p11, an annexin II subunit,an auxiliary protein associated with the background K+ channel,TASK-1

TASK-1 belongs to the 2P domain K+ channel family and is the prototype of background K+ channels that set the resting membrane potential and tune action potential duration. Its activity is highly regulated by hormones and neurotransmitters. Although numerous auxiliary proteins have been described to modify biophysical, pharmacological and expression properties of different voltage- and Ca2+-sensitive K+ channels, none of them is known to modulate 2P domain K+ channel activity. We show here that p11 interacts specifically with the TASK-1 K+ channel. p11 is a subunit of annexin II, a cytoplasmic protein thought to bind and organize specialized membrane cytoskeleton compartments. This association with p11 requires the integrity of the last three C-terminal amino acids, Ser-Ser-Val, in TASK-1. Using series of C-terminal TASK-1 deletion mutants and several TASK-1-GFP chimeras, we demonstrate that association with p11 is essential for trafficking of TASK-1 to the plasma membrane. p11 association with the TASK-1 channel masks an endoplasmic reticulum retention signal identified as Lys-Arg-Arg that precedes the Ser-Ser-Val sequence.     

New structure and function in plant K+ channels: KCO1, an outward rectifier with a steep Ca2+ dependency.

Potassium (K+) channels mediating important physiological functions are characterized by a common pore-forming (P) domain. We report the cloning and functional analysis of the first higher plant outward rectifying K+ channel (KCO1) from Arabidopsis thaliana. KCO1 belongs to a new class of ''two-pore'' K+ channels recently described in human and yeast. KCO1 has four putative transmembrane segments and tandem calcium-binding EF-hand motifs. Heterologous expression of KCO1 in baculovirus-infected insect (Spodoptera frugiperda) cells resulted in outwardly rectifying, K+-selective currents elicited by depolarizing voltage pulses in whole-cell measurements. Activation of KCO1 was strongly dependent on the presence of nanomolar concentrations of cytosolic free Ca2+ [Ca2+]cyt. No K+ currents were detected when [Ca2+]cyt was adjusted to <150 nM. However, KCO1 strongly activated at increasing [Ca2+]cyt, with a saturating activity observed at approximately 300 nM [Ca2+]cyt. KCO1 single channel analysis on excised membrane patches, resulting in a single channel conductance of 64 pS, confirmed outward rectification as well as Ca2+-dependent activation. These data suggest a direct link between calcium-mediated signaling processes and K+ ion transport in higher plants. The identification of KCO1 as the first plant K+ outward channel opens a new field of structure-function studies in plant ion channels.     

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.     

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.     

The heterogeneity of ion channels in chromaffin granule membranes

Chromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic ⁸⁶Rb⁺ flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 ± 10 pS, 220 ± 8 pS, 152 ± 8 pS and 13 ± 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 ± 8 pS and 31 ± 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0.     

Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and phosphatidylinositol bisphosphate (PIP(2)): a study of channel rundown and reactivation

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6. 2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of K(ATP) channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP(2)), and phosphorylation. Upon excision of inside-out patches into a Ca(2+)- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6. 2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca(2+) accelerated this process, suggesting a role for PIP(2) hydrolysis mediated by a Ca(2+)-dependent phospholipase C. PIP(2) could reactivate channel activity after a brief exposure to Ca(2+), but not after prolonged exposure. However, in both cases, PIP(2) reversed the increase in ATP sensitivity, indicating that PIP(2) lowers the ATP sensitivity by increasing P(o) as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca(2+) facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5-15 min, increased ATP sensitivity and loss of reactivation by PIP(2) and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP(2) responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP(2) and phosphorylation.     

Transformation of renal tubule epithelial cells by simian virus-40 is associated with emergence of Ca(2+)-insensitive K+ channels and altered mitogenic sensitivity to K+ channel blockers.

