Environmental KCl Causes an Upregulation of Apical Membrane Maxi K and ENaC Channels in Everted Ambystoma Collecting Tubule

Patch clamp methods were used to characterize the channels on the apical membrane of initial collecting ducts from Ambystoma tigrinum. Apical membranes were exposed by everting and perfusing fragments of the renal tubule in vitro. Tubules were dissected from two groups of animals; one maintained in tap water, and the other kept in a solution of 50 mm KCl from seven to nineteen days. Patches of apical membranes on tubules taken from animals exposed to tap water expressed low-conductance amiloride sensitive sodium channels (ENaC) in 22 of 49 patches. Only three maxi K channels were observed in this group. In animals exposed to KCl, low-conductance amiloride sensitive sodium channels, 3.7 ± 0.2 pS (36 of 45 patches) and high-conductance 98.3 ± 5.0 pS (19 of 45 patches) potassium channels were observed. The estimated density of apical maxi K channels increased dramatically from 0.08 to 0.76 channels/μ² in tubules taken from animals exposed to KCl. All but four of nineteen patches which contained maxi K channels also expressed the low conductance sodium channels. Therefore, at least 85% of the maxi K channels studied were in principal cells. We speculate that the increase in maxi K channel activity may represent a mechanism for enhancing the potassium secretory capacity of the initial collecting duct. As expected, exposure of the animals to 50 mm KCl prior to dissection of the initial collecting ducts also increased the estimated density of ENaC from 0.99 to 3.89 channels/μ². This upregulation of sodium channel activity is presumably related to the widely recognized effect of potassium loading to increase the plasma aldosterone level. Received: 25 August 1997/Revised: 13 November 1997     

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     

G Protein-mediated Activation of a Nonspecific Cation Current in Cultured Rat Retinal Pigment Epithelial Cells

We used whole-cell patch-clamp recording techniques to investigate G protein-activated currents in cultured rat retinal pigment epithelial (RPE) cells. Using 140 mm KCl intracellular and 130 mm NaCl extracellular solutions, rat RPE cells possessed both inward and outward K⁺ currents. Upon addition of the nonhydrolyzable guanine triphosphate analogue, guanosine-5′-O-(3-thiophosphate) (GTPγS, 0.1 mm), to the recording electrode, a nonspecific cation (NSC) current was elicited. The NSC current had a mean reversal potential of +5.7 mV in 130 mm extracellular NaCl with Cs⁺-aspartate in the pipette, and was not affected by alterations in the extracellular Ca²⁺ or Cl⁻ concentration. The GTPγS-activated current was found to be permeable to several monovalent cations (K⁺, Na⁺, choline, TRIS, and NMDG). Addition of fluoroaluminate, an activator of large molecular weight heterotrimeric GTP-binding proteins (G proteins), to the intracellular recording solution activated the NSC current. The G protein involved was pertussis toxin (PTX)-sensitive, since GTPγS failed to activate the NSC current in cells pretreated with PTX. Further investigation of second messenger molecules suggested that activation of the NSC current was not affected by alterations in intracellular Ca²⁺ or ATP. From these results, we conclude that a G protein-regulated NSC current is present in rat RPE cells. Activation of the NSC current may sufficiently depolarize RPE cells to activate outward K⁺ currents. This would provide a mechanism by which these cells could rid themselves of accumulated K⁺. Received: 25 January 1996/Revised: 24 April 1996     

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     

Flow-Dependent Activation of Maxi K+ Channels in Apical Membrane of Rabbit Connecting Tubule

The Ca²⁺-activated maxi K⁺ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean number of open channels (NP _o ) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the stretch-activation of Ca²⁺-permeable cation channels, because the maxi K⁺ channel was not stretch-activated in both the cell-attached configuration using Ca²⁺-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K⁺ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K⁺ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K⁺ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV _t ) when luminal K⁺ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV _t was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced ΔV _t to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV _t to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV _t was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV _t =−7.2 ± 0.5 mV in control vs.ΔV _t =−6.7 ± 0.6 mV in AA). These results suggest that the Ca²⁺-activated maxi K⁺ channel is responsible for flow-dependent K⁺ secretion by coupling with the stretch-activated Ca²⁺-permeable cation channel in the rabbit CNT. Received: 21 August 1997/Revised: 20 March 1998     

