Ion Channels Formed by NB,an Influenza B Virus Protein

The influenza B virus protein, NB, was expressed in Escherichia coli, either with a C-terminal polyhistidine tag or with NB fused to the C-terminus of glutathione S-transferase (GST), and purified by affinity chromatography. NB produced ion channel activity when added to artificial lipid bilayers separating NaCl solutions with unequal concentrations (150–500 mm cis, 50 mm trans). An antibody to a peptide mimicking the 25 residues at the C-terminal end of NB, and amantadine at high concentration (2–3 mm), both depressed ion channel activity. Ion channels had a variable conductance, the lowest conductance observed being approximately 10 picosiemens. At a pH of 5.5 to 6.5, currents reversed at positive potentials indicating that the channel was more permeable to sodium than to chloride ions (P_Na/P_Cl∼ 9). In asymmetrical NaCl solutions at a pH of 2.5, currents reversed closer to the chloride than to the sodium equilibrium potential indicating that the channel had become more permeable to chloride than to sodium ions (P_Cl/P_Na∼ 4). It was concluded that, at normal pHs, NB forms cation-selective channels. Received: 6 March 1995/Revised: 17 November 1995     

Nonselective Block by La3+ of Arabidopsis Ion Channels Involved in Signal Transduction

Lanthanide ions such as La³⁺ are frequently used as blockers to test the involvement of calcium channels in plant and animal signal transduction pathways. For example, the large rise in cytoplasmic Ca²⁺ concentration triggered by cold shock in Arabidopsis seedlings is effectively blocked by 10 mm La³⁺ and we show here that the simultaneous large membrane depolarization is similarly blocked. However, a pharmacological tool is only as useful as it is selective and the specificity of La³⁺ for calcium channels was brought into question by our finding that it also blocked a blue light (BL)-induced depolarization that results from anion channel activation and believed not to involve calcium channels. This unexpected inhibitory effect of La³⁺ on the BL-induced depolarization is explained by our finding that 10 mm La³⁺ directly and completely blocked the BL-activated anion channel when applied to excised patches. We have investigated the ability of La³⁺ to block noncalcium channels in Arabidopsis. In addition to the BL-activated anion channel, 10 mm La³⁺ blocked a cation channel and a stretch-activated channel in patches of plasma membrane excised from hypocotyl cells. In root cells, 10 mm La³⁺ inhibited the activity of an outward-rectifying potassium channel at the whole cell and single-channel level by 47% and 58%, respectively. We conclude that La³⁺ is a nonspecific blocker of multiple ionic conductances in Arabidopsis and may disrupt signal transduction processes independently of any effect on Ca²⁺ channels. Received: 28 July 1997/Revised: 13 November 1997     

Glycosylation Influences Voltage-Dependent Gating of Cardiac and Skeletal Muscle Sodium Channels

The role of glycosylation on voltage-dependent channel gating for the cloned human cardiac sodium channel (hH1a) and the adult rat skeletal muscle isoform (μl) was investigated in HEK293 cells transiently transfected with either hH1a or μl cDNA. The contribution of sugar residues to channel gating was examined in transfected cells pretreated with various glycosidase and enzyme inhibitors to deglycosylate channel proteins. Pretreating transfected cells with enzyme inhibitors castanospermine and swainsonine, or exo-glycosidase neuroaminidase caused 7 to 9 mV depolarizing shifts of V _1/2 for steady-state activation of hH1a, while deglycosylation with corresponding drugs elicited about the same amount of depolarizing shifts (8 to 9 mV) of V _1/2 for steady-state activation of μl. Elevated concentrations of extracellular Mg²⁺ significantly masked the castanospermine-elicited depolarizing shifts of V _1/2 for steady-state activation in both transfected hH1a and μl. For steady-state activation, deglycosylation induced depolarizing shifts of V _1/2 for hH1a (10.6 to 12 mV), but hyperpolarizing shifts for μl (3.6 to 4.4 mV). Pretreatment with neuraminidase had no significant effects on single-channel conductance, the mean open time, and the open probability. These data suggest that glycosylation differentially regulates Na channel function in heart and skeletal muscle myocytes. Received: 8 April 1999/Revised: 18 June 1999     

