Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells

The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K _Ca ) channel and (iii) a small K⁺ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na⁺ and K⁺, but not permeable to Cl⁻ or N-methyl-d-glucamine. Channel activity required the presence of Ca²⁺ at the cytosolic face, but was detected at Ca²⁺ concentrations as low as 10⁻⁷ m (open probability (P _o ) = 0.11 ± 0.03, n= 8). Gadolinium decreased P _o of the NSC channel from both the external and cytosolic side (IC₅₀∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K⁺ could be fitted to the I-V relationship in asymmetrical K⁺ and Na⁺ solutions. The channel was impermeable to Cl⁻ and N-methyl-d-glucamine. P _o of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca²⁺. TEA (20 mm), charybdotoxin (100 nm) and Ba²⁺ (1 mm) but not amiloride (1 mm) reduced P _o from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P _o and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca²⁺ activated, voltage-insensitive and involved in both constitutive K⁺ and Na⁺ reabsorption from endolymph while the BK channel might participate in the K⁺ pathway under stimulated conditions that produce an elevated intracellular Ca²⁺ or depolarized membrane potential. Received: 14 October 1999/Revised: 10 December 1999     

The Essential Role of Cytosolic Cl− in Ca2+ Regulation of an Amiloride-Sensitive Channel in Fetal Rat Pneumocyte

An amiloride-sensitive, Ca²⁺-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na⁺ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca²⁺ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (P _o ) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca²⁺. The P _o of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca²⁺ to 0.26 ± 0.05 (n= 8). The cytosolic Ca²⁺ concentration ([Ca²⁺] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl⁻ concentration ([Cl⁻] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca²⁺] _c from 100 to 20 nm itself had no significant effects on the P _o if the [Cl⁻] _c was reduced to 20 mm; the P _o was 0.58 ± 0.10 at 100 nm [Ca²⁺] _c and 0.55 ± 0.09 at 20 nm [Ca²⁺] _c (n= 8) with 20 mm [Cl⁻] _c in inside-out patches. On the other hand, the P _o (0.28 ± 0.10) at 20 nm [Ca²⁺] _c with 40 mm [Cl⁻] _c was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca²⁺] _c with 20 mm [Cl⁻] _c , suggesting that reduction of [Cl⁻] _c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca²⁺ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl⁻] _c . Received: 11 August 2000/Revised: 4 December 2000     

A Calcium-activated and nucleotide-sensitive nonselective cation channel in M-1 mouse cortical collecting duct cells

We recently reported that M-1 mouse cortical collecting duct cells show nonselective cation (NSC) channel activity (Proc. Natl. Acad. Sci. USA 89:10262–10266, 1992). In this study, we further characterize the M-1 NSC channel using single-channel current recordings in excised inside-out patches. The M-1 NSC channel does not discriminate between Na⁺, K⁺, Rb⁺, Cs⁺, and Li⁺. It has a linear I-V relation with a conductance of 22.7±0.5 pS (n=78) at room temperature. The P_cation/ P_anion ratio is about 60 and there is no measurable conductance for NMDG, Ca²⁺, Ba²⁺, and Mn²⁺. Cytoplasmic calcium activates the M-1 NSC channel at a threshold of 10^–6 m and depolarization increases channel activity (NP _o ). Cytoplasmic application of adenine nucleotides inhibits the M-1 NSC channel. At doses of 10^–4 m and 10^–3 m, ATP reduces NP _o by 23% and 69%, respectively.Furthermore, since ADP (10^–3 m) reduces NP _o by 93%, the inhibitory effect of adenine nucleotides is not dependent on the presence of a -phosphoryl group and therefore does not involve protein phosphorylation. The channel is not significantly affected by 8-Br-cGMP (10^–4 m) or by cGMP-dependent protein kinase (10^–7 m) in the presence of 8-Br-cGMP (10^–5 m) and ATP (10^–4 m). The NSC channel is not sensitive to amiloride (10^–4 m cytoplasmic and/or extracellular) but flufenamic acid (10^–4 m) produces a voltage-dependent block, reducing NP _o by 35% at depolarizing voltages and by 80% at hyperpolarizing voltages.We conclude that the NSC channel of M-1 mouse cortical collecting duct cells belongs to an emerging family of calcium-activated and nucleotide-sensitive nonselective cation channels. It does not contribute to amiloride-sensitive sodium absorption and is unlikely to be a major route for calcium entry. The channel is normally quiescent but may be activated under special physiological conditions, e.g., during volume regulation.The expert technical assistance of U. Fink and I. Doering-Hirsch is gratefully acknowledged. We thank A. Rabe and Dr. J. Disser for programming the computer software.This work was supported by a grant from the Deutsche Forschungsge-meinschaft (DFG grant Fr 233/9-1) and a grant from the National Institutes of Health (NIH grant DK-17433).     