Large-Conductance Cation Channels in the Envelope of Nuclei from Rat Cerebral Cortex

Eucaryotic nuclei are surrounded by a double-membrane system enclosing a central cisterna which is continuous with the endoplasmic reticulum and serves as a calcium store for intracellular signaling. The envelope regulates protein and nucleic acid traffic between the nucleus and the cytoplasm via nuclear pores. These protein tunnels cross through both nuclear membranes and are permeable for large molecules. Surprisingly, patch clamp recordings from isolated nuclei of different cell species have revealed a high resistance of the envelope, enabling tight seals and the resolution of single ion channel activity. Here we present for the first time single-channel recordings from nuclei prepared from neuronal tissue. Nuclei isolated from rat cerebral cortex displayed spontaneous long-lasting large conductances in the nucleus-attached mode as well as in excised patches. The open times are in the range of seconds and channel activity increases with depolarization. The single-channel conductance in symmetrical K⁺ is 166 pS. The channels are selective for cations with P _K/P _Na= 2. They are neither permeable to, nor gated by Ca²⁺. Thus, neuronal tissue nuclei contain a large conductance ion channel selective for monovalent cations which may contribute to ionic homeostasis in the complex compartments surrounding these organelles. Received: 12 November 1996/Revised: 18 February 1997     

Voltage-Induced Activation of Mechanosensitive Cation Channels of Leech Neurons

The voltage dependence of stretch-activated cation channels in leech central neurons was studied in cell-free configurations of the patch-clamp technique. We established that stretch-activated channels excised from identified cell bodies of desheathed ganglia, as well as from neurons in culture, were slowly and reversibly activated by depolarizing membrane potentials. Negative pressure stimuli, applied to the patch pipette during a slow periodical modulation of membrane potential, enhanced channel activity, whereas positive pressures depressed it. Voltage-induced channel activation was observed, with soft glass pipettes, both in inside-out and outside-out membrane patches, at negative and positive reference potentials, respectively. The results presented in this study demonstrate that membrane depolarization induces slow activation of stretch-activated channels of leech central neurons. This phenomenon is similar to that found in Xenopus oocytes, however, some peculiar features of the voltage dependence in leech stretch-activated channels indicate that specific membrane-glass interactions might not necessarily be involved. Moreover, following depolarization, stretch-activated channels in membrane patches from neurons in culture exhibited significantly shorter delay to activation (sec) than their counterparts from neurons of freshly isolated ganglia (hundreds of sec).     

Expression of Cyclic Nucleotide-Gated Cation Channels in Airway Epithelial Cells

Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with hαCNC1. Whole cell currents stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 or A549 cells are carried by inward sodium and outward potassium with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 are inhibited by l-cis-diltiazem with an IC₅₀ of 154 μm, by 2′,4′-dichlorobenzamil with an IC₅₀ of 50 μm and by amiloride with an IC₅₀ of 133 μm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an IC₅₀ of 87 μm, by 2′4′-dichlorobenzamil with an IC₅₀ of 38 μm and by amiloride with an IC₅₀ of 32 μm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both αCNC1 and βCNC subunits are present in A549 cells and the presence of the βCNC subunit, may as previously reported, increase the affinity of these channel blockers compared to the hαCNC1 subunit alone. The mRNA of two other isoforms of this channel, CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC₅₀ of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption via cyclic nucleotide-gated cation channels in airway cells. Received: 24 February/Revised: 28 May 1999     

