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     

Thermoreception of Paramecium: Different Ca2+ Channels Were Activated by Heating and Cooling

A Paramecium cell responded to heat and cold stimuli, exhibiting increased frequency of directional changes in its swimming behavior. The increase in the frequency of directional changes was maintained during heating, but was transient during cooling. Although variations were large, as expected with this type of electrophysiological recording, results consistently showed a sustained depolarization of deciliated cells in response to heating. Depolarizations were also consistently observed upon cooling. However, these depolarizations were transient and not continuous throughout the cooling period. These depolarizations were lost or became small in Ca²⁺-free solutions. In a voltage-clamped cell, heating induced a continuous inward current and cooling induced a transient inward current under conditions where K⁺ currents were suppressed. The heat-induced inward current was not affected significantly by replacing extracellular Ca²⁺ with equimolar concentrations of Ba²⁺, Sr²⁺, Mg²⁺, or Mn²⁺, and was lost upon replacing with equimolar concentration of Ni²⁺. On the other hand, the cold-induced inward current was not affected significantly by Ba²⁺, or Sr²⁺, however the decay of the inward current was slowed and was lost or became small upon replacing with equimolar concentrations of Mg²⁺, Mn²⁺, or Ni²⁺. These results indicate that Paramecium cells have heat-activated Ca²⁺ channels and cold-activated Ca²⁺ channels and that the cold-activated Ca²⁺ channel is different from the heat-activated Ca²⁺ channel in the ion selectivity and the calcium-dependent inactivation. Received: 9 September 1998/Revised: 22 January 1999     

Cold-sensitive Ca2+ Influx in Paramecium

The concentration of intracellular calcium, [Ca²⁺] _i , in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red. Cooling of a deciliated Paramecium caused a transient increase in [Ca²⁺] _i at the anterior region of the cell. Increase in [Ca²⁺] _i was not observed at any region in Ca²⁺-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K⁺ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing extracellular Ca²⁺. The cold-induced inward current was lost upon replacing extracellular Ca²⁺ with equimolar concentration of Co²⁺, Mg²⁺ or Mn²⁺, but it was not affected significantly by replacing with equimolar concentration of Ba²⁺ or Sr²⁺. These results indicate that Paramecium cells have Ca²⁺ channels that are permeable to Ca²⁺, Ba²⁺ and Sr²⁺ in the anterior soma membrane and the channels are opened by cooling. Received: 1 April 1996/Revised: 23 July 1996     

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     

Inhibition of K/HCO(3) cotransport in squid axons by quaternary ammonium ions

Previous squid-axon studies identified a novel K/HCO₃ cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K⁺ and HCO⁻ ₃ out of the cell, tending to lower intracellular pH (pH_i). With an inwardly directed K/HCO₃ gradient, the cotransporter mediates a net uptake of alkali (i.e., K⁺ and HCO⁻ ₃ influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA⁺) inhibit the inwardly directed cotransporter by interacting at the intracellular K⁺ site. We computed the equivalent HCO⁻ ₃ influx (J _HCO3) mediated by the cotransporter from the rate of pH_i increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pH_i 8.0, using a dialysis fluid (DF) free of K⁺, Na⁺ and Cl⁻. Our standard artificial seawater (ASW) also lacked Na⁺, K⁺ and Cl⁻. After halting dialysis, we introduced an ASW containing 437 mm K⁺ and 0.5% CO₂/12 mm HCO⁻ ₃, which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pH_i decrease, due to CO₂ influx, followed by a slower plateau-phase pH_i increase, due to inward cotransport of K⁺ and HCO⁻ ₃. With no QA⁺ in the DF, J _HCO3 was ∼58 pmole cm⁻² sec⁻¹. With 400 mm tetraethylammonium (TEA⁺) in the DF, J _HCO3 was virtually zero. The apparent K _i for intracellular TEA⁺ was ∼78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K⁺ channels. Introducing 100 mm inhibitor into the DF reduced J _HCO3 to ∼20 pmole cm⁻² sec⁻¹ for tetramethylammonium (TMA⁺), ∼24 for TEA⁺, ∼10 for tetrapropylammonium (TPA⁺), and virtually zero for tetrabutylammonium (TBA⁺). The apparent K _i value for TBA⁺ is ∼0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA⁺), with an apparent K _i of ∼91 μm. Thus, trans-side quaternary ammonium ions inhibit K/HCO₃ influx in the potency sequence PPTEA⁺ > TBA⁺ > TPA⁺ > TEA⁺≅ TMA⁺. The identification of inhibitors of the K/HCO₃ cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter. Received: 21 November 2000/Revised: 14 May 2001     