Ca<Superscript>2+</Superscript>-activated nonselective cation channels in rat neonatal atrial myocytes

A nonselective cation channel activated by intracellular Ca²⁺ was identified in inside-out membrane patches taken from cultured rat atrial myocytes. Ca²⁺ (0.01–1.00 mM) reversibly activated the channel in a concentration-dependent manner. The channel often showed a quick and irreversible rundown within a few minutes after patch excision. The I-V relationship of the channel was linear between –100 and +100 mV. The single channel conductance was 26.0 ± 0.5 pS and its open probability was weakly voltage-dependent. Ion-substitution experiments showed that the channel was permeable to monovalent cations (P_x/P_Cs: Li⁺ (1.5) = K⁺ (1.5)> Na⁺ (1.2) > Rb⁺ (1.1) > Cs⁺ (1.0)) but not to Cl^– (P_Cl/P_Cs < 0.01) and Ca²⁺ (P_Ca/P_Cs =0.02 ± 0.01). Present address: A.B. Zhainazarov UF Center for Smell and Taste, McKnight Brain Institute, University of Florida, 100 S Newell Dr., L1-131, P.O. Box 100015, Gainesville, FL 32610-0015, USA     

ADH Action on Whole-Cell Currents by Cytosolic Ca2+-dependent Pathways in Aldosterone-treated A6 Cells

We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca²⁺ concentrations ([Ca²⁺] _c , 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca²⁺] _c , basal conductances mainly consisted of Cl⁻ conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca²⁺] _c to 2 nm abolished the basal Cl⁻ conductance. AVT evoked Cl⁻ conductances at 2 as well as 30 nm [Ca²⁺] _c . In addition to Cl⁻ conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca²⁺] _c but not at 2 nm [Ca²⁺] _c . Thus, cytosolic Ca²⁺ regulates NSC and Cl⁻ conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca²⁺] _c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells. Received: 6 May 1996/Revised: 28 June 1996     

Single calcium-dependent cation channels in mouse pancreatic acinar cells

Summary The Ca²⁺-activated nonselective cation channel in mouse pancreatic acini has been studied with the help of patch-clamp single-channel current recording in both the cell-attached conformation and in excised inside-out membrane patches. In intact resting mouse pancreatic acinar cells no unitary activity was observed. Adding saponin to the bath solution to disrupt the plasma membrane (apart from the isolated patch membrane from which current recording was made) evoked unitary inward current steps when the free ionized Ca²⁺ concentration in the bath ([Ca²⁺] _i ) was 5×10^–8 m or above. When an electrically isolated patch membrane was excised and the internal aspects of the plasma membrane were exposed to the bath solution, channel activation could be obtained when [Ca²⁺] _i was 10^–7 m or above. However, with the passage of time the total inward current declined and about 1 min after excision no unitary current steps could be observed. At this stage Ca²⁺ in micromolar concentration was needed to open the channels and several hundred micromoles of Ca²⁺ per liter were required for maximal channel activation. Our results indicate that the Ca²⁺-activated nonselective cation channel is more sensitive to internal Ca²⁺ than hitherto understood and that it may therefore play a role under physiological conditions in intact cells.     