Transcellular Chloride Pathways in Ambystoma Proximal Tubule

The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl⁻ absorption linked to Na⁺ were investigated in isolated perfused tubules using Cl⁻-selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl⁻ (a _i ^Cl ) is 11.3 ± 0.5 mm, the basolateral (V ₁ ), apical (V ₂ ), and transepithelial (V ₃ ) potential differences are −68 ± 1.2 mV, +62 ± 1.2 mV and −6.4 ± 0.3 mV, respectively. When Na⁺ absorption is decreased by removal of organic substrates from the lumen, a _i ^Cl falls by 1.3 ± 0.3 mm and V ₂ hyperpolarizes by +11.4 ± 1.7 mV. Subsequent removal of Na⁺ from the lumen causes a _i ^Cl to fall further by 2.3 ± 0.4 mm and V ₂ to hyperpolarize further by +15.3 ± 2.4 mV. The contribution of transporters and channels to the observed changes of a _i ^Cl was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on a _i ^Cl . The effects of luminal HCO⁻ ₃ removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO₃ or Cl-OH. The effects of basolateral HCO⁻ ₃ removal, of basolateral Na⁺ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO₃ exchange. Several lines of evidence favor conductive Cl⁻ transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the a _i ^Cl by 2.4 ± 0.6 mm or 2.9 ± 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO⁻ ₃-free Ringer, the equilibrium potential for Cl⁻ does not differ from the membrane potential V ₂ . Finally, the logarithmic changes in a _i ^Cl in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl⁻ channels at the apical and basolateral cell membranes of the proximal tubule. Received: 14 November 1997/Revised: 6 July 1998     

Families of Proteins Forming Transmembrane Channels

Channel-forming proteins/peptides fall into over 100 currently recognized families, most of which are restricted to prokaryotes or eukaryotes, but a few of which are ubiquitous. These proteins fall into three major currently recognized classes: (i) α-helix-type channels present in bacterial, archaeal and eukaryotic cytoplasmic and organellar membranes, (ii) β-barrel-type porins present in the outer membranes of Gram-negative bacterial cells, mitochondria and chloroplasts, and (iii) protein/peptide toxins targeted to the cytoplasmic membranes of cells other than those that synthesize the toxins. High-resolution 3-dimensional structural data are available for representative proteins/peptides of all three of these channel-forming types. Each type exhibits distinctive features that distinguish them from the other channel protein types and from carriers. Structural, functional, and evolutionary aspects of transmembrane channel-formers are discussed. Received: 10 September 1999/Revised: 11 February 2000     

A Component of Platypus (Ornithorhynchus anatinus) Venom Forms Slow-Kinetic Cation Channels

The lipid bilayer technique is used to examine the biophysical properties of anion and cation channels frequently formed by platypus (Ornithorhynchus anatinus) venom (OaV). The OaV-formed anion channel in 250/50 mm KCl cis/trans has a maximum conductance of 857 ± 23 pS (n= 5) in 250/50 mm KCl cis/trans. The current-voltage relationship of this channel shows strong inward rectification. The channel activity undergoes time-dependent inactivation that can be removed by depolarizing voltage steps more positive than the reversal potential for chloride, E _Cl , (+40 mV). The reversal potential of the OaV-formed slow current activity in 250/50 mm KCl cis/trans is close to the potassium equilibrium potential (E _K ) of −40 mV. The conductance values for the slow channel are 22.5 ± 2.6 pS and 41.38 ± 4.2 pS in 250/50 and 750/50 mm cis/trans, respectively. The gating kinetics of the slow ion channels are voltage-dependent. The channel open probability (P _o ) is between 0.1 and 0.8 at potentials between 0 and +140 mV. The channel frequency (F _o ) increases with depolarizing voltages between 0 and +140 mV, whereas mean open time (T _o ) and mean closed time (T _c ) decrease. Ion substitution experiments of the cis solution show that the channel has conductance values of 21.47 ± 2.3 and 0.53 ± 0.1 pS in 250 mm KCl and choline Cl, respectively. The amplitude of the single channel current is dependent on [K⁺] _cis and the current reversal potential (E _rev ) responds to increases in [K⁺] _cis by shifting to more negative voltages. The increase in current amplitude as a function of increasing [K⁺] _cis can be best described by a third order polynomial fit. At +140 mV, the values of the maximal single channel conductance (γ _max ) and the concentration for half maximal γ (K _s ) are 38.6 pS and 380 mm and decline to 15.76 pS and 250 mm at 0 mV, respectively. The ion selectivity of the channel to K⁺, Na⁺, Cs⁺ and choline⁺ was determined in ion substitution experiments. The permeability values for P _K+ :P _Na+ :P _Cs+ :P _choline+ were 1:1:0.63:0.089, respectively. On the other hand, the activity of the slow channel was eliminated (Fig. 7B). The slow channel was reversibly inhibited by [TEA⁺] _trans and the half-maximal inhibitory concentration (K _i ) was ∼48 mm. Received: 26 April 1999/Revised: 19 July 1999     

