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

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

External Protons Enhance the Activity of the Hyperpolarization-activated K Channels in Guard Cell Protoplasts of Vicia faba

Hyperpolarization-activated K channels (K _H channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell mode, we characterized the effect of varying the external pH between 4.4–8.1 on the activity of the K _H channels in isolated guard cell protoplasts from Vicia faba leaves. Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the K _H channels by 30–150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by ∼60% between pH 8.1 and 4.4 with an apparent pK _a of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing of these charges was ∼30 ? and their apparent dissociation constant was 10^−4.6. The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence of all four transition rate constants. Received: 26 April 1996/Revised: 29 June 1996     

Characterization of a Chloride-Selective Channel from Rough Endoplasmic Reticulum Membranes of Rat Hepatocytes: Evidence for a Block by Phosphate

We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with a mean conductance of 164 ± 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl⁻ than for K⁺, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with an open probability value ranging from 0.9 at −20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability sequence for test halides determined from reversal potentials was Br⁻ > Cl⁻ > I⁻≈ F⁻. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br⁻ and Cl⁻ have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel activity was not affected by 50 μm DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl⁻. Our results suggest that channel block involves the interaction of HPO²⁻ ₄ with a site located at 70% of the membrane span. Received: 10 January 1997/Revised: 29 May 1997     

Apical Nonspecific Cation Channels in Everted Collecting Tubules of Potassium-Adapted Ambystoma

We observed intermediate conductance channels in approximately 20% of successful patch-clamp seals made on collecting tubules dissected from Ambystoma adapted to 50 mm potassium. These channels were rarely observed in collecting tubules taken from animals which were maintained in tap water. Potassium-adaptation either leads to an increase in the number of channels present or activates quiescent channels. In cell-attached patches the conductance averaged 30.3 ± 2.4 (9) pS. Since replacement of the chloride in the patch pipette with gluconate did not change the conductance, the channel carries cations, not anions. Notably, channel activity was observed at both positive and negative pipette voltages. When the pipette was voltage clamped at 0 mV or positive voltages, the current was directed inward, consistent with the movement of sodium into the cell. The pipette voltage at which the polarity of the current reversed (movement of potassium into the pipette) was −29.6 ± 6.5(9) mV. Open probability at 0 mV pipette voltage was 0.08 ± 0.03 and was unaffected when the apical membrane was exposed to either 2 × 10⁻⁶ or 2 × 10⁻⁵ m of amiloride. Exposure of the basolateral surface of the tubule to a saline containing 15 mm potassium caused a significant increase (P less than 0.001) in the open probability of these channels to 0.139 ± 0.002 without affecting the conductance of the apical channel. These data illustrate the presence of an intermediate conductance, poorly selective, amiloride-insensitive cation channel in native vertebrate collecting tubule. We postulate that, at least in amphibia, this channel may be used to secrete potassium. Received: 14 January 2000/Revised: 16 June 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     

The Transient Outward Current of Isolated Bovine Adrenal Zona Fasciculata Cells: Comparison between Standard and Perforated Patch Recording Methods

Voltage-clamp experiments were performed on single bovine adrenal fasciculata cells in short-term primary culture using either standard (broken membrane) or perforated whole-cell patch clamp recording. The membrane current measured with the perforated method was dominated by a very stable transient outward current. By contrast, the transient outward current recorded using the standard method was unstable. The reversal potential of the transient outward current varied linearly with the logarithm of [K⁺] _e with a slope of 47 mV per decade. The onset of activation was sigmoidal and was fitted with a power function where n= 4. Time constants ranged from 1 to 4 msec with a maximum at −25 mV. The steady-state activation curve spanned the voltage range −50 to +80 mV without reaching a clear maximum. During a pulse, the current decayed in a biexponential manner. Time constants τ₁ and τ₂ were voltage-dependent and ranged from 50 to 200 msec respectively for a voltage step at +50 mV. The steady-state inactivation was dependent on the conditioning pulse duration. Using short conditioning pulses (1.2 sec), the curve which spanned the voltage range −40 to −20 mV, was 15 mV more positive than that obtained with longer conditioning pulses (60 sec). Time constants of this ``very slow inactivation' process (τ<sub>vs</sub>) determined for voltage steps at −60 and −50 mV were 15 and 10 sec respectively. A ``facilitation process' of the peak current was observed when the duration or the amplitude of conditioning pulses were increased in the voltage range −100 to −50 mV. Recovery from inactivation followed a biexponential time course which seemed a mixture of both inactivation processes. In some experimental conditions, isolated cells were able to produce overshooting action potentials. These results are discussed in relation with the membrane electrogenesis of this cell type. Received: 14 November 1994/Revised: 24 October 1995     

The Effect of an Extracellular Mucilage on the Response to Osmotic Shock in the Charophyte Alga Lamprothamnium papulosum