Further Characteristics of the Ca2+-inactivated Cl− Channel in Xenopus laevis Oocytes

Removal of extracellular Ca²⁺ activates ion channels in the plasma membrane of defolliculated oocytes of the South Africa clawed toad Xenopus laevis. At present, there is controversy about the nature of the Ca²⁺-inactivated ion channels. Recently, we identified one of these channels as a Ca²⁺-inactivated Cl⁻ channel (CaIC) using single channel analysis. In this work we confirm and extend previous observations on the CaIC by presenting a decisive extension of the regulation and inhibition profile. CaIC current is reversibly blocked by the divalent and trivalent cations Zn²⁺ (half-maximal blocker concentration, K_1/2= 8 μm), Cu²⁺ (K_1/2= 120 μm) and Gd³⁺ (K_1/2= 20 μm). Furthermore, CaIC is inhibited by the specific Cl⁻ channel blocker NPPB (K_1/2≈ 3 μm). Interestingly, CaIC-mediated currents are further sensitive to the cation channel inhibitor amiloride (500 μm) but insensitive to its high affinity analogue benzamil (100 μm). An investigation of the pH-dependence of the CaIC revealed a reduction of currents in the acidic range. Using simultaneous measurements of membrane current (I _m ), conductance (G _m ) and capacitance (C _m ) we demonstrate that Ca²⁺ removal leads to instant activation of CaIC already present in the plasma membrane. Since C _m remains constant upon Ca²⁺ depletion while I _m and G _m increase drastically, no exocytotic transport of CaIC from intracellular pools and functional insertion into the plasma membrane is involved in the large CaIC currents. A detailed overview of applicable blockers is given. These blockers are useful when oocytes are utilized as an expression system for foreign proteins whose investigations require Ca²⁺-free solutions and disturbances by CaIC currents are unwanted. We further compare and discuss our results with data of Ca²⁺-inactivated cation channels reported by other groups. Received: 18 June 1999/Revised: 13 August 1999     

Mechanism of Shrinkage Activation of Nonselective Cation Channels in M-1 Mouse Cortical Collecting Duct Cells

It has previously been shown that osmotic cell shrinkage activates a nonselective cation (NSC) channel in M-1 mouse cortical collecting duct cells [54] and in a variety of other cell types [20]. In the present study we further characterized the shrinkage-activated NSC channel in M-1 cells and its mechanism of activation using whole-cell current recordings. Osmotic cell shrinkage induced by addition of 100 mm sucrose to the bath solution caused a 20-fold increase in whole-cell inward currents from −10.8 ± 1.5 pA to −211 ± 10.2 pA (n= 103). A similar response was observed when cell shrinkage was elicited using a hypo-osmotic pipette solution. This indicates that cell shrinkage and not extracellular osmolarity per se is the signal for current activation. Cation substitution experiments revealed that the activated channels discriminate poorly between monovalent cations with a selectivity sequence NH₄ (1.2) ≥ Na⁺ (1) ≈ K⁺ (0.9) ≈ Li⁺ (0.9). In contrast there was no measurable permeability for Ca²⁺ or Ba²⁺ and the cation-to-anion permeability ratio was about 14. The DPC-derivatives flufenamic acid, 4-methyl-DPC and DCDPC were the most effective blockers followed by LOE 908, while amiloride and bumetanide were ineffective. The putative channel activator maitotoxin had no effect. Current activation was dependent upon the presence of intracellular ATP and Mg²⁺ and was inhibited by staurosporine (1 μm) and calphostin C (1 μm). Moreover, cytochalasin D (10 μm) and taxol (2 μm) reduced the current response to cell shrinkage. These findings suggest that the activation mechanism of the shrinkage-activated NSC channel involves protein kinase mediated phosphorylation steps and cytoskeletal elements. Received: 3 May 2000/Revised: 6 July 2000     

