Effects of Lipophilic Ions on Outer Hair Cell Membrane Capacitance and Motility

The outer hair cell (OHC) from the mammalian organ of Corti possesses a bell-shaped voltage-dependent capacitance function. The nonlinear capacitance reflects the activity of membrane bound voltage sensors associated with membrane motors that control OHC length. We have studied the effects of the lipophilic ions, tetraphenylborate (TPB⁻) and tetraphenylphosphonium (TPP⁺), on nonlinear capacitance and motility of isolated guinea-pig OHCs. Effects on supporting cells were also investigated. TPB⁻ produced an increase in the peak capacitance (Cm _pk ) and shifted the voltage at peak capacitance (V _pkCm ) to hyperpolarized levels. Washout reversed the effects. Perfusion of 0.4 μm TPB⁻ caused an average increase in Cm _pk of 16.3 pF and V _pkCm shift of 13.6 mV. TPP⁺, on the other hand, only shifted V _pkCm in the positive direction, with no change in Cm _pk . The contributions from native OHC and TPB⁻-induced capacitance were dissected by a double Boltzmann fitting paradigm, and by blocking native OHC capacitance. While mechanical response studies indicate little effect of TPB⁻ on the motility of OHCs which were in normal condition or treated with salicylate or gadolinium, the voltage at maximum mechanical gain (V _{δ Lmax} ) was shifted in correspondence with native V _pkCm , and both changed in a concentration-dependent manner. Both TPB⁻-induced changes in Cm _pk and V _pkCm were affected by voltage prepulses and intracellular turgor pressure. TPB⁻ induced a voltage-dependent capacitance in supporting cells whose characteristics were similar to those of the OHC, but no indication of mechanical responses was noted. Our results indicate that OHC mechanical responses are not simply related to quantity of nonspecific nonlinear charge moved within the membrane, but to the effects of motility voltage-sensor charge movement functionally coupled to a mechanical effector. Received: 14 May 1998/Revised: 24 August 1998     

Distortion Component Analysis of Outer Hair Cell Motility-Related Gating Charge

The underlying Boltzmann characteristics of motility-related gating currents of the outer hair cell (OHC) are predicted to generate distortion components in response to sinusoidal transmembrane voltages. We studied this distortion since it reflects the mechanical activity of the cell that may contribute to peripheral auditory system distortion. Distortion components in the OHC electrical response were analyzed using the whole-cell voltage clamp technique, under conditions where ionic conductances were blocked. Single or double-sinusoidal transmembrane voltage stimulation was delivered at various holding voltages, and distortion components of the current responses were detected by Fourier analysis. Current response magnitude and phase of each distortion component as a function of membrane potential were compared with characteristics of the voltage-dependent capacitance, obtained by voltage stair-step transient analysis or dual-frequency admittance analysis. The sum distortion was most prominent among the distortion components at all holding voltages. Notches in the sum (f1+f2), difference (f2−f1) and second harmonic (2f) components occur at the voltage where peak voltage-dependent capacitance resides (V _pkCm ). Rapid phase reversals also occurred at V _pkCm , but phase remained fairly stable at more depolarized and hyperpolarized potentials. Thus, it is possible to extract Boltzmann parameters of the motility-related charge movement from these distortion components. In fact, we have developed a technique to follow changes in the voltage dependence of OHC motility and charge movement by tracking the voltage at phase reversal of the f2−f1 product. When intracellular turgor pressure was changed, V _pkCm and distortion notch voltages shifted in the same direction. These data have important implications for understanding cochlear nonlinearity, and more generally, indicate the usefulness of distortion analysis to study displacement currents. Received: 31 December 1998/Revised: 12 March 1999     

Gating and Permeation in Ion Channels Formed by Gramicidin A and Its Dioxolane-linked Dimer in Na+ and Cs+ Solutions