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     

Extracellular GTP Causes Membrane-Potential Oscillations through the Parallel Activation of Mg2+ and Na+ Currents in Paramecium tetraurelia

Paramecium tetraurelia responds to extracellular GTP (≥ 10 nm) with repeated episodes of prolonged backward swimming. These backward swimming events cause repulsion from the stimulus and are the behavioral consequence of an oscillating membrane depolarization. Ion substitution experiments showed that either Mg²⁺ or Na⁺ could support these responses in wild-type cells, with increasing concentrations of either cation increasing the extent of backward swimming. Applying GTP to cells under voltage clamp elicited oscillating inward currents with a periodicity similar to that of the membrane-potential and behavioral responses. These currents were also Mg²⁺- and Na⁺-dependent, suggesting that GTP acts through Mg²⁺-specific (I _Mg) and Na⁺-specific (I _Na) conductances that have been described previously in Paramecium. This suggestion is strengthened by the finding that Mg²⁺ failed to support normal behavioral or electrophysiological responses to GTP in a mutant that specifically lacks I _Mg (``eccentric'), while Na<sup>+</sup> failed to support GTP responses in ``fast-2,' a mutant that specifically lacks I _Na. Both mutants responded normally to GTP if the alternative cation was provided. As I _Mg and I _Na are both Ca²⁺-dependent currents, the characteristic GTP behavior could result from oscillations in intracellular Ca²⁺ concentration. Indeed, applying GTP to cells in the absence of either Mg²⁺ or Na⁺ revealed a minor inward current with a periodicity similar to that of the depolarizations. This current persisted when known voltage-dependent Ca²⁺ currents were blocked pharmacologically or genetically, which implies that it may represent the activation of a novel purinergic-receptor–coupled Ca²⁺ conductance. Received: 28 October 1996/Revised: 24 December 1996     

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

In the E₁ state of the Na,K-ATPase all cations present in the cytoplasm compete for the ion binding sites. The mutual effects of mono-, di- and trivalent cations were investigated by experiments with the electrochromic fluorescent dye RH421. Three sites with significantly different properties could be identified. The most unspecific binding site is able to bind all cations, independent of their valence and size. The large organic cation Br₂-Titu³⁺ is bound with the highest affinity (<μm), among the tested divalent cations Ca²⁺ binds the strongest, and Na⁺ binds with about the same equilibrium dissociation constant as Mg²⁺ (∼0.8 mm). For alkali ions it exhibits binding affinities following the order of Rb⁺≃ K⁺ > Na⁺ > Cs⁺ > Li⁺. The second type of binding site is specific for monovalent cations, its binding affinity is higher than that of the first type, for Na⁺ ions the equilibrium dissociation constant is < 0.01 mm. Since binding to that site is not electrogenic it has to be close to the cytoplasmic surface. The third site is specific for Na⁺, no other ions were found to bind, the binding is electrogenic and the equilibrium dissociation constant is 0.2 mm. Received: 7 August 2000/Revised: 14 November 2000     