The Impermeant Ion Methylammonium Blocks K+ and NH+ 4 Currents through KAT1 Channel Differently: Evidence for Ion Interaction in Channel Permeation

The permeation properties of KAT1, an inward rectifying potassium channel from plant cells, were investigated with different ions in the external medium. With either K⁺, NH⁺ ₄ or methylammonium (MA) in the external solution, the channel, expressed in Xenopus oocytes, appeared permeable to K⁺ and, to a lesser extent, to NH⁺ ₄ but not to the slightly bigger, methylated analogue of NH⁺ ₄, MA. Substituting NH⁺ ₄ for K⁺ shifted the voltage dependency of channel activation further negative and hastened activation kinetics. This suggests that channel operation depends on the transported substrate. In mixed solution (50 mm K⁺, 50 mm MA) MA inhibited K⁺ current in a voltage-independent manner. The maximum block did not exceed 50% of the K⁺ current. In contrast, when NH⁺ ₄ was the permeant ion (50 mm NH⁺ ₄, 50 mm MA) MA caused a voltage-dependent, slowly developing open channel block, achieving complete inhibition at very negative voltages. The latter block could be partially overcome by the addition of K⁺ in the external solution. The data support a model in which ions, after entering the channel pore, compete with different affinities for binding sites on their permeation pathway. Received: 6 October 1997/Revised: 28 January 1998     

I. Ion Channels in Human THP-1 Monocytes

The THP-1 human monocytic leukemia cell line is a useful model of macrophage differentiation. Patch clamp methods were used to identify five types of ion channels in undifferentiated THP-1 monocytes. (i) Delayed rectifier K⁺ current, I _DR, was activated by depolarization to potentials positive to −50 mV, inactivated with a time constant of several hundred msec, and recovered from inactivation with a time constant ∼21 sec. I _DR was inhibited by 4-aminopyridine (4-AP), tetraethylammonium (TEA⁺), and potently by charybdotoxin (ChTX). (ii) Ca-activated K⁺ current (I _SK) dominated whole-cell currents in cells studied with 3–10 μm [Ca²⁺] _i . I _SK was at most weakly voltage-dependent, with reduced conductance at large positive potentials, and was inhibited by ChTX and weakly by TEA⁺, Cs⁺, and Ba²⁺, but not 4-AP or apamin. Block by Cs⁺ and Ba²⁺ was enhanced by hyperpolarization. (iii) Nonselective cation current, I _cat, appeared at voltages above +20 mV. Little time-dependence was observed, and a panel of channel blockers was without effect. (iv) Chloride current, I _Cl, was present early in experiments, but disappeared with time. (v) Voltage-activated H⁺ selective current is described in detail in a companion paper (DeCoursey & Cherny, 1996. J. Membrane Biol. 152:2). The ion channels in THP-1 cells are compared with channels described in other macrophage-related cells. Profound changes in ion channel expression that occur during differentiation of THP-1 cells are described in a companion paper (DeCoursey et al., 1996. J. Membrane Biol. 152:2). Received: 19 September 1995/Revised: 14 March 1996     

A Patch-Clamp Study of Ion Channels in Protoplasts Prepared from the Marine Alga Valonia utricularis

