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     

Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes

Vasopressin stimulates the activity of the epithelial Na channel (ENaC) through the cAMP/PKA pathway in the cortical collecting tubule, or in similar amphibian epithelia, but the mechanism of this regulation is not yet understood. This stimulation by cAMP could not be reproduced with the rat or Xenopus ENaC expressed in Xenopus oocyte. Recently, it was shown that the α-subunit cloned from the guinea-pig colon (αgp) could confer the ability to be activated by the membrane-permeant cAMP analogue 8-chlorophenyl-thio-cAMP (cpt-cAMP) to channels produced by expression of αgp, βrat and γrat ENaC subunits. In this study we investigate the mechanism of this activation. Forskolin treatment, endogenous production of cAMP by activation of coexpressed β adrenergic receptors, or intracellular perfusion with cAMP did not increase the amiloride-sensitive Na current, even though these maneuvers stimulated CFTR (cystic fibrosis transmembrane conductance regulator)-mediated Cl currents. In contrast, extracellular 8-cpt-cAMP increased αgp, βrat and γrat ENaC activity but had no effect on CFTR. Swapping intracellular domains between the cpt-cAMP-sensitive αgp and the cpt-cAMP-resistant αrat-subunit showed that neither the N-terminal nor the C-terminal of α ENaC was responsible for the effect of cpt-cAMP. The mechanisms of activation of ENaC by cpt-cAMP and of CFTR by the cAMP/PKA pathway are clearly different. cpt-cAMP seems to increase the activity of ENaC formed by αgp and βγrat by interacting with the extracellular part of the protein. Received: 19 January 2001/Revised: 27 April 2001     

Interconverting Gating Modes of a Nonselective Cation Channel from the Tapeworm Echinococcus granulosus Reconstituted on Planar Lipid Bilayers

A 107-pS (symmetrical 150 mm KCl), nonselective cation channel was reconstituted from a microsomal membrane fraction of the larval stage of the tapeworm Echinococcus granulosus. Most of the time, it displayed a high open probability (>0.95) irrespective of either the applied voltage, Ca²⁺, Ba²⁺, or tetraethylammonium concentration. Nevertheless, in contrast with this ``leaklike' behavior, less frequently this ``all-the-time-open' channel reversibly entered two different kinetic modes. One of them was characterized by lower P _o values and some voltage sensitivity (V _?≅ 129 mV, and an equilibrium constant for channel closing changing e-fold per 63-mV change) the kinetic analysis revealing that it resulted from the appearance of voltage-sensitivity in the mean closed times and a sixfold increase in the equilibrium constant for channel closing at 0 mV. The other mode was characterized by a very fast open-close activity leading to poorly resolved current levels and a P _o around 0.6–0.7 which, occasionally and in a voltage-sensitive manner, entered a long-lived nonconducting state. However, the rare nature of these mode-shifting transitions precluded a more detailed analysis of their kinetics. The conductive properties of the channel were not affected by these switches. Model gating alone does not seem to ensure any physiological role of this channel and, instead, some other channel changes must occur if this phenomenon were to be of regulatory importance in vivo. Thus, mode-shifting might constitute an alternative target for channel activity modulation also in tapeworms. Received: 30 August 1996/Revised: 31 January 1997     

The Functional Surface Charge Density of a Fast K Channel in the Myelinated Axon of Xenopus laevis

The action of Mg²⁺ on the putative xKv1.1 channel in the myelinated axon of Xenopus laevis was analyzed in voltage clamp experiments. The main effect was a shift in positive direction of the open probability curve (16 mV at 20 mm Mg²⁺), calculated from measurements of the instantaneous current at Na reversal potential after 50–100 msec steps to different potentials. The shift was measured at an open probability level of 25% to separate it from shifts of other K channel populations in the nodal region. The results could be explained in terms of screening effects on fixed charges located on the surface of the channel protein. Using the Grahame equation the functional charge density was estimated to −0.45 e nm⁻². Analyzing this value, together with previously estimated values from other K channels, with reference to the charge of different extracellular loops of the channel protein, we conclude that the loop between the transmembrane S5 segment and the pore forming P segment determines the functional charge density of voltage-gated K channels. Received: 11 December 1997/Revised: 24 April 1998     

Divalent Cation Effects on the Shaker K Channel Suggest a Pentapeptide Sequence as Determinant of Functional Surface Charge Density