III. Ion Channel Expression in PMA-differentiated Human THP-1 Macrophages

Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K⁺ currents, I _DR, present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K⁺ channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K⁺ channel (I _IR) and a Ca²⁺-activated maxi-K channel (I _BK). I _IR was a classical inward rectifier, conducting large inward currents negative to E _K and very small outward currents. I _IR was blocked in a voltage-dependent manner by Cs⁺, Na⁺, and Ba²⁺, block increasing with hyperpolarization. Block by Na⁺ and Ba²⁺ was time-dependent, whereas Cs⁺ block was too fast to resolve. Rb⁺ was sparingly permeant. In cell-attached patches with high [K⁺] in the pipette, the single I _IR channel conductance was ∼30 pS and no outward current could be detected. I _BK channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the conductance was ∼200–250 pS and at potentials positive to ∼100 mV a negative slope conductance of the unitary current was observed, suggesting block by intracellular Na⁺. I _BK was activated at large positive potentials in cell-attached patches; in inside-out patches the voltage-activation relationship was shifted to more negative potentials by increased [Ca²⁺]. Macroscopic I _BK was blocked by external TEA⁺ with half block at 0.35 mm. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K⁺ channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold. Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed. Received: 19 September 1995/Revised: 14 March 1996     

An Energy-Barrier Model for the Permeation of Monovalent and Divalent Cations Through the Maxi Cation Channel in the Plasma Membrane of Rye Roots

The depolarization-activated, high-conductance ``maxi' cation channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to a wide variety of monovalent and divalent cations. The permeation of K⁺, Na⁺, Ca²⁺ and Ba²⁺ through the pore could be simulated using a model composed of three energy barriers and two ion binding sites (a 3B2S model), which assumed single-file permeation and the possibility of double cation occupancy. The model had an asymmetrical free energy profile. Differences in permeation between cations were attributed primarily to differences in their free energy profiles in the regions of the pore adjacent to the extracellular solution. In particular, the height of the central free energy peak differed between cations, and cations differed in their affinities for ion binding sites. Significant ion repulsion occurred within the pore, and the mouths of the pore had considerable surface charge. The model adequately described the diverse current vs. voltage (I/V) relationships obtained over a wide variety of experimental conditions. It described the phenomena of non-Michaelian unitary conductance vs. activity relationships for K⁺, Na⁺ and Ca²⁺, differences in selectivity sequences obtained from measurements of conductance and permeability ratios, changes in relative cation permeabilities with solution composition, and the complex effects of Ba²⁺ and Ca²⁺ on K⁺ currents through the channel. The model enabled the prediction of unitary currents and ion fluxes through the maxi cation channel under physiological conditions. It could be used, in combination with data on the kinetics of the channel, as input to electrocoupling models allowing the relationships between membrane voltage, Ca²⁺ influx and Ca²⁺ signaling to be studied theoretically. Received: 29 April 1998/Revised: 20 November 1998     

Ni2+ Slows the Activation Kinetics of High-Voltage-Activated Ca2+ Currents in Cortical Neurons: Evidence for a Mechanism of Action Independent of Channel-Pore Block

The effects of Ni²⁺ were evaluated on slowly-decaying, high-voltage-activated (HVA) Ca²⁺ currents expressed by pyramidal neurons acutely dissociated from guinea-pig piriform cortex. Whole-cell, patch-clamp recordings were performed with Ba²⁺ as the charge carrier. Ni²⁺ blocked HVA Ba²⁺ currents (I _Bas) with an EC₅₀ of approximately 60 μm. Additionally, after application of nonsaturating Ni²⁺ concentrations, residual currents activated with substantially slower kinetics than both total and Ni²⁺-sensitive I _Bas. None of the pharmacological components of slowly decaying, HVA currents activated with kinetics significantly different from that of total currents, indicating that the effect of Ni²⁺ on I _Bas kinetics cannot be attributed to the preferential inhibition of a fast-activating component. The effect of Ni²⁺ on I _Ba amplitude was voltage-independent over the potential range normally explored in our experiments (−60 to +20 mV), hence the Ni²⁺-dependent decrease of I _Ba activation rate is not due to a voltage- and time-dependent relief from block. Moreover, Ni²⁺ significantly reduced I _Ba deactivation speed upon repolarization, which also is not compatible with a depolarization-dependent unblocking mechanism. The dependence on Ni²⁺ concentration of the I _Ba activation-rate reduction was remarkably different from that found for I _Ba block, with an EC₅₀ of ∼20 μm and a Hill coefficient of ∼1.73 vs.∼1.10. These results demonstrate that Ni²⁺, besides inhibiting the I _Bas under study probably by exerting a blocking action on the pore of the underlying Ca²⁺ channels, also interferes with Ca²⁺-channel gating kinetics, and strongly suggest that the two effects depend on Ni²⁺ occupancy of binding sites at least partly distinct. Received: 13 July 2000/Revised: 9 November 2000     