We compared the pattern of K+ channels and the mitogenic sensitivity to K+ channel blocking agents in primary cultures of rabbit proximal tubule cells (PC.RC) (Ronco et al., 1990) and two derived SV-40-transformed cell lines exhibiting specific functions of proximal (RC.SV1) and more distal (RC.SV2) tubule cells (Vandewalle et al., 1989). First, K+ channel equipment surveyed by the patch-clamp technique was modified after SV-40 transformation in both cell lines; although a high conductance Ca(2+)-activated K+ channel [K+200 (Ca2+)] remained the most frequently recorded K+ channel, the transformed state was characterized by emergence of three Ca(2+)-insensitive K+ channels (150, 50, and 30 pS), virtually absent from primary culture, contrasting with reduced frequency of two Ca(2+)-sensitive K+ channels (80 and 40 pS). Second, quinine (Q), tetraethylammonium ion (TEA) and charybdotoxin (CTX), at concentrations not affecting cell viability, all decreased 3H-TdR incorporation and cell growth in PC.RC cultures, but only TEA had similar effects in transformed cells. The latter were further characterized by paradoxical effects of Q that induced a marked increase in thymidine incorporation. Q also exerted contrasting effects on channel activity: it inhibited the [K+200 (Ca2+)] when the channel was highly active, with a Ki (0.2 mM) similar to that measured for 3H-TdR incorporation in PC.RC cells (0.3 mM), but increased the mean current through poorly active channels. TEA blocked all K+ channels with conductance greater than or equal to 50 pS, including the [K+200 (Ca2+)], in a range of concentrations that substantially affected cell proliferation. The unique effect of TEA on SV-40-transformed cells might be related to broad inhibition of K+ channels.     

重组人白细胞介素—1β对小鼬有骨髓基质细胞K^＋通道的影响

In the present study, both cell-attached and inside-out mode of patch clamp technique were applied to detect the effect of recombinant human interleukin-1 beta (IL-1 beta) on K+ channels of mice bone marrow stromal cells. A 16.7 pS voltage-dependent K+ channel resembling the delayed rectifier K+ channel in excitable cells was identified and characterized. Under stimulation of IL-1 beta (1000 U/ml), the single channel conductance increased to 26.1 +/- 3.6 pS (P < 0.01). IL-1 beta also markedly increased the open time constant and the open probability, but decreased the closed time constant as compared to the same patches without IL-1 beta. In addition, IL-1 beta induced multi-channel activities in about 80 percent patches by inducing more K+ channels of the same type open. These results suggest that activation of K+ channels plays a role in signal transduction of interleukin-1.     

Properties of single potassium channels in guinea pig hepatocytes

The patch-clamp technique of cell-attached and inside-out configurations was used to study the single potassium channels in isolated guinea pig hepatocytes. The single potassium channels in isolated guinea pig hepatocytes were recorded at different K⁺ concentrations. A linear single-channel current-voltage relationship was obtained at the voltage range of -80 to -20 mV with slope conductance of 70 ± 6 pS (n = 10). Under symmetrical high K⁺ concentration of 148 mM in the cell-attached patch membrane, the I-V curve exhibited a mild inward rectification at potentials positive to +20 mV. The values of reversal potential was +5 ± 2 mV (n = 10). When the external potassium concentration ([K⁺]₀) was decreased to 74 mM and 20 mM, the slope conductance was decreased to 48 ± 2 pS (n = 4) and 24 ± 3 pS (n = 3), respectively. The reversal potential was changed by 58 mV for a tenfold change in [K⁺]₀, indicating that this channel was highly selective for K⁺. Open probabilities (P₀) of the channel were 73-93% without apparent voltage dependence. The distributions of open time of the channels were fitted to two exponentials, while those of closed time were fitted to three exponentials, exhibiting no voltage dependence. The success rate of K⁺ channel activity to be recorded was 28% at room temperature, and there were no increases in the success rate nor in the channel opening probabilities at a temperature of 34-36°C. P₀ in inside-out patches was not changed by application of 1 μM Ca²⁺ nor 1 mM Mg²⁺ to the internal side of patch membranes. It is concluded that a novel type of the K⁺ channels in guinea pig hepatocytes had different properties of slope conductance, channel kinetics, and sensitivity to [Ca²⁺]_i, from those in other species. © 1994 Wiley-Liss, Inc.     

Single K+ currents during differentiation of embryonic muscle cells in vitro

After 3-7 days in culture, chicken myotubes possess five types of K+ channel: two high-conductance channels of 195 and 105 pS which are sensitive to tetraethylammonium (TEA), an ATP-sensitive channel of 64 pS and two low-conductance channels of 40 and 15 pS which are insensitive to TEA and ATP. The same population of channels is to be found in EGTA-treated muscle cells with blocked fusion and, with the exception of the ATP-sensitive channel, also in 1-day-old myoblasts. There are differences between myoblasts and myotubes in the percentage of incidence of individual channel types. High-conductance K+ channels are most frequently to be observed in myotubes, but they are rare in myoblasts and EGTA-treated cells where low-conductance K+ channels predominate.     