The effects of the divalent cations strontium and magnesium on Shaker K channels expressed in Xenopus oocytes were investigated with a two-electrode voltage-clamp technique. 20 mm of the divalent cation shifted activation (conductance vs. potential), steady-state inactivation and inactivation time constant vs. potential curves 10–11 mV along the potential axis. The results were interpreted in terms of the surface charge theory, and the surface charge density was estimated to be −0.27 e nm⁻². A comparison of primary structure data and experimental data from the present and previous studies suggests that the first five residues on the extracellular loop between transmembrane segment 5 and the pore region constitutes the functional surface charges. The results further suggest that the surface charge density plays an important role in controlling the activation voltage range. Received: 12 November 1997/Revised: 1 June 1998     

The Antifungal Imidazole Clotrimazole and its Major In Vivo Metabolite are Potent Blockers of the Calcium-Activated Potassium Channel in Murine Erythroleukemia Cells

Clotrimazole (CLT), a member of the antifungal imidazole family of compounds, has been found to inhibit both calcium (Ca²⁺)-activated ⁸⁶Rb and potassium (K) fluxes of human red cells and to inhibit red cell binding of ¹²⁵I-charybdotoxin (ChTX) [11]. We have now used patch-clamp techniques to demonstrate reversible inhibition of whole cell K_Ca2+ currents in murine erythroleukemia (MEL) cells by submicromolar concentrations of CLT. Inhibition was equivalent whether currents were elicited by bath application of the Ca²⁺ ionophore A23187 or by dialyzing cells with a pipette solution containing micromolar concentrations of free Ca²⁺. The extent of inhibition of whole cell MEL K_Ca2+ currents was voltage-dependent, decreasing with increasing test potential. We also determined the single channel basis of the CLT inhibition in MEL cells by demonstrating the inhibition of a calcium-activated, ChTX-sensitive K channel by CLT in outside-out patches. The channel was also blocked by the des-imidazolyl metabolite of CLT, 2-chlorophenyl-bisphenyl-methanol (MET II) [15], thus demonstrating that the imidazole ring is not required for the inhibitory action of CLT. Single K_Ca2+ channels were also evident in inside-out patches of MEL cells. Block of K current by CLT was not unique to MEL cells. CLT also inhibited a component of the whole cell K current in PC12 cells. Channel specificity of block by CLT was determined by examining its effects on other types of voltage-sensitive currents. CLT block showed the following rank order of potency: K currents in PC12 cells > Ca²⁺ currents in PC12 cells ≫ Na currents in sympathetic neurons. These results demonstrate that direct inhibition of single K_Ca2+ by CLT can be dissociated from inhibition of cytochrome P-450 in MEL cells. Received: 10 September 1996/Revised: 12 December 1996     

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     

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).     

Cation Permeability and Selectivity of a Root Plasma Membrane Calcium Channel

Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential and their contribution to cation toxicities. The selectivity of the rca channel, a Ca²⁺-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K⁺, Na⁺, Ca²⁺ and Mg²⁺ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure of the rca channel appeared to differ from that of L-type calcium channels from animal cell membranes since its ion binding sites had a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the rca channel. It described the apparent submillimolar K _m for the relationship between unitary conductance and Ca²⁺ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca²⁺ on K⁺ and Na⁺ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of the rca channel for Ca²⁺ influx under physiological conditions. Received: 23 August 1999/Revised: 12 November 1999     

Nitric Oxide Inhibition of the Rat Olfactory Cyclic Nucleotide-Gated Cation Channel

The effects of nitric oxide (NO) and other cysteine modifying agents were examined on cyclic nucleotide-gated (CNG) cation channels from rat olfactory receptor neurons. The NO compounds, S-nitroso-cysteine (SNC) and 3-morpholino-sydnonomine (SIN-1), did not activate the channels when applied for up to 10 min. The cysteine alkylating agent, N-ethylmaleimide (NEM), and the oxidising agent, dithionitrobensoate (DTNB), were also without agonist efficacy. Neither SNC nor DTNB altered the cAMP sensitivity of the channels. However, 2-min applications of SIN-1, SNC and DTNB inhibited the cAMP-gated current to approximately 50% of the control current level. This inhibition showed no spontaneous reversal for 5 min but was completely reversed by a 2-min exposure to DTT. The presence of cAMP protected the channels against NO-induced inhibition. These results indicate that inhibition is caused by S-nitrosylation of neighboring sulfhydryl groups leading to sulfhydryl bond formation. This reaction is favored in the closed channel state. Since recombinantly expressed rat olfactory α and β CNG channel homomers and α/β heteromers are activated and not inhibited by cysteine modification, the results of this study imply the existence of a novel subunit or tightly bound factor which dominates the effect of cysteine modification in the native channels. As CNG channels provide a pathway for calcum influx, the results may also have important implications for the physiological role of NO in mammalian olfactory receptor neurons. Received: 30 March 1998/Revised: 17 June 1998     

Architecture of the Neuronal Nicotinic Acetylcholine Receptor Ion Channel at the Binding Site of bis-Ammonium Blockers