The association of two gramicidin A (gA) peptides via H-bonds in lipid bilayers causes the formation of an ion channel that is selective for monovalent cations only. In this study, two gAs were covalently linked with a dioxolane group (SS dimer). Some functional properties of natural gA channels were compared to that synthetic dimer in Na⁺- or Cs⁺-containing solutions. The SS dimer remained in the open configuration most of the time, while natural gA channels had a relatively brief mean open time. Single channel conductances to Na⁺ (g _Na ) or Cs⁺ (g _Cs ) in the SS dimer were smaller than in natural gA. However, g _Na was considerably more attenuated than g _Cs . This probably results from a tight solvation of Na⁺ by the dioxolane linker in the SS channel. In Cs⁺ solutions, the SS had frequent closures. By contrast, in Na⁺ solutions the synthetic dimer remained essentially in the open state. The mean open times of SS channels in different solutions (T _open,Na > T _open,Cs > T _open,H ) were inversely proportional to the single channel conductances (g _H > g _Cs > g _Na ). This suggests that ion occupancy inside the pore stabilizes the open configuration of the gA dimer. The mean closed time of the SS dimer was longer in Cs⁺ than in H⁺ solutions. Possible mechanisms for these effects are discussed. Received: 17 September 1999/Revised: 21 December 1999     

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

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

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     

Mouse VDAC Isoforms Expressed in Yeast: Channel Properties and Their Roles in Mitochondrial Outer Membrane Permeability

The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane. The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions. The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3). These proteins and different versions of VDAC3 were expressed in yeast cells (S. cerevisiae) missing the major yeast VDAC gene and studied using different approaches. When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol). In contrast, electrophysiological studies on purified proteins showed very different channel properties. VDAC1 is the prototypic version whose properties are highly conserved among other species. VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity. VDAC3 can also form channels in planar phospholipid membranes. It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV). Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed. These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria. The roles of these different VDAC proteins are discussed. Received: 19 June 1998/Revised: 1 April 1999     

Subunit Regulation of the Human Brain α1E Calcium Channel

The α₁ subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α₂δ and β subunits. α_1E channels directed with the expression of Ba²⁺ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α_1E with α_2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α_1E with β_2a shifted the peak current relationship by −10 mV, and strongly reduced Ba²⁺ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba²⁺ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β_2a and the neuronal α_2bδ subunits increased by ≈10-fold whole-cell Ba²⁺ currents although coinjection with either β_2a or α_2bδ alone failed to significantly increase α_1E peak currents. Coexpression with β_2a and α_2bδ yielded Ba²⁺ currents with inactivation kinetics similar to the β_2a induced currents, indicating that the neuronal α_2bδ subunit has little effect on α_1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit genes. The slower inactivation was unique to α_1E/β_2a currents. Coexpression with β_1a, β_1b, β₃, and β₄, yielded faster-inactivating Ba²⁺ currents than currents recorded from the α_1E subunit alone. Furthermore, α_1E/α_2bδ/β_1a; α_1E/α_2bδ/β_1b; α_1E/α_2bδ/β₃; α_1E/α_2bδ/β₄ channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The β subunit-induced changes in the properties of α_1E channel were comparable to modulation effects reported for α_1C and α_1A channels with β₃≈β_1b > β_1a≈β₄≫β_2a inducing fastest to slowest rate of whole-cell inactivation. Received: 27 March 1997/Revised: 10 July 1997     

pH-Sensitive Gating Kinetics of the Maxi-K Channel in the Tonoplast of Chara australis

The most frequently observed K⁺ channel in the tonoplast of Characean giant internodal cells with a large conductance (ca. 170 pS; Lühring, 1986; Laver & Walker, 1987) behaves, although inwardly rectifying, like animal maxi-K channels. This channel is accessible for patch–clamp techniques by preparation of cytoplasmic droplets, where the tonoplast forms the membrane delineating the droplet. Lowering the pH of the bathing solution, that virtually mimicks the vacuolar environment, from an almost neutral level to values below pH 7, induced a significant but reversible decrease in channel activity, whereas channel conductance remained largely unaffected. Acidification (pH 5) on both sides of the membrane decreased open probability from a maximum of 80% to less than 20%. Decreasing pH at the cytosolic side inhibited channel activity cooperatively with a slope of 2.05 and a pK _a 6.56. In addition, low pH at the vacuolar face shifted the activating voltage into a positive direction by almost 100 mV. This is the first report about an effect of extraplasmatic pH on gating of a maxi-K channel. It is suggested that the Chara maxi-K channel possesses an S4-like voltage sensor and negatively charged residues in neighboring transmembrane domains whose S4-stabilizing function may be altered by protonation. It was previously shown that gating kinetics of this channel respond to cytosolic Ca²⁺ (Laver & Walker, 1991). With regard to natural conditions, pH effects are discussed as contributing mainly to channel regulation at the vacuolar membrane face, whereas at the cytosolic side Ca²⁺ affects the channel. An attempt was made to ascribe structural mechanisms to different states of a presumptive gating reaction scheme. Received: 8 May 1998/Revised: 18 September 1998     