The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant ``mother cells' (Wang, J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma 196:123–134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species. Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl<sup>−</sup> channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K<sup>+</sup> channel (VKC1, Valonia K <sup>+</sup> Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl<sup>−</sup> media, VAC1 was most frequently observed and had a single channel conductance of 36 ± 7 pS (n= 4) in the outside-out and 33 ± 5 pS (n= 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0 ± 4 mV (n= 4, outside-out) and 9 ± 7 mV (n= 10, cell-attached) close to the Nernst potential of Cl<sup>−</sup> and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl<sup>−</sup> media (bath/pipette; E <sub>Cl−</sub> −20 ± 7 mV (n= 4); Nernst potential −28 mV). Consistent with a selectivity for Cl<sup>−</sup>, VAC1 was inhibited by 100 μM DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann fits of the channel activity under symmetrical 150 mm Cl<sup>−</sup> conditions yielded a midpoint potential of −12 ± 5 mV (n= 4, outside-out) and −3 ± 6 mV (n= 9, cell-attached) and corresponding apparent minimum gating charges of 15 ± 3 (n= 4) and 18 ± 5 (n= 9). The midpoint potential shifted to more negative values in the presence of a Cl<sup>−</sup> gradient. VAC2 was activated by voltages more negative than E <sub>Cl−</sub> and was always observed together with VAC1, but less frequently. It showed a ``flickering' gating. The single channel conductance was 99 ± 10 pS (n= 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel conductance of the main conductance state was 36 ± 5 pS (n= 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance state had a single channel conductance of 124 ± 27 pS (n= 6). In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl⁻ conductance of the plasmalemma of the turgescent ``mother cells' and that this channel (as well as VAC2) can operate in the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related (as the stretch-activated channels) to processes involved in turgor regulation. Received: 24 June 1999/Revised: 2 September 1999     

Proton Inhibition of Sodium Channels: Mechanism of Gating Shifts and Reduced Conductance

Extracellular acidosis affects both permeation and gating of the expressed rat skeletal muscle Na⁺ channel (μ1). Reduction of the extracellular pH produced a progressive decrease in the maximal whole-cell conductance and a depolarizing shift in the whole-cell current-voltage relationship. A smaller depolarizing shift in the steady-state inactivation curve was observed. The pK of the reduction of maximal conductance was 6.1 over the pH range studied. An upper limit estimate of the pK of the shift of the half-activation voltage was 6.1. The relative reduction in the maximal whole-cell conductance did not change with higher [Na⁺] _o . The conductance of single fenvalerate-modified Na⁺ channels was reduced by extracellular protons. Although the single-channel conductance increased with higher [Na⁺] _o , the maximal conductances at pH 7.6, 7.0 and 6.0 did not converge at [Na⁺] _o up to 280 mm, inconsistent with a simple electrostatic effect. A model incorporating both Na⁺ and H⁺ binding in the pore and cation binding to a Gouy-Chapman surface charge provided a robust fit to the single-channel conductance data with an estimated surface charge density of 1e⁻/439?². Neither surface charge nor proton block alone suffices to explain the effects of extracellular acidosis on Na⁺ channel permeation; both effects play major roles in mediating the response to extracellular pH. Received: 14 May 1996/Revised: 19 September 1996     

Ionic Channels of the Sugar Beet Tonoplast are Regulated by a Multi-ion Single-file Permeation Mechanism

Ionic channels of the sugar beet tonoplast were studied using the patch-clamp technique. At micromolar concentrations of cytosolic calcium, several (at least four) distinct single-channel current levels were routinely identified. On the basis of channel voltage dependence, kinetic properties and conductance of single openings, the largest channel (103 ± 2 pS in symmetric 150 mm KCl) corresponds to the slow vacuolar (SV) channel already identified by Hedrich and Neher (1987). The majority of the whole-vacuole current was ascribed to this time-dependent slow-activating channel elicited by positive vacuolar potentials. The channel of intermediate amplitude (41 ± 1 pS in 150 mm KCl) did not show any voltage dependence and delay in the activation upon the application of voltage steps to both positive and negative transmembrane potentials. Owing to its voltage independence this channel was denominated FV1. The opening probability of the SV-type channel increased by increasing the cytoplasmic calcium concentration, while the activity of the FV1 channel did not increase appreciably by changing the calcium concentration in the range from 6 μm to 1 mm. All the channels identified showed a linear current-voltage characteristic in the range ±100 mV and at least the three most conductive ones displayed potassium selectivity properties. Substitution of potassium with tetramethylammonium (TMA) on the cytosolic side demonstrated that both the SV and FV1 channels are impermeable to TMA influx into the vacuole and support the potassium selectivity properties of these two channels. Moreover, the single channel conductances of all the channels identified increased as a function of the potassium concentration and reached a maximum conductivity at [K⁺] ∼0.5 m. This behavior can be explained by a multi-ion occupancy single-file permeation mechanism. Received: 26 December 1995/Revised: 10 July 1996     