Two Distinct K+ Channels in Lamprey (Lampetra fluviatilis) Erythrocyte Membrane Characterized by Single Channel Patch Clamp

Two channels, distinguished by using single-channel patch-clamp, carry out potassium transport across the red cell membrane of lamprey erythrocytes. A small-conductance, inwardly rectifying K⁺-selective channel was observed in both isotonic and hypotonic solutions (osmolarity decreased by 50%). The single-channel conductance was 26 ± 3 pS in isotonic (132 mm K⁺) solutions and 24 ± 2 pS in hypotonic (63 mm K⁺) solutions. No outward conductance was found for this channel, and the channel activity was completely inhibited by barium. Cell swelling activated another inwardly rectifying K⁺ channel with a larger inward conductance of 65 pS and outward conductance of 15 pS in the on-cell configuration. In this channel, rectification was due to the block of outward currents by Mg²⁺ and Ca²⁺ ions, since when both ions were removed from the cytosolic side in inside-out patches the conductance of the channel was nearly ohmic. In contrast to the small-conductance channel, the swelling-activated channel was observed also in the presence of barium in the pipette. Neither type of channel was dependent on the presence of Ca²⁺ ions on the cytosolic side for activity. Received: 18 July 1997/Revised: 30 January 1998     

Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording

Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 ± 25 pS and 350 ± 14 pS in 400 mm symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca²⁺ and glutamate^?. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mech anosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.     

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.     

Properties of Mg(2+)-dependent cation channels in human leukemia K562 cells

The endogenous Mg(2+)-inhibited cation (MIC) current was recently described in different cells of hematopoietic lineage and was implicated in the regulation of Mg2+ homeostasis. Here we present a single channel study of endogenously expressed Mg(2+)-dependent cation channels in the human myeloid leukemia K562 cells. Inwardly directed unitary currents were activated in cell-attached experiments in the absence of Ca2+ and Mg2+ in the pipette solution. The current-voltage (I-V) relationships displayed strong inward rectification and yielded a single channel slope conductance of approximately 30 pS at negative potentials. The I-V relationships were not altered by patch excision into divalent-free solution. Channel open probability (P(o)) and mean closed time constant (tau(C)) were strongly voltage-dependent, indicating that gating mechanisms may underlie current inward rectification. Millimolar concentrations of Ca2+ or Mg2+ applied to the cytoplasmic side of the membrane produced slow irreversible inhibition of channel activity. The Mg(2+)-dependent cation channels described in this study differ from the MIC channels described in human T-cells, Jurkat, and rat basophilic leukemia (RBL) cells in their I-V relationships, kinetic parameters and dependence on intracellular divalent cations. Our results suggested that endogenously expressed Mg(2+)-dependent cation channels in K562 cells and the MIC channels in other hematopoietic cells might be formed by different channel proteins.     

Characterization of potassium channels in pancreatic beta cells from ob/ob mice

The patch-clamp technique in the cell-attached mode was used to study the K channels present in the membrane of cultured pancreatic beta cells from ob/ob mice. Three types of K+ channels were regularly observed, with conductances of 64, 20 and 146 pS. The conduction and kinetic properties of the 64 pS channel were similar to those of the ATP-sensitive potassium channel from normal beta cells. Furthermore, glucose blocked the activity of this channel at the same concentrations as that reported for normal cells. The 20 pS and the 146 pS were insensitive to glucose. The latter K+ channel appears to be similar to the large conductance voltage-activated potassium channels described in normal rodent beta cells. Thus, potassium channels in ob/ob pancreatic beta cells in culture are in most respects normal. Other factors may account for the abnormal electrical response to glucose of ob/ob pancreatic islets, such as reversible impairment of their function in vivo or defects not related to potassium permeability.     

Biophysical properties and metabolic regulation of a TASK-like potassium channel in rat carotid body type 1 cells

The single channel properties of TASK-like oxygen-sensitive potassium channels were studied in rat carotid body type 1 cells. We observed channels with rapid bursting kinetics, active at resting membrane potentials. These channels were highly potassium selective with a slope conductance of 14-16 pS, values similar to those reported for TASK-1. In the absence of extracellular divalent cations, however, single channel conductance increased to 28 pS in a manner similar to that reported for TASK-3. After patch excision, channel activity ran down rapidly. Channel activity in inside-out patches was markedly increased by 2 and 5 mM ATP and by 2 mM ADP but not by 100 microM ADP or 1 mM AMP. In cell-attached patches, both cyanide and 2,4-dinitrophenol strongly inhibited channel activity. We conclude that 1) whilst the properties of this channel are consistent with it being a TASK-like potassium channel they do not precisely conform to those of either TASK-1 or TASK-3, 2) channel activity is highly dependent on cytosolic factors including ATP, and 3) changes in energy metabolism may play a role in regulating the activity of these background K+ channels.     