We have used current/voltage (I/V) analysis to investigate the role played by extracellular mucilage in the cellular response to osmotic shock in Lamprothamnium papulosum. Cells lacking extracellular mucilage originated in a brackish environment (1/3 seawater). These were compared, first with cells coated with thick (∼50 μm) extracellular mucilage, collected from a marine lake, and second, with equivalent mucilaginous marine cells, treated with heparinase enzyme to disrupt the mucilage layer. Histochemical stains Toluidine Blue and Alcian Blue at low pH identified the major component of the extracellular mucilage as sulfated polysaccharides. Treating mucilage with heparinase removed the capacity for staining with cationic dyes at low pH, although the mucilage was not removed, and remained as a substantial unstirred layer. Cells lacking mucilage responded to hypotonic shock with depolarization (by ∼95 mV), cessation of cyclosis, due to transient opening of Ca²⁺ channels, and opening of Ca²⁺-activated Cl⁻ channels and K⁺ channels. Cell conductance transiently increased tenfold, but after 60 min was restored to the conductance prior to hypotonic shock. Mucilaginous cells depolarized by a small amount (∼18 mV), but Ca²⁺ channels failed to open in large enough numbers for cyclosis to cease. Likewise most Ca²⁺-activated Cl⁻ channels failed to open and conductance increased only ∼1.2-fold above the prehypotonic level. After 60 min conductance was less than the conductance prior to hypotonic shock. Heparinased mucilaginous cells recovered several aspects of the hypotonic response in cells lacking mucilage. These cells depolarized (by ∼103 mV); cyclosis ceased, indicating that Ca²⁺ channels had opened, and conductance increased to ∼4 times the value prior to hypotonic shock, indicating that Ca²⁺-activated Cl⁻ channels opened. However, after 60 min, these cells had neither restored membrane potential (and remained at positive values), nor decreased their conductance. It was not possible to determine whether K⁺ channels had opened. The heparinased cells recovered the normal hypotonic response of mucilaginous cells when heparinase was washed out. Apical seawater cells, which lacked mucilage, were unaffected by heparinase treatment. The results demonstrate that the presence of extracellular sulfated polysaccharide mucilage impacts upon the electrophysiology of the response to osmotic shock in Lamprothamnium cells. The role of such sulfated mucilages in marine algae and animal cells is compared and discussed. Received: 24 March 1998/Revised: 28 April 1999     

K+-Sensitive Gating of the K+ Outward Rectifier in Vicia Guard Cells

The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K⁺ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K⁺ current was monitored under voltage clamp in 0.1–30 mm K⁺ and in equivalent concentrations of Rb⁺, Cs⁺ and Na⁺. From a conditioning voltage of −200 mV, clamp steps to voltages between −150 and +50 mV in 0.1 mm K⁺ activated current through outward-rectifying K⁺ channels (I _{K, out}) at the plasma membrane in a voltage-dependent fashion. Increasing [K⁺] _o shifted the voltage-sensitivity of I _{K, out} in parallel with the equilibrium potential for K⁺ across the membrane. A similar effect of [K⁺] _o was evident in the kinetics of I _{K, out} activation and deactivation, as well as the steady-state conductance- (g _K ⁻) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near −130 mV in 0.1 mm K⁺, −80 mV in 1.0 mm K⁺, and −20 mV in 10 mm K⁺. Similar data were obtained with Rb⁺ and Cs⁺, but not with Na⁺, consistent with the relative efficacy of cation binding under equilibrium conditions (K⁺≥ Rb⁺ > Cs⁺ > > Na⁺). Changing Ca²⁺ or Mg²⁺ concentrations outside between 0.1 and 10 mm was without effect on the voltage-dependence of g _K or on I _{K, out} activation kinetics, although 10 mm [Ca²⁺] _o accelerated current deactivation at voltages negative of −75 mV. At any one voltage, increasing [K⁺] _o suppressed g _K completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K⁺ was sensitive to voltage, varying from 0.5 to 20 mm with clamp voltages near −100 to 0 mV, respectively. These, and additional data indicate that extracellular K⁺ acts as a ligand and alters the voltage-dependence of I _{K, out} gating; the results implicate K⁺-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of two K⁺ ions outside affects the distribution between closed states of the channel. Received: 27 November 1996/Revised: 4 March 1997     

Two Different Conductances Contribute to the Anion Currents in Coffea arabica Protoplasts