Ion Channel Permeable for Divalent and Monovalent Cations in Native Spinach Thylakoid Membranes

A cation-selective channel was characterized in isolated patches from osmotically swollen thylakoids of spinach (Spinacea oleracea). This channel was permeable for K⁺ as well as for Mg²⁺ and Ca²⁺ but not for Cl⁻. When K⁺ was the main permeant ion (symmetrical 105 mm KCl) the conductance of the channel was about 60 pS. The single channel conductance for different cations followed a sequence K⁺ > Mg²⁺≥ Ca²⁺. The permeabilities determined by reversal potential measurements were comparable for K⁺, Ca²⁺, and Mg²⁺. The cation channel displayed bursting behavior. The total open probability of the channel increased at more positive membrane potentials. Kinetic analysis demonstrated that voltage dependence of the total open probability was determined by the probability of bursts formation while the probability to find the channel in open state within a burst of activity was hardly voltage-dependent. The cation permeability of intact spinach thylakoids can be explained on the single channel level by the data presented here. Received: 26 December 1995/Revised: 17 April 1996     

Hyperpolarization-activated Ca2+-permeable Channels in the Plasma Membrane of Tomato Cells

The hyperpolarization of the electrical plasma membrane potential difference has been identified as an early response of plant cells to various signals including fungal elicitors. The hyperpolarization-activated influx of Ca²⁺ into tomato cells was examined by the application of conventional patch clamp techniques. In both whole cell and single-channel recordings, clamped membrane voltages more negative than −120 mV resulted in time- and voltage-dependent current activation. Single-channel currents saturated with increasing activities of Ca²⁺ and Ba²⁺ from 3 to 26 mm and the single channel conductance increased from 4 pS to 11 pS in the presence of 20 mm Ca²⁺ or Ba²⁺, respectively. These channels were 20–25 and 10–13 times more permeable to Ca²⁺ than to K⁺ and to Cl⁻, respectively. Channel currents were strongly inhibited by 10 μm lanthanum and 50% inhibited by 100 μm nifedipine. This evidence suggests that hyperpolarization-activated Ca²⁺-permeable channels provide a mechanism for the influx of Ca²⁺ into tomato cells. Received: 13 February 1996/Revised: 12 August 1996     

Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K⁺, Na⁺, Cs⁺ and Li⁺; organic cations: TMA and TEA, and divalents: Ba²⁺, Ca²⁺, Mg²⁺ and Mn²⁺. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs⁺, Na⁺ and K⁺ had chord conductances in the range of 35–55 pS with the exception of Li⁺, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K⁺, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba²⁺ and 15 pS for Ca²⁺ at -80 mV and 10 pS for Mg²⁺ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K⁺ currents much like the divalents. As expected, the channel K⁺ conductance increased with K⁺ concentration saturating at ~ 45 pS and the K_D of K⁺ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.     