Involvement of protein kinase A activation and phospholipase A(2) inhibition in the adenosine-activated basolateral 50 pS K(+) channels in the thick ascending limb of the rat kidney

The present study was designed to investigate the role of protein kinase A (PKA) and phospholipase A(2) (PLA(2)) in the stimulating effect of adenosine on the basolateral 50 pS K(+) channels in the thick ascending limb (TAL) of the rat kidney. Under the anatomic microscope, the TAL was dissected. The current of 50 pS K(+) channels were recorded by patch clamp technology. The protein expression of phosphorylated PKA and phosphorylated PLA(2) were examined by Western blot. The results showed that cyclohexyladenosine (CHA), an analog of adenosine, increased the 50 pS K(+) channel activity (P < 0.05). In the presence of H8, an antagonist of PKA, CHA did not affect the 50 pS K(+) channel activity. In the presence of AACOCF3 (an antagonist of PLA(2)), CHA did not further increase the 50 pS K(+) channel activity. CHA increased phosphorylation level of PKA, whereas inhibited phosphorylation of PLA(2) in the TAL of the rat kidney (P < 0.01). Furthermore, after blocking the PLA(2) with AACOCF3, CHA still increased the expression of phosphorylated PKA. On the contrary, CHA did not obviously change the expression of phosphorylated PLA(2) after H8 pretreatment. The results suggest that the stimulation of basolateral 50 pS K(+) channels by CHA is mediated by the activation of PKA followed by the inhibition of PLA(2) in the TAL of the rat kidney.     

Multiple types of voltage-dependent Ca2+-activated K+ channels of large conductance in rat brain synaptosomal membranes

K+-selective ion channels from a mammalian brain synaptosomal membrane preparation were inserted into planar phospholipid bilayers on the tips of patch-clamp pipettes, and single-channel currents were measured. Multiple distinct classes of K+ channels were observed. We have characterized and described the properties of several types of voltage-dependent, Ca2+-activated K+ channels of large single-channel conductance (greater than 50 pS in symmetrical KCl solutions). One class of channels (Type I) has a 200-250-pS single-channel conductance. It is activated by internal calcium concentrations greater than 10(-7) M, and its probability of opening is increased by membrane depolarization. This channel is blocked by 1-3 mM internal concentrations of tetraethylammonium (TEA). These channels are similar to the BK channel described in a variety of tissues. A second novel group of voltage-dependent, Ca2+-activated K+ channels was also studied. These channels were more sensitive to internal calcium, but less sensitive to voltage than the large (Type I) channel. These channels were minimally affected by internal TEA concentrations of 10 mM, but were blocked by a 50 mM concentration. In this class of channels we found a wide range of relatively large unitary channel conductances (65-140 pS). Within this group we have characterized two types (75-80 pS and 120-125 pS) that also differ in gating kinetics. The various types of voltage-dependent, Ca2+-activated K+ channels described here were blocked by charybdotoxin added to the external side of the channel. The activity of these channels was increased by exposure to nanomolar concentrations of the catalytic subunit of cAMP-dependent protein kinase. These results indicate that voltage-dependent, charybdotoxin-sensitive Ca2+-activated K+ channels comprise a class of related, but distinguishable channel types. Although the Ca2+-activated (Type I and II) K+ channels can be distinguished by their single-channel properties, both could contribute to the voltage-dependent Ca2+-activated macroscopic K+ current (IC) that has been observed in several neuronal somata preparations, as well as in other cells. Some of the properties reported here may serve to distinguish which type contributes in each case. A third class of smaller (40-50 pS) channels was also studied. These channels were independent of calcium over the concentration range examined (10(-7)-10(-3) M), and were also independent of voltage over the range of pipette potentials of -60 to +60 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+-dependent K+ channels from rat olfactory cilia characterized in planar lipid bilayers

Olfactory cilia contain cyclic nucleotide-gated and Ca2+-dependent Cl- conductances that underlie excitatory chemotransduction, and a Ca2+-dependent K+ (KCa) conductance, apparently involved in inhibitory transduction. Previous single-channel patch-clamp studies on olfactory cilia revealed four different KCas, with different conductances and kinetics. Here, we further characterized these channels in planar bilayers, where blockers could be properly tested. All four ciliary KCas were observed: The 16 pS channel, K0.5,Ca=40 microM and apamin-sensitive; the 30 and 50 pS channel, K0.5,Ca=59 microM, clotrimazole-sensitive and charybdotoxin-insensitive; the 60 pS channel, clotrimazole-sensitive and charybdotoxin-insensitive; and the 210 pS channel, K0.5,Ca=63 microM, blocked by charybdotoxin and iberiotoxin. The presence of the 16 and 210 pS channels was confirmed by immunoblotting.