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     

Chemical Gating of Heteromeric and Heterotypic Gap Junction Channels

Gap junction channels contain two hemichannels (connexons), each being a connexin (Cx) hexamer. In cells expressing multiple connexins, heteromeric connexons are believed to form, whereas cell pairs expressing different connexins generate heterotypic channels. To define gating behavior of heteromeric and heterotypic channels, CO₂-induced gating was tested in Xenopus oocyte pairs expressing Cx32, or 5R/N (Cx32 mutant), as well as in pairs in which one oocyte (mx) expressed a 50/50 mixture of Cx32 and 5R/N and the other either the mixture (mx), Cx32 (32) or 5R/N (R/N). In 5R/N, replacement of 5 C-terminus arginines with asparagines greatly increased CO₂ sensitivity. In response to 3 and 15 min CO₂ exposures, junctional conductance (G _j ) decreased to 85% and 47%, in 32–32 pairs, and to 7% and 0.9%, in R/N-R/N pairs, respectively. In mx-mx and mix-32 pairs, G _j decreased to similar values (33% and 35%, respectively) with 15 min CO₂. The sensitivity of mx-R/N pairs was similar to that of heterotypic 32-R/N pairs, as G _j dropped to 36% and 38%, respectively, with 3 min CO₂. Monoheteromeric (mx-32 and mx-R/N) and biheteromeric (mx-mx) channels behaved as if Cx32 were dominant, suggesting that hemichannel sensitivity is not an average of the sensitivities of its connexin monomers. In contrast, heterotypic channels behaved as if the two hemichannels of a cell-cell channel had no influence on each other. Received: 15 May 1997/Revised: 8 December 1997     

Role and Source of ATP for Activation of Nonselective Cation Channels by AlF Complex in Guinea Pig Chromaffin Cells

Intracellular dialysis with the solution containing the G protein activator, AlF complex, induced an inward nonselective cation current (I _NS) at −55 mV in chromaffin cells. Amplitudes of I _NS induced by dialysis with ATP-free AlF solutions progressively diminished as cells were pretreated with cyanide, a mitochondrial inhibitor. After a 10-min pretreatment, generation of I _NS by the AlF complex depended on exogenous ATP delivered from pipette solution. The relationship between amplitudes of I _NS and concentrations of MgATP was well expressed by a rectangular hyperbola with an EC₅₀ of 0.265 mm. This result suggests that the cyanide treatment almost depleted ATP near the plasma membrane. On the other hand, a similar cyanide treatment of adrenal medullary preparations did not induce a marked decrease in cellular ATP content. GTP, ITP, or UTP could not substitute for ATP in generation of I _NS by the AlF complex. Similarly, the substitution of ATP with non- or poorly hydrolyzable ATP analogues did not aid in generating I _NS. Bath application of the kinase inhibitor, H-7 (100 μm), suppressed AlF-induced I _NS in a manner depending on intracellular Mg²⁺. We conclude that ATP is a prerequisite for generation of I _NS as a phosphoryl donor and that mitochondria is the main source of ATP. Received: 17 April 1996/Revised: 26 July 1996     