Leukotriene D4-induced Ca2+ Mobilization in Ehrlich Ascites Tumor Cells

Stimulation of Ehrlich ascites tumor cells with leukotriene D₄ (LTD₄) within the concentration range 1–100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca²⁺ concentration, [Ca²⁺] _i . The Ca²⁺ peak time, i.e., the time between addition of LTD₄ and the highest measured [Ca²⁺] _i value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration of LTD₄ (100 nm), the highest measured increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium is 260 ± 14 nm and the EC₅₀ value for LTD₄-induced Ca²⁺ mobilization is estimated at 10 nm. Neither the peptido-leukotrienes LTC₄ and LTE₄ nor LTB₄ are able to mimic or block the LTD₄-induced Ca²⁺ mobilization, hence the receptor is specific for LTD₄. Removal of Ca²⁺ from the experimental buffer significantly reduces the size of the LTD₄-induced increase in [Ca²⁺] _i . Furthermore, depletion of the intracellular Ins(1,4,5)P₃-sensitive Ca²⁺ stores by addition of the ER-Ca²⁺-ATPase inhibitor thapsigargin also reduces the size of the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium, and completely abolishes the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-free medium containing EGTA. Thus, the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells involves an influx of Ca²⁺ from the extracellular compartment as well as a release of Ca²⁺ from intracellular Ins(1,4,5)P₃-sensitive stores. The Ca²⁺ peak times for the LTD₄-induced Ca²⁺ influx and for the LTD₄-induced Ca²⁺ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35 min in six out of eight experiments, respectively. Stimulation with LTD₄ also induces a transient increase in Ins(1,4,5)P₃ generation in the Ehrlich cells, and the Ins(1,4,5)P₃ peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4,5)P₃ content seems to increase before the LTD₄-induced Ca²⁺ release from the intracellular stores but after the LTD₄-induced Ca²⁺ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD₄-induced increase in Ins(1,4,5)P₃ as well as the LTD₄-induced increase in [Ca²⁺] _i , indicating that a U73122-sensitive phospholipase C is involved in the LTD₄-induced Ca²⁺ mobilization in Ehrlich cells. The LTD₄-induced Ca²⁺ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD₄-activated Ca²⁺ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion, LTD₄ acts in the Ehrlich cells via a specific receptor for LTD₄, which upon stimulation initiates an influx of Ca²⁺, through yet unidentified Ca²⁺ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P₃ formation and finally release of Ca²⁺ from the intracellular Ins(1,4,5)P₃-sensitive stores. Received: 9 February 1996/Revised: 15 August 1996     

Calcium-Sensitive Nonselective Cation Channel Identified in the Epithelial Cells Isolated from the Endolymphatic Sac of Guinea Pigs

We identified a Ca²⁺-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 ± 1.3 pS (n= 8) in our standard solution. The relative ionic permeability of the channel was in the order K⁺= Na⁺ > Ca²⁺≫ Cl⁻. This channel was weakly voltage-dependent but was strongly activated by Ca²⁺ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca²⁺ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 μm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μm), or ATP (1 mm), which increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), activated the channel at an estimated [Ca²⁺]_i from 0.6 μm to 10 μm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca²⁺ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na⁺ under certain pathological conditions that will increase [Ca²⁺]_i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells. Received: 24 October 2000/Revised: 10 April 2001     

Inositol 1,3,4,5-tetrakisphosphate is essential for sustained activation of the Ca2+-dependent K+ current in single internally perfused mouse lacrimal acinar cells