The Estimation of Rapid Rate Constants from Current-Amplitude Frequency Distributions of Single-Channel Recordings

A method is described for estimating rapid rate constants from the distributions of current amplitude observed in single-channel electrical recordings. It has the advantages over previous, similar approaches that it can accommodate both multistate kinetic models and adjustable filtering of the data using an 8-pole Bessel filter. The method is conceptually straightforward: the observed distributions of current amplitude are compared with theoretical distributions derived by combining several simplifying assumptions about the underlying stochastic process with a model of the filter and electrical noise. Parameters are estimated by approximate maximum likelihood. The method was used successfully to estimate rate constants for both a simple two-state kinetic model (the transitions between open and closed states during the rapid gating of an outward-rectifying K⁺-selective channel in the plasma membrane of Acetabularia) and a complex multistate kinetic model (the blockade of the maxi cation channel in the plasma membrane of rye roots by verapamil). For the two-state model, parameters were estimated well, provided that they were not too fast or too slow in relation to the sampling rate. In the three-state model the precision of estimates depended in a complex way on the values of all rate parameters in the model. Received: 4 October 1996/Revised: 2 September 1997     

Sequence Analyses and Phylogenetic Characterization of the ZIP Family of Metal Ion Transport Proteins

Several novel but similar heavy metal ion transporters, Zrt1, Zrt2, Zip1-4 and Irt1, have recently been characterized. Zrt1, Zrt2 and Zip1-4 are probably zinc transporters in Saccharomyces cerevisiae and Arabidopsis thaliana whereas Irt1 appears to play a role in iron uptake in A. thaliana. The family of proteins including these functionally characterized transporters has been designated the Zrt- and Irt-related protein (ZIP) family. In this report, ZIP family proteins in the current databases were identified and multiply aligned, and a phylogenetic tree for the family was constructed. A family specific signature sequence was derived, and the available sequences were analyzed for residues of potential functional significance. A fully conserved intramembranous histidyl residue, present within a putative amphipathic, α-helical, transmembrane spanning segment, was identified which may serve as a part of an intrachannel heavy metal ion binding site. The occurrence of a proposed extramembranal metal binding motif (H X H X H) was examined in order to evaluate its potential functional significance for various members of the family. The computational analyses reported in this topical review should serve as a guide to future researchers interested in the structure-function relationships of ZIP family proteins. Received: 31 March 1997/Revised: 14 May 1998     

Regulation of Metabolite Flux through Voltage-Gating of VDAC Channels

The mitochondrial outer membrane channel, VDAC, is thought to serve as the major permeability pathway for metabolite flux between the cytoplasm and mitochondria. The permeability of VDAC to citrate, succinate, and phosphate was studied in channels reconstituted into planar phospholipid membranes. All ions showed large changes in permeability depending on whether the channel was in the open or in the low conductance, ``closed' state, with the closed state always more cation selective. This was especially true for the divalent and trivalent anions. Additionally, the anion flux when the voltage was zero was shown to decrease to 5–11% of the open state flux depending on the anion studied. These results give the first rigorous examination of the ability of metabolites to permeate through VDAC channels and indicate that these channels can control the flux of these ions through the outer membrane. This lends more evidence to the growing body of experiments that suggest that the outer mitochondrial membrane has a much more important role in controlling mitochondrial activity than has been thought historically. Received: 4 November 1996/Revised: 8 January 1997     

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     

Characteristics of a Low Affinity Passive Ca2+ Influx Component in Rat Parotid Gland Basolateral Plasma Membrane Vesicles