Effects of Na+ on the Predominant K+ Channel in the Tonoplast of Chara: Decrease of Conductance by Blocks in 100 Nanosecond Range and Induction of Oligo- or Poly-subconductance Gating Modes

We present three mechanisms by which Na⁺ inhibits the open channel currents of the predominant K⁺ channel in the tonoplast of Chara corallina: (i) Fast block, i.e., short (100 ns range) interruptions of the open channel current which are determined by open channel noise analysis, (ii): Oligo-subconductance mode, i.e., a gating mode which occurs preferentially in the presence of Na⁺; this mode comprises a discrete number (here 3) of open states with smaller conductances than normal, and (iii): Polysubconductance mode, i.e., a gating mode with a nondiscrete, large number (>30) of states with smaller conductances than the main open channel conductance. This novel mode has also been observed only in the presence of Na⁺. Received: 16 November 1999/Revised: 8 February 2000     

Slow Gating of Gap Junction Channels and Calmodulin

Certain COOH-terminus mutants of connexin32 (Cx32) were previously shown to form channels with unusual transjuctional voltage (V _j ) sensitivity when tested heterotypically in oocytes against Cx32 wild type. Junctional conductance (G _j ) slowly increased by severalfold or decreases to nearly zero with V _j positive or negative, respectively, at mutant side, and V _j positive at mutant side reversed CO₂-induced uncoupling. This suggested that the CO₂-sensitive gate might be a V _j -sensitive slow gate. Based on previous data for calmodulin (CaM) involvement in gap junction function, we have hypothesized that the slow gate could be a CaM-like pore plugging molecule (cork gating model). This study describes a similar behavior in heterotypic channels between Cx32 and each of four new Cx32 mutants modified in cytoplasmic-loop and/or COOH-terminus residues. The mutants are: ML/NN+3R/N, 3R/N, ML/NN and ML/EE; in these mutants, N or E replace M105 and L106, and N replace R215, R219 and R220. This study also reports that inhibition of CaM expression strongly reduces V _j and CO₂ sensitivities of two of the most effective mutants, suggesting a CaM role in slow and chemical gating. Received: 19 April 2000/Revised: 11 August 2000     

Multiple Mechanosensitive Ion Channels from Escherichia coli,Activated at Different Thresholds of Applied Pressure

Mechanosensitive ion channels from Escherichia coli were studied in giant proteoliposomes reconstituted from an inner membrane fraction, or in giant round cells in which the outer membrane and the cell wall had been disrupted by a lysozyme-EDTA treatment and a mild osmotic shock. Patch-clamp experiments revealed the presence in these two preparations of an array of different conductances (100 to 2,300 pS in 0.1 m KCl) activated by stretch. The electrical activity induced by stretch in the native membrane was complex, due to the activation of several different conductances. In contrast, patches of proteoliposomes generally contained clusters of identical conductances, which differed from patch to patch. These experiments are consistent with the notion that these different conductances correspond to different proteins in the plasma membrane of E. coli, which segregate into clusters of identical channels on dilution involved in reconstitution in proteoliposomes. These conductances could be grouped into three subfamilies of poorly selective channels. In both preparations, the higher the conductance, the higher was the negative pressure needed for activation. We discuss the putative role of these channels as parts of a multicomponent osmoregulatory system. Received: 23 May 1995/Revised: 31 January 1996     