Summary We have examined the effects of various inositol polyphosphates, alone and in combination, on the Ca²⁺-activated K⁺ current in internally perfused, single mouse lacrimal acinar cells. We used the patch-clamp technique for whole-cell current recording with a set-up allowing exchange of the pipette solution during individual experiments so that control and test periods could be directly compared in individual cells. Inositol 1,4,5-trisphosphate (Ins 1,4,5 P₃) (10–100 m) evoked a transient increase in the Ca²⁺-sensitive K⁺ current that was independent of the presence of Ca²⁺ in the external solution. The transient nature of the Ins 1,4,5 P₃ effect was not due to rapid metabolic breakdown, as similar responses were obtained in the presence of 5mm 2,3-diphosphoglyceric acid, that blocks the hydrolysis of Ins 1,4,5 P₃, as well as with the stable analoguedl-inositol 1,4,5-trisphosphorothioate (Ins 1,4,5 P(S)₃) (100 m). Ins 1,3,4 P₃ (50 m) had no effect, whereas 50 m Ins 2,4,5 P₃ evoked responses similar to those obtained by 10 m Ins 1,4,5 P₃. A sustained increase in Ca²⁺-dependent K⁺ current was only observed when inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5 P₄) (10 m) was added to the Ins 1,4,5 P₃ (10 m)-containing solution and this effect could be terminated by removal of external Ca²⁺. The effect of Ins 1,3,4,5 P₄ was specifically dependent on the presence of Ins 1,4,5 P₃ as it was not found when 10 m concentrations of Ins 1,3,4 P₃ or Ins 2,4,5 P₃ were used. Ins 2,4,5 P₃ (but not Ins 1,3,4 P₃) at the higher concentration of 50 m did, however, support the Ins 1,3,4,5 P₄-evoked sustained current activation. Ins 1,3,4 P₃ could not evoke sustained responses in combination with Ins 1,4,5 P₃ excluding the possibility that the action of Ins 1,3,4,5 P₄ could be mediated by its breakdown product Ins 1,3,4 P₃. Ins 1,3,4,5 P₄ also evoked a sustained response when added to an Ins 1,4,5 P(S)₃-containing solution. Ins 1,3,4,5,6 P₅ (50 m) did not evoke any effect when administered on top of Ins 1,4,5 P₃. In the absence of external Ca²⁺, addition of Ins 1,3,4,5 P₄ to an Ins 1,4,5 P₃-containing internal solution evoked a second transient K⁺ current activation. Readmitting external Ca²⁺ in the continued presence internally of Ins 1,4,5 P₃ and Ins 1,3,4,5 P₄ made the response reappear. We conclude that both Ins 1,4,5 P₃ and Ins 1,3,4,5 P₄ play crucial and specific roles in controlling intracellular Ca²⁺ homeostasis.     

Ruthenium red selectively depletes inositol 1,4,5-trisphosphate-sensitive calcium stores in permeabilized rabbit pancreatic acinar cells

Rabbit pancreatic acinar cells, permeabilized by saponin treatment, rapidly accumulated 3.5 nmol of Ca²⁺/mg protein in an energy-dependent pool when incubated at an ambient free Ca²⁺ concentration of 100 nm. Maximal loading of the internal stores was reached at 10 min and remained unchanged thereafter. Complete inhibition of the Ca²⁺ pump with thapsigargin revealed that this plateau was the result of a steady-state between slow Ca²⁺ efflux and ATP-driven Ca²⁺ uptake. Sixty percent of the pool could be released by Ins(1,4,5)P₃, whereas GTP released another twenty percent. The striking finding of this study is that the energy-dependent store could also be released by ruthenium red. Uptake experiments in the presence of ruthenium red revealed that the dye, at concentrations below 100 m, selectively reduced the size of the Ins(1,4,5)P₃-releasable pool. Ruthenium red had no effect on the half-maximal stimulatory concentration of Ins(1,4,5)P₃. At concentrations beyond 100 m, the dye also affected the GTP-releasable pool. Comparison with thapsigargin revealed that ruthenium red released Ca²⁺ from stores loaded to steady-state at a rate markedly faster than can be explained by inhibition of the ATPase alone. From the data presented, we concluded that ruthenium red selectively releases Ca²⁺ from the Ins(1,4,5)P₃-sensitive store by activating a Ca²⁺ release channel, whereas Ca²⁺ release from the GTP-sensitive store is predominantly caused by inhibition of the Ca²⁺ pump. The postulated ruthenium red-sensitive Ca²⁺ release channel might be similar to the ryanodine-receptor in muscle.The research of Dr. P.H.G.M. Willems has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences.     

Gating and Conduction Properties of a Sodium-activated Cation Channel from Lobster Olfactory Receptor Neurons