Structure-activity relationships of 56 pentamethylenbis-ammonium compounds, the blockers of the neuronal nicotinic acetylcholine receptor (nAChR) ion channel, have been studied to estimate the cross-sectional dimensions of the channel pore. The cat superior cervical sympathetic ganglion in situ and isolated guinea pig ileum were used to evaluate the potency of the compounds to block ganglionic transmission. Minimum-energy conformations of each compound were calculated by the molecular mechanics method. A topographic model of the binding site of the blockers was proposed. It incorporates two narrowings, a large and a small one. The small narrowing is located between the large one and the cytoplasmic end of the pore. The cross-sectional dimensions of the large and small narrowings estimated from the dimensions of the blockers are 6.1 × 8.3 ? and 5.5 × 6.4 ?, respectively, the distance between the narrowings along the pore being approximately 7 ?. Most potent blockers would occlude the pore via binding to the channel at the levels of both narrowings. Less potent blockers are either too large or too small to bind to both narrowings simultaneously: large blockers would occlude the pore at the level of large narrowing, while small blockers would pass the large narrowing and occlude the pore at the level of small narrowing only. A comparison of the topographic model with a molecular five-helix bundle model of nAChR pore predicts Serine and Threonine rings to be the most probable candidates for the large and small narrowings, respectively. Received: 6 September 1995/Revised: 12 March 1996     

Activation of Divalent Cation Influx into S. cerevisiae Cells by Hypotonic Downshift

Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells from YPD medium containing 0.8 m sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca²⁺ and other divalent cations into the cell. For cells grown in YPD at 37°C, this hypotonic downshift increases Ca²⁺ accumulation 6.7-fold. Hypotonic downshift-induced Ca²⁺ accumulation and steady-state Ca²⁺ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg²⁺. The Ca²⁺-influx pathway responsible for hypotonic-induced Ca²⁺ influx may account for about 10–35% of Ca²⁺ accumulation by cells growing in YPD. Ca²⁺ influx is not required for cells to survive a hypotonic downshift. Hypotonic downshift greatly reduces the ability of S. cerevisiae cells to survive a 5-min exposure to 10 mm Cd²⁺ suggesting that mutants resistant to acute Cd²⁺ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx. Received: 14 January 1997/Revised: 20 June 1997     

Single-Channel Analysis of a Delayed Rectifier K+ Channel in Xenopus Myelinated Nerve

Single-channel properties of a delayed rectifier voltage-gated K⁺ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E₅₀, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105 mm K⁺, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C. In Na⁺-rich solution with 2.5 mm extracellular K⁺γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K⁺ over Na⁺. γ depended on extracellular K⁺ concentration (K _D = 19.6 mm) and temperature (Q ₁₀= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested with a half-maximal inhibiting concentration (IC₅₀) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC₅₀= 6.8 nm) and mast cell degranulating peptide (MCDP, IC₅₀= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential. Received: 2 June 1995/Revised: 13 October 1995     

Modulation of Recombinant GABA Receptor/Channel Subunits by Domain-specific Antibodies in Xenopus Oocytes

To study interaction of specific antibodies with the GABA receptor/channel, antisera were raised against the extracellular domains of the GABA_A receptor/channel β₂ subunit, γ₂ subunit and the GABA_C receptor/channel ρ₁ subunit. The specificity of the antibodies was characterized by immunocytochemistry and by Western blotting of transfected FDC-P1 cells expressing recombinant GABA receptor/channel subunits. The effects of the antibodies on whole-cell currents in Xenopus laevis oocytes expressing homomeric recombinant GABA receptor/channel β₂, γ₂, and ρ₁ were studied using two-microelectrode voltage clamp. In the absence of GABA, anti-α₂, anti-γ₂, and anti-ρ₁ antisera elicited whole-cell currents in oocytes expressing β₂, γ₂, and ρ₁ subunits, respectively. The effect of antibody on channel activation was concentration-dependent. The whole-cell currents induced by anti-β₂ and anti-γ₂ were several-fold greater than those induced by application of 100 μm GABA. In Xenopus oocytes expressing recombinant ρ₁ subunits, GABA-induced whole-cell currents were inhibited by the anti-ρ₁ antibody. In contrast, the GABA-induced whole-cell currents were potentiated several-fold by anti-β₂ and anti-γ₂ antibodies in Xenopus oocytes expressing homomeric β₂ and γ₂ subunits. Our studies indicate that antibodies specific to the N-terminal domain of GABA receptor/channel subunits can modulate the neurotransmitter receptor function. Received: 2 February 2001/Revised: 11 April 2001     