Divalent cations modify adsorption of 5′-AMP onto precipitated calcium phosphate: A model for cation modulation of adsorptive processes in primitive aqueous environments

The adsorption of 5′-AMP onto precipitated calcium phosphate (CaPi) requires the presence of soluble calcium and this dependence exhibits a Michaelian-like behavior. This result suggests that the formation of a complex between 5′-AMP and free Ca²⁺ (CaAMP) is a prelude to the adsorption of the nucleotide in the solid matrix. At concentrations one order of magnitude higher, Mn²⁺ and Mg²⁺ can substitute for soluble Ca²⁺ in the adsorption of 5′-AMP onto solid CaPi. However, when added simultaneously with 5′-AMP to a heterogeneous mixture that contains CaPi and soluble Ca²⁺, Mn²⁺ and Mg²⁺ inhibit the adsorption of 5′-AMP in a concentration-dependent manner. This suggests the formation of complexes that are much less effective for 5′-AMP adsorption than the CaAMP complex. On the other hand, Mn²⁺ and Mg²⁺ cannot promote desorption of the nucleotide attached to the precipitate in the presence of soluble Ca²⁺ if they are added after adsorption has attained equilibrium. Although desorption of 5′-AMP can be obtained by a sequential dilution of the soluble phase with buffer and no nucleotide in a process that obeys a Langmuir equation, the lack of effect of Mn²⁺ or Mg²⁺ when adsorption has attained its maximal value suggests strong interactions between the CaAMP complex and the solid matrix when adsorption equilibrium is reached. The divalent cations present in the matrix also participate with different selectivity in the attachment of the CaAMP complex, indicating that a cation-exchange mechanism could have acted in the modulation of adsorptive/desorptive processes involving biomonomers and phosphate surfaces in primitive aqueous environments. Received: 11 December 1995 / Accepted: 5 April 1996     

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     

Calcium and Magnesium: Low Passive Permeability and Tubular Secretion in the Mouse Medullary Thick Ascending Limb of Henle's Loop (MTAL)

Recent studies from our laboratory have shown that in the mouse and rat nephron Ca²⁺ and Mg²⁺ are not reabsorbed in the medullary part of the thick ascending limb (mTAL) of Henle's loop. The aim of the present study was to investigate whether the absence of transepithelial Ca²⁺ and Mg²⁺ transport in the mouse mTAL is due to its relative low permeability to divalent cations. For this purpose, transepithelial ion net fluxes were measured by electron probe analysis in isolated perfused mouse mTAL segments, when the transepithelial potential difference (PD_te.) was varied by chemical voltage clamp, during active NaCl transport inhibition by luminal furosemide. The results show that transepithelial Ca²⁺ and Mg²⁺ net fluxes in the mTAL are not driven by the transepithelial PD_te. At zero voltage, a small but significant net secretion of Ca²⁺ into the tubular lumen was observed. With a high lumen-positive PD_te generated by creating a transepithelial bath-to-lumen NaCl concentration gradient, no Ca²⁺ and Mg²⁺ reabsorption was noted; instead significant and sustained Ca²⁺ and Mg²⁺ net secretion occurred. When a lumen-positive PD_te was generated in the absence of apical furosemide, but in the presence of a transepithelial bath-to-lumen NaCl concentration gradient, a huge Ca²⁺ net secretion and a lesser Mg²⁺ net secretion, not modified by ADH, were observed. Replacement of Na⁺ by K⁺ in the lumen perfusate induced, in the absence of PD_te changes, important but reversible net secretions of Ca²⁺ and Mg²⁺. In conclusion, our results indicate that the passive permeability of the mouse mTAL to divalent cations is very low and not influenced by ADH. This nephron segment can secrete Ca²⁺ and Mg²⁺ into the luminal fluid under conditions which elicit large lumen-positive transepithelial potential differences. Given the impermeability of this epithelium to Ca²⁺ and Mg²⁺, the secretory processes would appear to be of cellular origin. Received: 30 January 1996/Revised: 24 April 1996     

Relationship between Extra- and Intracellular Calcium in Distal Segments of the Renal Tubule. Role of the Ca2+ Receptor RaKCaR