The gating and conduction properties of a channel activated by intracellular Na⁺ were studied by recording unitary currents in inside-out patches excised from lobster olfactory receptor neurons. Channel openings to a single conductance level of 104 pS occurred in bursts. The open probability of the channel increased with increasing concentrations of Na⁺. At 210 mm Na⁺, membrane depolarization increased the open probability e-fold per 36.6 mV. The distribution of channel open times could be fit by a single exponential with a time constant of 4.09 msec at −60 mV and 90 mm Na⁺. The open time constant was not affected by the concentration of Na⁺, but was increased by membrane depolarization. At 180 mm Na⁺ and −60 mV, the distribution of channel closed times could be fit by the sum of four exponentials with time constants of 0.20, 1.46, 8.92 and 69.9 msec, respectively. The three longer time constants decreased, while the shortest time constant did not vary with the concentration of Na⁺. Membrane depolarization decreased all four closed time constants. Burst duration was unaffected by the concentration of Na⁺, but was increased by membrane depolarization. Permeability for monovalent cations relative to that of Na⁺ (P _X /P _Na ), calculated from the reversal potential, was: Li⁺ (1.11) > Na⁺ (1.0) > K⁺ (0.54) > Rb⁺ (0.36) > Cs⁺ (0.20). Extracellular divalent cations (10 mm) blocked the inward Na⁺ current at −60 mV according to the following sequence: Mn²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺ > Ba²⁺. Relative permeabilities for divalent cations (P _Y /P _Na ) were Ca²⁺ (39.0) > Mg²⁺ (34.1) > Mn²⁺ (15.5) > Ba²⁺ (13.8) > Na⁺ (1.0). Both the reversal potential and the conductance determined in divalent cation-free mixtures of Na⁺ and Cs⁺ or Li⁺ were monotonic functions of the mole fraction, suggesting that the channel is a single-ion pore that behaves as a multi-ion pore when the current is carried exclusively by divalent cations. The properties of the channel are consistent with the channel playing a role in odor activation of these primary receptor neurons. Received: 17 September 1996/Revised: 15 November 1996     

High-conductance K+ channel in pancreatic islet cells can be activated and inactivated by internal calcium

Summary The Ca²⁺-activated K⁺ channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage (I/V) relationship showed outward rectification and the null potential was more negative than –40 mV. In symmetrical K⁺-rich solutions the single-channelI/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca²⁺-free solution containing 2mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities (P) above 0.1. Raising the free Ca²⁺ concentration in contact with the membrane inside ([Ca²⁺]_i) to 1.5×10^–7 m had little effect on the relationship between membrane potential andP. When [Ca²⁺]_i was increased to 3×10^–7 m and 6×10^–7 m smaller potential changes were required to open the channels. Increasing [Ca²⁺]_i further to 8×10^–7 m again activated the channels, but the relationship between membrane potential andP was complex. Changing the membrane potential from –50 mV to +20 mV increasedP from near 0 to 0.6 but further polarization to +50 mV decreasedP to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca²⁺]_i=1 and 2 m. In this situation a membrane potential change from –70 to +20 mV increasedP from near 0 to about 0.7 but further polarization to +80 mV reducedP to less than 0.1. The high-conductance K⁺ channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca²⁺]_i within the range 0.1 to 1 m which suggests a physiological role for this channel in regulating the membrane potential and Ca²⁺ influx through voltage-activated Ca²⁺ channels.     

Involvement of Ca2+ in 1-Aminocyclopropane-1-Carboxylic Acid-Stimulated Pollen Germination

The role of 1-aminocyclopropane-1-carboxylic acid (ACC) in pollen germination was investigated in several plant species. It was found that ACC stimulated in vitro pollen germination in all five species of plants tested. EGTA and phenothiazine inhibited the increase in the germination rate induced by ACC. Free Ca²⁺ levels in the cytosol ([Ca²⁺]_cyt) in ungerminated and germinated pollen were 136 and 287 nm, respectively. Adding 0.25 mm ACC to the germination medium increased the [Ca²⁺]_cyt in germinated pollen up to 450 nm. When pollen was treated with both 0.25 mm ACC and 3.6 μm inositol 1,4,5-trisphosphate, the [Ca²⁺]_cyt increased to 850 nm, and pollen germination was also stimulated. In the presence of Li⁺, an inhibitor of inositol monophosphatase, the [Ca²⁺]_cyt was reduced to 155 nm, and the ACC-stimulated pollen germination was inhibited. The data provided evidence for the involvement of Ca²⁺ as a messenger in the stimulative effect of ACC on pollen germination. Received December 1, 1995; accepted February 18, 1998     

Calcium- and inositol polyphosphate-sensitivity of the calcium-sequestering endoplasmic reticulum in the photoreceptor cells of the honeybee drone

