1 to 1 m. The possible role of these two channels in the physiology of epidermal cells is discussed.This work was supported by NSF grant DCB-890 3744 to E.V.     

Conduction of Monovalent and Divalent Cations in the Slow Vacuolar Channel

The conduction properties of individual physiologically important cations Na+, K+, Mg2+, and Ca2+ were determined in the slowly activating (SV) channel of sugar beet vacuoles. Current-voltage relationships of the open channel were measured on excised tonoplast patches in a continuous manner by applying a +/-140 mV ramp-wave protocol. Applying KCl gradients of either direction across the patch we have determined that the relative Cl- to K+ permeability was < or =1%. Symmetrical increase of the concentration of tested cation caused an increase of the single channel conductance followed by saturation. Fitting of binding isotherms at zero voltage to the Michaelis-Menten equation resulted in values of maximal conductance of 300, 385, 18, and 13 pS, and of apparent dissociation constants of 64, 103, 0.04, and 0.08 mm for Na+, K+, Mg2+, and Ca2+, respectively. Deviations from the single-ion occupancy mechanism are documented, and alternative models of permeation are discussed. The magnitude of currents carried by divalent cations at low concentrations can be explained by an unrealistically wide (approximately 140 A) radius of the pore entrance. We propose instead a fixed negative charge in the pore vestibules, which concentrates the cations in their proximity. The conduction properties of the SV channel are compared with reported characteristics of voltage-dependent Ca2+-permeable channels, and consequences for a possible reduction of postulated multiplicity of Ca2+ pathways across the tonoplast are drawn.     

Two types of K+ channels in the apical membrane of rabbit proximal tubule in primary culture

The patch-clamp technique was used to investigate ionic channels in the apical membrane of rabbit proximal tubule cells in primary culture. Cell-attached recordings revealed the presence of a highly selective K+ channel with a conductance of 130 pS. The channel activity was increased with membrane depolarization. Experiments performed on excised patches showed that the channel activity depended on the free Ca2+ concentration on the cytoplasmic face of the membrane and that decreasing the cytoplasmic pH from 7.2 to 6.0 also decreased the channel activity. In symmetrical 140 mM KCl solutions the channel conductance was 200 pS. The channel was blocked by barium, tetraethylammonium and Leiurus quinquestriatus scorpion venom (from which charybdotoxin is extracted) when applied to the extracellular face of the channel. Barium and quinidine also blocked the channel when applied to the cytoplasmic face of the membrane. Another K+ channel with a conductance of 42 pS in symmetrical KCl solutions was also observed in excised patches. The channel was blocked by barium and apamin, but not by tetraethylammonium applied to the extracellular face of the membrane. Using the whole-cell recording configuration we determined a K+ conductance of 4.96 nS per cell that was blocked by 65% when 10 mM tetraethylammonium was applied to the bathing medium.     

Further characterization of the cation channel of a yeast vacuolar membrane in a planar lipid bilayer

A voltage-dependent and Ca2(+)-activated cation channel recently found in the vacuolar membrane of the yeast Saccharomyces cerevisiae was incorporated into planar lipid bilayers and further characterized in macroscopic and single channel levels. Single channel conductances for various cations were in the order: NH4+ greater than K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+, and were nearly consistent with the order of permeability ratio estimated from reversal potentials determined by macroscopic measurement. Up to 6 mM of Ca2+ added to the cis (cytoplasmic) side opened the channel, but higher concentrations closed the channel without affecting the single channel conductance. Ba2+ closed the channel without affecting the single channel conductance. Ba2+ closed the channel from the cis side. In addition to the above channel, a small cation-selective channel of about 40 pS was found.     

Regulation of the hyperpolarization-activated K+ channel in the lateral membrane of the cortical collecting duct [published erratum appears in J Gen Physiol 1995 Sep;106(3):579]

An intermediate-conductance K+ channel (I.K.), the activity of which is increased by hyperpolarization, was previously identified in the lateral membrane of the cortical collecting duct (CCD) of the rat kidney (Wang, W. H., C. M. McNicholas, A. S. Segal, and G. Giebisch. 1994. American Journal of Physiology. 266:F813-F822). The biophysical properties and regulatory mechanisms of this K+ channel have been further investigated with patch clamp techniques in the present study. The slope conductance of the channel in inside-out patches was 50 pS with 140 mM KCl in the pipette and 5 mM KCl, 140 mM NaCl (NaCl Ringer''s solution) in the bath. Replacement of the bath solution with symmetrical 140 mM KCl solution changed the slope conductance of the channel to 85 pS and shifted the reversal potential by 55 mV, indicating that the selectivity ratio of K+/Na+ was at least 10:1. Channel open probability (Po) in inside-out patches was 0.12 at 0 mV and was increased by hyperpolarization. The voltage-dependent Po was fitted with the Boltzmann''s equation: Po = 1/[1 + exp(V-V1/2)zF/RT], with z = 1.2 and V1/2 = -40 mV. Addition of 2 mM tetraethylammonium or 500 mM quinidine to the bath blocked the activity of the K+ channel in inside-out patches. In addition, decrease in the bath pH from 7.40 to 6.70 reduced Po by 30%. Addition of the catalytic subunit of protein kinase A (PKAc; 20 U/ml) and 100 microM [corrected] MgATP to the bath increased Po from 0.12 to 0.49 at 0 mV and shifted the voltage dependence curve of channel activity toward more positive potentials by 40 mV. Two exponentials were required to fit both the open-time and the closed-time histograms. Addition of PKAc increased the long open-time constant and shortened the long closed-time constant. In conclusion, PKA-mediated phosphorylation plays an important role in the regulation of the voltage dependence of the hyperpolarization-activated K+ channel in the basolateral membrane of CCD.     

Large-conductance calcium-activated anion channel characteristics in neuroblastoma cells

Large-conductance anion channel characteristics were investigated in neuroblastoma cells (N2A) by using different configurations of the patch-clamp technique. In excised patches, the channel was induced by depolarising potentials in 90% of experiments, had a conductance of 340 pS in symmetrical 135 mmol/l NaCl and exhibited the typical bell-shape activity. Neither the channel induction nor the channel activity was affected by rising the Ca2+ concentration on the cytopasmic side of membranes. In cell-attached configuration the maximal channel activity was shifted towards more positive potentials in comparison to that of excised patches and an increase in intracellular Ca2+, obtained by extracellular application of the Ca2+-ionophore A23187 in the presence of 0.2 micromol/l Ca2+, induced single-channel currents in 80% of patches compared to 31% of cell-attached experiments showing channel activity in normal conditions. In turn, application of 2 micromol/l Ca2+ induced channel activity in 100% of patches. The reversal potential of the channel in cell-attached patches was around -10 mV as the resting potential of cells eliciting channel activity. For cells where channel activity was not detected in cell-attached mode, the resting potential was around -45 mV. Channel activity could be restored in most whole-cell recordings in the presence of 2 micromol/l or more intracellular Ca2+ concentrations. The Ca2+-induction and the relation between channel activity and cell resting potential seem to suggest a role of the large-conductance anion channel in resting potential modulation during some basic functions of the neuroblastoma cell proliferation.