We have previously reported the presence of two Ca²⁺ influx components with relatively high (K_Ca= 152 ± 79 μm) and low (K_Ca= 2.4 ± 0.9 mm) affinities for Ca²⁺ in internal Ca²⁺ pool-depleted rat parotid acinar cells [Chauthaiwale et al. (1996) Pfluegers Arch. 432:105–111]. We have also reported the presence of a high affinity Ca²⁺ influx component with K_Ca= 279 ± 43 μm in rat parotid gland basolateral plasma membrane vesicles (BLMV). [Lockwich, Kim & Ambudkar (1994) J. Membrane Biol. 141:289–296]. The present studies show that a low affinity Ca²⁺ influx component is also present in BLMV with K_Ca= 2.3 ± 0.41 mm (V_max= 16.36 ± 4.11 nmoles of Ca²⁺/mg protein/min). Our data demonstrate that this low affinity component is similar to the low affinity Ca²⁺ influx component that is activated by internal Ca²⁺ store depletion in dispersed parotid gland acini by the following criteria: (i) similar K_Ca for calcium flux, (ii) similar IC₅₀ for inhibition by Ni²⁺ and Zn²⁺; (iii) increase in K_Ca at high external K⁺, (iv) similar effects of external pH. The high affinity Ca²⁺ influx in cells is different from the low affinity Ca²⁺ influx component cells in its sensitivity to pH, KCl, Zn²⁺ and Ni²⁺. The low and high affinity Ca²⁺ influx components in BLMV can also be distinguished from each other based on the effects of Zn²⁺, Ni²⁺, KCl, and dicyclohexylcarbodiimide. In aggregate, these data demonstrate the presence of a low affinity passive Ca²⁺ influx pathway in BLMV which displays characteristics similar to the low affinity Ca²⁺ influx component detected in parotid acinar cells following internal Ca²⁺ store depletion. Received: 19 March 1997/Revised: 25 November 1997     

Electrocoupling of Ion Transporters in Plants: Interaction with Internal Ion Concentrations

There are five major electroenzymes in the plasmalemma of plant cells: a driving electrogenic pump, inward and outward rectifying K⁺ channels, a Cl⁻-2H⁺ symporter, and Cl⁻-channels. It has been demonstrated previously (Gradmann, Blatt & Thiel 1993, J. Membrane Biol. 136:327–332) how voltage-gating of these electroenzymes causes oscillations of the transmembrane voltage (V) at constant substrate concentrations. The purpose of this study is to examine the interaction of the same transporter ensemble with cytoplasmic concentrations of K⁺ and Cl⁻. The former model system has been extended to account for changing internal concentrations. Constant-field theory has been applied to describe the influence of ion concentrations on current-voltage relationships of the active channels. The extended model is investigated using a reference set of model parameters. In this configuration, the system converges to stable slow oscillations with intrinsic changes in cytoplasmic K⁺ and Cl⁻ concentrations. These slow oscillations reflect alternation between a state of salt uptake at steady negative values of V and a state of net salt loss at rapidly oscillating V, the latter being analogous to the previously reported oscillations. By switching off either concentration changes or gating, it is demonstrated that the fast oscillations are mostly due to the gating properties of the Cl⁻ channel, whereas the slow oscillations are controlled by the effect of the Cl⁻ concentration on the current. The sensitivity of output results y (e.g., frequency of oscillations) to changes of the model parameters x (e.g., maximum Cl⁻ conductance) has been investigated for the reference system. Further examples are presented where some larger changes of specific model parameters cause fundamentally different behavior, e.g., convergence towards a stable state of only the fast oscillations without intrinsic concentration changes, or to a steady-state without any oscillations. The main and general result of this study is that the osmotic status of a plant cell is stabilized by the ensemble of familiar electroenzymes through oscillatory interactions with the internal concentrations of the most abundant ions. This convergent behavior of the stand-alone system is an important prerequisite for osmotic regulation by means of other physiological mechanisms, like second messengers and gating modifiers. Received: 23 February/Revised: 16 July 1998     

Potentiation by Isoproterenol on Carbachol-induced K+ and Cl− Currents and Fluid Secretion in Rat Parotid

Isoproterenol (IPR) and 8-(4-chlorophenylthio)-cyclic AMP (cpt-cAMP) enhanced carbachol (CCh)-induced fluid secretion from rat parotid glands, but had no effect by themselves. The enhancement by IPR was blocked by propranolol. In dispersed parotid acinar cells, IPR and cpt-cAMP potentiated CCh-induced K⁺ and Cl⁻ currents (I _K and I _Cl). IPR at the concentration of 0.1 μm significantly potentiated the CCh-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺] _i ), but 1 mm cpt-cAMP did not. The incidence of the potentiation by IPR in CCh-induced Mn²⁺ entry was 31% and that by cpt-cAMP was 21%. The potentiation by IPR in the ionic currents and the [Ca²⁺] _i was suppressed by propranolol. These results suggest that the CCh-induced fluid secretion from rat parotid glands is enhanced by IPR through the potentiation of I _K and I _Cl mainly by the increased cyclic AMP level and partially by the potentiated Ca²⁺ influx and [Ca²⁺] _i increase, and that IPR is more effective than cpt-cAMP in the enhancement of the CCh-induced [Ca²⁺] _i increase. Received: 6 October 1997/Revised: 16 April 1998     