Summary The photoreceptor cells in the honeybee drone contain an elaborate Ca²⁺-sequestering endoplasmic reticulum (ER). We measured Ca-oxalate formation within the ER of permeabilized retinal slices with a microphotometer and studied the kinetics of Ca²⁺-uptake into the ER and the properties of Ins(1,4,5)P₃-induced Ca²⁺-release.The ATP-dependent Ca²⁺-uptake mechanism has a high affinity for Ca²⁺: Uptake rate was half maximal at Ca²⁺ _free 0.6 M.Addition of Ins(1,4,5)P₃ caused a persistent depression of Ca-oxalate formation due to Ca²⁺ -release from the ER. The Ins(1,4,5)P₃-dependent Ca²⁺-release mechanism has a high affinity (half maximal rate with 0.2 M Ins(1,4,5)P₃) and a high specificity for Ins(1,4,5)P₃: Ins(2,4,5)P₃ was 6 times, Ins(1,3,4,5)P₄ was 15 times less potent in inducing Ca²⁺-release. 3 M Ins(1,4)P₂ had no detectable effect. The sensitivity for Ins(1,4,5)P₃ was maximal between 280 nM and 1.6 M Ca²⁺ _free and decreased at higher and lower Ca²⁺-concentrations.Our data show that the ER in invertebrate photoreceptor cells is an effective Ca²⁺ -sink and an Ins(1,4,5)P₃-sensitive Ca²⁺-source. We support the idea (Payne et al. 1988) that the ER-network close to the photoreceptive membrane, the submicrovillar cisternae (SMC), are the light- and Ins(1,4,5)P₃-sensitive Ca²⁺-stores.Abbreviations ER endoplasmic reticulum - Ins(1,4,5)P ₃ D-inositol 1,4,5-trisphosphate - Ins(1,3,4)P ₃ D-inositol 1,3,4-trisphosphate - Ins(2,4,5)P ₃ D-inositol 2,4,5-trisphosphate - Ins(1,4)P ₂ D-inositol 1,4-bisphosphate - Ins(1,3,4,5)P ₄ D-inositol 1,3,4,5-tetrakisphosphate - SMC submicrovillar cisternae - [Ca ²⁺]_i intracellular free Ca²⁺-concentration     

Single-Channel Characterization of the Pharmacological Properties of the K(Ca2+) Channel of Intermediate Conductance in Bovine Aortic Endothelial Cells

The pharmacological profile of a voltage-independent Ca²⁺-activated potassium channel of intermediate conductance (IK(Ca²⁺)) present in bovine aortic endothelial cells (BAEC) was investigated in a series of inside-out and outside-out patch-clamp experiments. Channel inhibition was observed in response to external application of ChTX with a half inhibition concentration of 3.3 ± 0.3 nm (n= 4). This channel was insensitive to IbTX, but channel block was detected following external application of MgTX and StK leading to the rank order toxin potency ChTX > StK > MgTX >>IbTX. A reduction of the channel unitary current amplitude was also measured in the presence of external TEA, with half reduction occurring at 23 ± 3 mm TEA (n= 3). The effect of TEA was voltage insensitive, an indication that TEA may bind to a site located on external side of the pore region of this channel. Similarly, the addition of d-TC to the external medium caused a reduction of the channel unitary current amplitude with half reduction at 4.4 ± 0.3 mm (n= 4). In contrast, application of d-TC to the bathing medium in inside-out experiments led to the appearance of long silent periods, typical of a slow blocking process. Finally, the IK(Ca²⁺) in BAEC was found to be inhibited by NS1619, an activator of the Ca²⁺-activated potassium channel of large conductance (Maxi K(Ca²⁺)), with a half inhibition value of 11 ± 0.8 μm (n= 4). These results provide evidence for a pharmacological profile distinct from that reported for the Maxi K(Ca²⁺) channel, with some features attributed to the voltage-gated K_V1.2 potassium channel. Received: 6 November 1997/Revised: 19 February 1998     

Large-conductance Calcium-activated Potassium Channels of Cultured Rat Melanotrophs