Characterization of Whole Cell Chloride Conductances in a Mouse Inner Medullary Collecting Duct Cell Line mIMCD-3

The chloride conductance of inner medullary collecting duct cells (mIMCD-3 cell line) has been investigated using the whole cell configuration of the patch clamp technique. Seventy-seven percent of cells were chloride selective when measured with a NaCl-rich bathing solution and a TEACl-rich pipette solution. Seventy-five percent of chloride-selective cells (90/144) had whole cell currents which exhibited an outwardly-rectifying (OR) current-voltage (I/V) relationship, while the remaining cells exhibited a linear (L) I/V relationship. The properties of the OR and L chloride currents were distinct. OR currents (mean current densities at ±60 mV of 66 ± 5 pA/pF and 44 ± 3 pA/pF), were time- and voltage-independent with an anion selectivity (from calculated permeability ratios) of SCN⁻ (2.3), NO⁻ ₃ (1.8), ClO⁻ ₄ (1.7), Br⁻ (1.7), I⁻ (1.6), Cl⁻ (1.0), HCO⁻ ₃ (0.5), gluconate⁻ (0.2). Bath additions of NPPB, flufenamate, glibenclamide (all 100 μm) and DIDS (500 μm) produced varying degrees of block of OR currents with NPPB being the most potent (IC₅₀ of approximately 50 μm) while DIDS was the least effective. Linear chloride currents had similar current densities to the OR chloride currents and were also time- and voltage-independent. The anion selectivity sequence was SCN⁻ (2.5), NO⁻ ₃ (1.9), Br⁻ (1.4), I⁻ (1.1), Cl⁻ (1.0), ClO⁻ ₄ (0.5), HCO⁻ ₃ (0.5), gluconate⁻ (0.3). In contrast to the OR conductance, glibenclamide was the most potent and DIDS the least potent blocker of L currents. An IC₅₀ of >100 μm was observed for NPPB block. Neither OR of L chloride currents were affected by acutely or chronically increased intracellular cAMP and were not affected when intracellular Ca²⁺ levels were increased or decreased. The molecular identity and physiological role of OR and linear currents in mIMCD-3 cells are discussed. Received: 13 June 1995/Revised: 15 September 1995     

Large Conductance Ca2+-activated K+ Channels in the Soma of Rat Motoneurones

Properties of large conductance Ca²⁺-activated K⁺ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K⁺ _o //155 mm K⁺ _i ) and 231 ± 4 pS in external high-K _o solution (155 mm K⁺ _o //155 mm K⁺ _i ). The channels were activated by depolarization and by an increase in internal Ca²⁺ concentration. Potentials of half-maximum channel activation (E₅₀) were −13, −34, −64 and −85 mV in the presence of 10⁻⁶, 10⁻⁵, 10⁻⁴ and 10⁻³ m internal Ca²⁺, respectively. Using an internal solution containing 10⁻⁴ m Ca²⁺, averaged K_Ca currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged K_Ca currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with a 50% blocking concentration (IC₅₀) of 0.17 ± 0.02 mm. K_Ca channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that K_Ca channels activated by Ca²⁺ entry during the action potential play an important role in the excitability of motoneurones. Received: 7 November 1996/Revised: 29 October 1997     

Role of Protein Phosphatase in the Regulation of Na+-K+-ATPase by Vasopressin in the Cortical Collecting Duct