Characterization of volume-sensitive organic osmolyte efflux and anion current in Xenopus oocytes

Cell swelling activates an outwardly rectifying anion current in numerous mammalian cell types. An extensive body of evidence indicates that the channel responsible for this current is the major pathway for volume regulatory organic osmolyte loss. Cell swelling also activates an outwardly rectifying anion current in Xenopus oocytes. Unlike mammalian cells, oocytes allow the direct study of both swelling-activated anion current and organic osmolyte efflux under nearly identical experimental conditions. We therefore exploited the unique properties of oocytes in order to examine further the relationship between anion channel activity and swelling-activated organic osmolyte transport. Swelling-activated anion current and organic osmolyte efflux were studied in parallel in batches of oocytes obtained from single frogs. The magnitude of swelling-activated anion current and organic osmolyte efflux exhibited a positive linear correlation. In addition, the two processes had similar pharmacological characteristics and activation, rundown and reactivation kinetics. The present study provides further strong support for the concept that the channel responsible for swelling-activated Cl⁻ efflux and the outwardly rectifying anion conductance is also the major pathway by which organic osmolytes are lost from vertebrate cells during regulatory volume decrease. Received: 22 April 1996/Revised: 18 December 1996     

A Novel Large Conductance, Nonselective Cation Channel in Pheochromocytoma (PC12) Cells

A new type of nonselective cation channel was identified and characterized in pheochromocytoma (PC12) cells using inside-out and cell-attached patch-clamp recordings. The channel shows a large unitary conductance (274 pS in symmetric 145 mm K⁺) and selectivity for Na⁺≈ K⁺ > Li⁺, and is practically impermeable to Cl⁻. The channel activity-voltage relationship is bell-shaped, showing maximal activation at ≈−10 mV. The overall activity of this channel is unmodified by [Na⁺] _ic , or [Ca⁺⁺] _ic . However, increases in [Ca⁺⁺] _ic lead to a decrease in the unitary current amplitude. In addition, overall activity is mildly increased when suction is applied to the back of the patch pipette. Together, these characteristics distinguish the present channel from all other large conductance nonselective cation channels reported so far in a variety of preparations. The frequency of appearance of this channel type is similar in undifferentiated and NGF-treated PC12 cells (≈8–27% of patches). The combination of large conductance, permeability to Na⁺, and existence of conducting states at negative potentials, may provide a significant pathway for inward current and depolarization in PC12 cells. Received: 14 February 1997/Revised: 28 July 1997     

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     

Stoichiometry of the Large Conductance Bacterial Mechanosensitive Channel of E. coli. A Biochemical Study

MscL, a 15 kDa transmembrane protein, is the only component involved in the formation of a 3 nS channel in the inner membrane of Escherichia coli that opens in response to mechanical or osmotic stress. While previous data had suggested that the functional MscL complex might be a hexamer, a recent crystallographic study of the MscL homologue from M. tuberculosis reveals a pentameric structure. The present work further examines the stoichiometry of the E. coli MscL using a variety of biochemical approaches. Detergent-purified 6His-MscL in solution and MscL in the membrane could be chemically crosslinked with the products displaying ladderlike patterns on SDS gels. Three crosslinking agents (EDC, DMS, and DMA) used at saturating concentrations invariably generated pentamers as the largest product. DSS produced additional bands corresponding to larger complexes although the pentamer band appeared to be the predominant product at high levels of crosslinker. It is not clear whether these extra bands reflect a difference in the crosslinking chemistry of DSS or whether its spacer arm is the longest of those used, or a combination of both facts. For the detergent-solubilized 6His-MscL both sedimentation equilibrium and gel chromatography showed the presence of multiple species. Thus the longer spacer arm could permit both intra- and intercomplex linkages. Nonetheless, the patterns obtained with all agents are consistent with and strongly suggest a pentameric organization for the MscL channel. Expression of MscL as genetically engineered double or triple subunit tandems yields low numbers of functional channels as compared to expressed monomers. The double-tandem assemblies must have an even number of subunits and crosslinking in the membrane confirmed hexamerization. Gel chromatography clearly demonstrated that the channels formed from the double tandems were larger than those formed from WT MscL, consistent with the native channel being pentameric. The observation that both double and triple tandems form channels of normal conductance implies that the pentameric assembly is to some degree independent of the number of subunit repeats in the polypeptide precursor. The channel is thus a pentameric core with the `extra' subunits left out of the functional complex. From sedimentation equilibrium and size-exclusion chromatography, we also conclude that MscL complexes are not in a dynamic equilibrium with monomers, but are pre-assembled; and thus, their gating properties must result from changes in the conformation of the entire complex induced by the mechanical stress. Received: 26 February 1999/Revised: 10 June 1999     

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