The effect of extracellular calcium ([Ca²⁺] _e ) on cytosolic calcium ([Ca²⁺] _i ) was investigated in thick ascending limbs and collecting ducts from the rat kidney, using the fluorescent dye fura-2. In cortical collecting ducts, basolateral but not apical changes in [Ca²⁺] _e were associated with parallel changes in [Ca²⁺] _i . Basal [Ca²⁺] _i was hardly modified by nifedipine and verapamil but was decreased by 60% by basolateral La³⁺. Increasing peritubular [Ca²⁺] _e triggered Ca²⁺ release from intracellular stores. This effect was not reproduced by agonists of the renal Ca²⁺-receptor RaKCaR, e.g., Ba²⁺, Mg²⁺, Gd³⁺, and neomycin, but was reproduced by Ni²⁺. Ni²⁺-induced mobilization of intracellular Ca²⁺ was larger in the inner medullary collecting duct, a segment which poorly responds to increasing [Ca²⁺] _e . In the cortical thick ascending limb, removing basolateral Ca²⁺ hardly altered [Ca²⁺] _i but increasing [Ca²⁺] _e or adding Ba²⁺, Mg²⁺, Gd³⁺ and neomycin released intracellular calcium. These data demonstrate that (1) basolateral influx of calcium occurs in cortical collecting ducts, under basal conditions; (2) this influx occurs through nonvoltage gated channels, permeable to Ba²⁺, insensitive to verapamil and nifedipine, and blocked by La³⁺; (3) increasing [Ca²⁺] _e stimulates the influx and triggers intracellular calcium release, independently of the phospholipase C-coupled receptor RaKCaR; (4) RaKCaR is functionally expressed in thick ascending limbs; (5) another membrane receptor, sensitive to Ni²⁺ but not to Ca²⁺ is present in the collecting duct. Received: 12 July 1996/Revised: 28 October 1996     

Separate Swelling- and Ca2+-activated Anion Currents in Ehrlich Ascites Tumor Cells

A Ca²⁺-activated (I _Cl,Ca) and a swelling-activated anion current (I _Cl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca²⁺] _i ), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl⁻ concentration. I _Cl,Ca current density increased with increasing [Ca²⁺] _i , and this current was abolished by lowering [Ca²⁺] _i to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA). In contrast, activation of I _Cl,vol did not require an increase in [Ca²⁺] _i . The kinetics of I _Cl,Ca and I _Cl,vol were different: at depolarized potentials, I _Cl,Ca as activated in a [Ca²⁺] _i - and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I _Cl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of I _Cl,vol was dependent on the extracellular Mg²⁺ concentration. The anion permeability sequence for both currents was I ⁻ > Cl⁻ > gluconate. I _Cl,Ca was inhibited by niflumic acid (100 μm), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 μm) and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS, 100 μm), niflumic acid being the most potent inhibitor. In contrast, I _Cl,vol was unaffected by niflumic acid (100 μm), but abolished by tamoxifen (10 μm). Thus, in Ehrlich cells, separate chloride currents, I _Cl,Ca and I _Cl,vol, are activated by an increase in [Ca²⁺] _i and by cell swelling, respectively. Received: 12 November 1997/Revised: 5 February 1998     

Non-covalent (iso)guanosine-based ionophores for alkali(ne earth) cations

Different (iso)guanosine-based self-assembled ionophores give distinctly different results in extraction experiments with alkali(ne earth) cations. A lipophilic guanosine derivative gives good extraction results for K⁺, Rb⁺, Ca²⁺, Sr²⁺, and Ba²⁺ and in competition experiments it clearly favors the divalent Sr²⁺ (and Ba²⁺) cations. 1,3-Alternate calix[4]arene tetraguanosine hardly shows any improvement in the extraction percentages compared to its reference compound 1,3-alternate calix[4]arene tetraamide. This indicates that one G-quartet does not provide efficient cation complexation under these conditions. In the case of the lipophilic isoguanosine derivative there is a cation size dependent affinity for the monovalent cations (Cs⁺ ? Rb⁺ ? K⁺), but not for the divalent cations (Ca²⁺ > Ba²⁺ > Sr²⁺ > Mg²⁺). In competition experiments the isoguanosine derivative, unlike guanosine, does not discriminate between monovalent and divalent cations, giving an almost equal extraction of Cs⁺ and Ba²⁺.