Inhibition of Vacuolar Ion Channels by Polyamines

In this work, direct effects of cytosolic polyamines on the two principle vacuolar ion channels were studied by means of patch-clamp technique. Fast and slow activating vacuolar channels were analyzed on membrane patches isolated from vacuoles of the red beet taproot. The potency of the fast and of the slow vacuolar channel blockage by polyamines decreased with a decrease of the polycation charge, spermine⁴⁺ > spermidine³⁺ > putrescine²⁺. In contrast to the inhibition of the fast vacuolar channel, the blockage of the slow vacuolar channel by polyamines displayed a pronounced voltage-dependence. Hence, in the presence of high concentration of polyamines the slow vacuolar channel was converted into a strong inward rectifier as evidenced by its unitary current-voltage characteristic. The blockage of the slow vacuolar channel by polyamines was relieved at a large depolarization, in line with the permeation of polyamines through this channel. The voltage-dependence of blockage was analyzed in terms of the conventional model, assuming a single binding site for polyamines within the channel pore. Taking advantage of a simple linear structure of naturally occurring polyamines, conclusions on a possible architecture of the slow vacuolar channel pore were drawn. The role of common polyamines in regulation of vacuolar ion transport was discussed. Received: 1 May 1998/Revised: 25 September 1998     

Large Conductance Ca2+-Activated K+ Channels in Human Meningioma Cells

Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K⁺ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC₅₀= 5.3 mm). Raising the internal Ca²⁺ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V _1/2) of the K⁺ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca²⁺-activated (BK _Ca ) K⁺ channel with a conductance of 296 pS (130 mm K⁺ at both sides of the patch). V _1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca²⁺, respectively, at the internal face of the patch. In cell-attached patches the open probability (P _o ) of BK _Ca channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K⁺ currents with 10 nm Ca²⁺ in the pipette. Application of 20 μm cytochalasin D increased P _o of BK _Ca channels in cell-attached patches within minutes. These data suggest that the activation of BK _Ca channels in meningioma cells does not only depend on voltage and internal Ca²⁺ but is also controlled by the cytoskeleton. Received 18 June 1999/Revised: 18 January 2000     

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     

IP3-Gated Channels and their Occurrence Relative to CNG Channels in the Soma and Dendritic Knob of Rat Olfactory Receptor Neurons

Olfactory receptor neurons respond to odorants with G protein-mediated increases in the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) and/or inositol-1,4,5-trisphosphate (IP3). This study provides evidence that both second messengers can directly activate distinct ion channels in excised inside-out patches from the dendritic knob and soma membrane of rat olfactory receptor neurons (ORNs). The IP3-gated channels in the dendritic knob and soma membranes could be classified into two types, with conductances of 40 +/- 7 pS (n = 5) and 14 +/- 3 pS (n = 4), with the former having longer open dwell times. Estimated values of the densities of both channels from the same inside-out membrane patches were very much smaller for IP3-gated than for CNG channels. For example, in the dendritic knob membrane there were about 1000 CNG channels x microm(-2) compared to about 85 IP3-gated channels x microm(-2). Furthermore, only about 36% of the dendritic knob patches responded to IP3, whereas 83% of the same patches responded to cAMP. In the soma, both channel densities were lower, with the CNG channel density again being larger ( approximately 57 channels x microm(-2)) than that of the IP3-gated channels ( approximately 13 channels x microm(-2)), with again a much smaller fraction of patches responding to IP3 than to cAMP. These results were consistent with other evidence suggesting that the cAMP-pathway dominates the IP3 pathway in mammalian olfactory transduction.     

Concentration Dependence of Sodium Permeation and Sodium Ion Interactions in the Cyclic AMP-gated Channels of Mammalian Olfactory Receptor Neurons

The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs) on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique. With a saturating concentration (100 μm) of adenosine 3′, 5′-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied at NaCl concentrations from 25 to 300 mm. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear, reversing close to 0 mV. When the external NaCl concentration was maintained at 150 mm and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the Na⁺ equilibrium potential indicating that P _Cl/P _Na≈ 0. However, at low external NaCl concentrations (≤100 mm) there was some significant chloride permeability. Our results further indicated that Na⁺ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K _ms in the range of 100–150 mm and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory CNG channels are significantly different from those of photoreceptor CNG channels. Received: 7 November 1996/Revised: 24 March 1997     