In the cortical collecting duct (CCD), arginin vasopressin (AVP) has been shown to increase the number and activity of basolateral Na⁺-K⁺-ATPase by recruiting or activating a latent pool of pumps. However, the precise mechanism of this phenomenon is still unknown. The aim of this study was to investigate whether this AVP-induced increase in basolateral Na⁺-K⁺-ATPase could depend on a dephosphorylation process. To this purpose, the effect of protein serine/threonine phosphatase (PP) inhibitors was examined on both the specific ³H-ouabain binding (to evaluate the number of pumps in the basolateral membrane) and the ouabain-dependent ⁸⁶Rb uptake (to evaluate pump functionality) in the presence or absence of AVP. In addition, the activity of two PP, PP1 and PP2A, was measured and the influence of AVP was examined on both enzymes. Experiments have been performed on mouse CCD isolated by microdissection. Results show that inhibition of PP2A prevents the AVP-induced increase in the number and activity of Na⁺-K⁺-ATPases, independent of an effect on the apical cell sodium entry. In addition, AVP rapidly increased the activity of PP2A without effect on PP1. These data suggest that PP2A is implied in the regulation of Na⁺-K⁺-ATPase activity by AVP in the CCD and that the AVP-dependent increase in the number of Na⁺-K⁺-ATPases is mediated by a PP2A-dependent dephosphorylation process. Received: 22 March 1996/Revised: 21 June 1996     

Polyamine Triggering of Exocytosis in Paramecium Involves an Extracellular Ca2+/(Polyvalent Cation)-Sensing Receptor, Subplasmalemmal Ca-Store Mobilization and Store-Operated Ca2+-Influx via Unspecific Cation Channels

The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca²⁺] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca²⁺ signals also occur when AED is applied in presence of the fast Ca²⁺ chelator, BAPTA. (ii) Extracellular La³⁺ application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca²⁺] _i transient (while injected La³⁺ causes a sustained fluorescence signal). (iii) Simply increasing [Ca²⁺] _o causes a similar rapid, short-lived [Ca²⁺] _i transient. All these phenomena, (i–iii), are compatible with activation of an extracellular ``Ca<sup>2+</sup>/(polyvalent cation)-sensing receptor' known from some higher eukaryotic systems, where this sensor (responding to Ca<sup>2+</sup>, La<sup>3+</sup> and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores (``alveolar sacs') are physically linked to the cell membrane and they can also be activated by the Ca²⁺ releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca²⁺ signal also in absence of Ca²⁺ _o we largely exclude a ``Ca<sup>2+</sup>-induced Ca<sup>2+</sup> release' (CICR) mechanism. Our finding of increased cortical Ca<sup>2+</sup> signals after store depletion and re-addition of extracellular Ca<sup>2+</sup> can be explained by a ``store-operated Ca²⁺ influx' (SOC), i.e., a Ca²⁺ influx superimposing store activation. AED stimulation in presence of Mn²⁺ _o causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOC-type Ca²⁺ influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca²⁺/polyamine-sensing receptor, (ii) release of Ca²⁺ from subplasmalemmal stores, (iii) and Ca²⁺ influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca²⁺] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca²⁺] microdomains (≤0.5 μm, ≤33 msec) upon stimulation. Received: 30 August 1999/Revised: 1 December 1999     

Effect of Mutation of Potassium-Efflux System,KefA, on Mechanosensitive Channels in the Cytoplasmic Membrane of Escherichia coli

The effect of a kefA mutation on the mechanosensitive channels in the cytoplasmic membrane of Escherichia coli was established by introducing a mutation of the kefA gene into wild-type E. coli by P1 transduction. The mutation of the kefA gene not only made the cells sensitive to K⁺ in the medium but also changed the mechanosensitive channel activity. The kefA mutation did not change the conductances of the two mechanosensitive channels in the cytoplasmic membrane of E. coli, but it prolonged the channel open time. Also, the kefA mutation made the cells more sensitive to pressure in comparison to wild-type cells. The high sensitivity to pressure of the kefA mutant was not modulated by betaine or by the potassium gradient across the membrane. The effect of the kefA mutation on mechanosensitive channels was not due to a membrane fluidity change. KefA might be a regulator for mechanosensitive channels. Received: 6 September 1995/Revised: 13 December 1995