Dinoflagellate nuclear SSU rRNA phylogeny suggests multiple plastid losses and replacements

Dinoflagellates are a trophically diverse group of protists with photosynthetic and non-photosynthetic members that appears to incorporate and lose endosymbionts relatively easily. To trace the gain and loss of plastids in dinoflagellates, we have sequenced the nuclear small subunit rRNA gene of 28 photosynthetic and four non-photosynthetic species, and produced phylogenetic trees with a total of 81 dinoflagellate sequences. Patterns of plastid gain, loss, and replacement were plotted onto this phylogeny. With the exception of the apparently early-diverging Syndiniales and Noctilucales, all non-photosynthetic dinoflagellates are very likely to have had photosynthetic ancestors with peridinin-containing plastids. The same is true for all dinoflagellates with plastids other than the peridinin-containing plastid: their ancestors have replaced one type of plastid for another, in some cases most likely through a non-photosynthetic intermediate. Eight independent instances of plastid loss and three of replacement can be inferred from existing data, but as more non-photosynthetic lineages are characterized these numbers will surely grow. Received: 25 September 2000 / Accepted: 24 April 2001     

Regulation of Cation-selective Channels in Liver Cells

In liver cells, cation-selective channels are permeable to Ca²⁺ and have been postulated to represent a pathway for receptor-mediated Ca²⁺ influx. This study examines the mechanisms involved in the regulation of these channels in a model liver cell line. Using patch-clamp recording techniques, it is shown that channel open probability is a saturable function of cytosolic [Ca²⁺], with half-maximal opening at 660 nm. By contrast, channel opening is not affected by membrane voltage or cytosolic pH. In intact cells, reduction of cytosolic [Cl⁻], a physiological response to Ca²⁺-mobilizing hormones and cell swelling, is also associated with an increase in channel opening. Finally, channel opening is inhibited by intracellular ATP through a mechanism that does not involve ATP hydrolysis. These findings suggest that opening of cation-selective channels is coupled to the metabolic state of the cell and provides a positive feedback mechanism for regulation of receptor-mediated Na⁺ and Ca²⁺ influx. Received: 8 October 1996     

Three Types of Membrane Excitations in the Marine Diatom Coscinodiscus wailesii

Three types of electrical excitation have been investigated in the marine diatom Coscinodiscus wailesii. I: Depolarization-triggered, transient Cl⁻ conductance, G _Cl (t), followed by a transient, voltage-gated K⁺ conductance, G _K , with an active state a and two inactive states i ₁ and i ₂ in series (a-i ₁-i ₂). II: Similar G _Cl (t) as in Type-I but triggered by hyperpolarization; a subsequent increase of G _K in this type is indicated but not analyzed in detail. III: Hyperpolarization-induced transient of a voltage-gated activity of an electrogenic pump (i ₂-a-i ₂), followed by G _Cl (t) as in Type-II excitations. Type-III with pump gating is novel as such. G _Cl (t) in all types seems to reflect the mechanism of InsP⁻ ₃ and Ca²⁺-mediated G _Cl (t) in the action potential in Chara (Biskup et al., 1999). The nonlinear current-voltage-time relationships of Type-I and Type-III excitations have been recorded under voltage-clamp using single saw-tooth command voltages (voltage range: −200 to +50 mV, typical slope: ±1 Vs⁻¹). Fits of the corresponding models to the experimental data provided numerical values of the model parameters. The statistical significance of these solutions is investigated. We suggest that the original function of electrical excitability of biological membranes is related to osmoregulation which has persisted through evolution in plants, whereas the familiar and osmotically neutral action potentials in animals have evolved later towards the novel function of rapid transmission of information over long distances. Received: 2 December 1999/Revised: 3 March 2000