Gating Kinetics of E. coli Poly-3-Hydroxybutyrate/Polyphosphate Channels in Planar Bilayer Membranes

Nonproteinaceous calcium channel complexes from Escherichia coli, composed of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP), exhibit two distinct gating modes (modes 1 and 2) in planar lipid bilayers. Here we report the kinetic characterization of the channel in mode 2, a mode characterized by two well-defined conductance levels, a fully open state (87 ± 3 pS), and a major subconductance state (56 ± 2 pS). Other subconductance states and full closures are rare (<0.5% of total time). Several kinetic properties of the channel showed asymmetric voltage-dependence indicating an asymmetry in the channel structure. Accordingly, single channels responded to potential change in one of two mirror-image patterns, postulated to arise from opposite orientations of the asymmetrical channel complex in the bilayer. The fraction of time spent in each conductance level was strongly voltage-sensitive. For channels reported in this study, presumably all oriented in the same direction, residence time in the fully open state increased as clamping potentials became more positive whereas residence time in the major subconductance state increased at more negative potentials. Analysis of open time distributions revealed existence of two kinetically distinct states for each level. The shorter time constants for both conductance states exhibited weak voltage-sensitivity; however, the longer time constants were strongly voltage-sensitive. A kinetic scheme, consistent with the complex voltage dependence of the channel, is proposed. Received: 1 February 1999/Revised: 2 April 1999     

Clustered Distribution of Calcium Sensitivities: An Indication of Hetero-tetrameric Gating Components in Ca2+-activated K+ Channels Reconstituted from Avian Nasal Gland Cells

High-conductance, Ca²⁺-activated K⁺ channels from the basolateral membrane of rabbit distal colon epithelial cells were reconstituted into planar phospholipid bilayers to examine the effect of Mg²⁺ on the single-channel properties. Mg²⁺ decreases channel current and conductance in a concentration-dependent manner from both the cytoplasmic and the extracellular side of the channel. In contrast to other K⁺ channels, Mg²⁺ does not cause rectification of current through colonic Ca²⁺-activated K⁺ channels. In addition, cytoplasmic Mg²⁺ decreases the reversal potential of the channel. The Mg²⁺-induced decrease in channel conductance is relieved by high K⁺ concentrations, indicating competitive interaction between K⁺ and Mg²⁺. The monovalent organic cation choline also decreases channel conductance and reversal potential, suggesting that the effect is unspecific. The inhibition of channel current by Mg²⁺ and choline most likely is a result of electrostatic screening of negative charges located superficially in the channel entrance. But in addition to charge, other properties appear to be necessary for channel inhibition, as Na⁺ and Ba²⁺ are no (or only weak) inhibitors. Mg²⁺ and possibly other cations may play a role in the regulation of current through these channels. Received: 25 August 1995/Revised: 16 November 1995     

NaF Potentiates a K+-selective Ion Channel in G292 Osteoblastic Cells

Clinical studies have established that NaF increases mineral content in bone, although the cellular mechanisms underlying its osteoinductive effects remain unclear. Because metabolic effects of fluoride have been linked to ion flux and alterations in membrane potential, we used patch-clamp recording techniques to examine the electrophysiological response of osteoblastic cells to NaF. In these experiments, we show that NaF increased the amplitude and P _open of a 73 pS potassium-selective ion channel. The effect of NaF depended on extracellular Ca²⁺ and could be blocked by a combination of calcium-channel blocking agents, suggesting that potentiation of channel activity was dependent on external calcium. Because all patches were in the cell-attached configuration, the effect of NaF was presumably indirect. Although the underlying cellular mechanisms remain unclear, our findings suggest that activity of calcium and/or potassium-selective channels via second messenger cascades may mediate many of the early events involved in the response of bone cells to inorganic fluoride. Received: 30 March 1995/Revised: 13 October 1995