A large conductance, Ca²⁺-activated K⁺ channel of the BK type was examined in cultured pituitary melanotrophs obtained from adult male rats. In cell-attached recordings the slope conductance for the BK channel was ≈190 pS and the probability (P _o ) of finding the channel in the open state at the resting membrane potential was low (<<0.1). Channels in inside-out patches and in symmetrical 150 mm K⁺ had a conductance of ≈260 pS. The lower conductance in the cell-attached recordings is provisionally attributed to an intracellular K⁺ concentration of ≈113 mm. The permeability sequence, relative to K⁺, was K⁺ > Rb⁺ (0.87) > NH⁺ ₄ (0.17) > Cs⁺≥ Na⁺ (≤0.02). The slope conductance for Rb⁺ was much less than for K⁺. Neither Na⁺ nor Cs⁺ carried measurable currents and 150 mm internal Cs⁺ caused a flickery block of the channel. Internal tetraethylammonium ions (TEA⁺) produced a fast block for which the dissociation constant at 0 mV (K _D (0 mV)) was 50 mm. The K _D (0 mV) for external TEA⁺ was much lower, 0.25 mm, and the blocking reaction was slower as evidenced by flickery open channel currents. With both internal and external TEA⁺ the blocking reaction was bimolecular and weakly voltage dependent. External charybdotoxin (40 nm) caused a large and reversible decrease of P _o . The P _o was increased by depolarization and/or by increasing the concentration of internal Ca²⁺. In 0.1 μm Ca²⁺ the half-maximal P _o occurred at ≈100 mV; increasing Ca²⁺ to 1 μm shifted the voltage for the half-maximal P _o to −75 mV. The Ca²⁺ dependence of the gating was approximated by a fourth power relationship suggesting the presence of four Ca²⁺ binding sites on the BK channel. Received: 23 October/Revised: 15 December 1995     

Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca²⁺ activation site was modified using N-bromoacetamide (NBA). 1mm NBA in the bath solution, at 2.5 m Ca²⁺ reduces the open probability,P _o , of the channel to <0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca²⁺-sensitive, requiring 25mm Ca²⁺ to raiseP _o to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca²⁺ (1mm) protects the channels from modification. Also presented is a second class of Ca²⁺-activated K⁺ channels which are normally present in MTAL cells which open infrequently at 10 m Ca²⁺ (P _o =0.01) but have aP _o of 0.08 at 1mm Ca²⁺. We can conclude (i) that NBA modifies the channel by shifting Ca²⁺-sensitivity to very high Ca²⁺, (ii) that NBA acts on a site involved in Ca²⁺ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.     

Activation and conductance properties of ryanodine-sensitive calcium channels from brain microsomal membranes incorporated into planar lipid bilayers

Summary Rat brain microsomal membranes were found to contain high-affinity binding sites for the alkaloid ryanodine (k _d 3nm.B _max 0.6 pmol per mg protein). Exposure of planar lipid bilayers to microsomal membrane vesicles resulted in the incorporation, apparently by bilayer-vesicle fusion, of at least two types of ion channel. These were selective for Cl^– and Ca²⁺, respectively. The reconstituted Ca²⁺ channels were functionally modified by 1 m ryanodine, which induced a nearly permanently open subconductance state. Unmodified Ca²⁺ channels had a slope conductance of almost 100 pS in 54mm CaHEPES and a Ca²⁺/TRIS⁺ permeability ratio of 11.0. They also conducted other divalent cations (Ba²⁺>Ca²⁺>Sr²⁺>Mg²⁺) and were markedly activated by ATP and its nonhydrolysable derivative AMPPCP (1mm). Inositol 1,4,5-trisphosphate (1–10 m) partially activated the same channels by increasing their opening rate. Brain microsomes therefore contain ryanodine-sensitive Ca²⁺ channels, sharing some of the characteristics of Ca²⁺ channels from striated but not smooth muscle sarcoplasmic reticulum. Evidence is presented to suggest they were incorporated into bilayers following the fusion of endoplasmic reticulum membrane vesicles, and their sensitivity to inositol trisphosphate may be consistent with a role in Ca²⁺ release from internal membrane stores.