The anion conductance of the plasma membrane of Coffea arabica protoplasts was isolated and characterized using the whole-cell patch clamp technique. Voltage pulse protocols revealed two components: a voltage-gated conductance (G _s ) and a voltage-independent one (G _l ). G _s is activated upon depolarization (e-fold activation every +36 mV) with time constants of 1 sec and 5 sec at all potentials. G _l and G _s also differ by their kinetic and biophysical properties. In bi-ionic conditions the current associated with G _s shows strong outward rectification and its permeability sequence is F⁻ > NO₃ ⁻ > Cl⁻. In the same conditions the current associated with G _l does not rectify and its permeability sequence is F⁻≫ NO₃ ⁻= Cl⁻. Furthermore, at potentials over +50 mV G _s , but not G _l , increases with a time constant of several minutes. Finally the gating of G _s is affected by stretch of the membrane, which leads to an increased activation and a reduced voltage sensitivity. Anion conductances similar to the ones described here have been found in many plant preparations but G _l -type components have been generally interpreted as the background activation of the slow voltage-gated channels (corresponding to G _s ). We show that in coffee protoplasts G _l and G _s are kinetically and biophysically distinct, suggesting that they correspond to two different molecular entities. Received: 25 November 1996/Revised: 9 April 1997     

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

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

The Sensitivity of the Human Kv1.3 (hKv1.3) Lymphocyte K+ Channel to Regulation by PKA and PKC is Partially Lost in HEK 293 Host Cells

cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (Δ459-523) form that lacks the putative PKA Ser⁴⁶⁸ phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering was observed in the case of the truncated channel. Some staining within the cell cytoplasm was found in both instances, suggesting an active process of biosynthesis. Analyses of the K⁺ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding potential (HP) of −80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to −40 mV and the current amplitude increased in a voltage-dependent manner. The parameters of activation were −5.7 and −9.9 mV (slope factor) and −35 mV (half activation, V _0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope factor) and −17.3 and −39.0 mV (V _0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials ranging from −30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at −30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mm KCl was 1.3 ± 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels were inhibited by TEA, 4-AP, Ni²⁺ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 ± 7.3% and 38.7 ± 9.4% inhibition of the full-length and the truncated channels, respectively. 8-BrcAMP induced a 39.4 ± 5.4% inhibition of the full-length channel but had no effect (8.6 ± 8.3%) on the truncated channel. Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel was partially sensitive to PKA and PKC. Cotransfecting the Kvβ1.2 subunit resulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kvβ2 subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics when transfected in the Kvβ-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced. Received: 18 June 1997/Revised: 7 October 1997     

Temperature Sensitivity of the Tonoplast Ca2+-Activated K+ Channel in Chara: The Influence of Reversing the Sign of Membrane Potential

A detailed temperature dependence study of a well-defined plant ion channel, the Ca²⁺-activated K⁺ channel of Chara corallina, was performed over the temperature range of their habitats, 5–36°C, at 1°C resolution. The temperature dependence of the channel unitary conductance at 50 mV shows discontinuities at 15 and 30°C. These temperatures limit the range within which ion diffusion is characterized by the lowest activation energy (E _a = 8.0 ± 1.6 kJ/mol) as compared to the regions below 15°C and above 30°C. Upon reversing membrane voltage polarity from 50 to −50 mV the pattern of temperature dependence switched from discontinuous to linear with E _a = 13.6 ± 0.5 kJ/mol. The temperature dependence of the effective number of open channels at 50 mV showed a decrease with increasing temperature, with a local minimum at 28°C. The mean open time exhibited a similar behavior. Changing the sign of membrane potential from 50 to −50 mV abolished the minima in both temperature dependencies. These data are discussed in the light of higher order phase transitions of the Characean membrane lipids and corresponding change in the lipid-protein interaction, and their modulation by transmembrane voltage. Received: 14 June 2000/Revised: 20 September 2000     

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     

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

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

Modeling the Current-Voltage Characteristics of Chara Membranes: I. The Effect of ATP Removal and Zero Turgor

We have obtained and modeled the electrical characteristics of the plasma membrane of Chara internodal cells: intact, without turgor and perfused with and without ATP. The cells were voltage and space-clamped to obtain the I/V (current-voltage) and G/V (conductance-voltage) profiles of the cell membrane. The intact cells yielded similar I/V characteristics with resting p.d.s of −221 ± 12 mV (cytoplasmic clamp, 5 cells) and −217 ± 12 mV (vacuolar clamp, 5 cells). The cut unperfused cells were depolarized at −169 ± 12 mV (7 cells) compared to the vacuole-clamped intact cells. The cells perfused with ATP fell into three groups: hyperpolarized group with resting p.d. −175 ± 12 mV (4 cells) and I/V profile similar to the intact and cut unperfused cells; depolarized group with resting p.d. of −107 ± 12 mV (6 cells) and I/V profiles close to linear; and excited cells with profiles showing a negative conductance region and resting p.d. at −59 ± 12 mV (5 cells). The cells perfused with medium containing no ATP showed upwardly concave I/V characteristics and resting p.d. at −81 ± 12 mV (6 cells). The I/V curves were modeled employing the ``Two-state' model for the H<sup>+</sup> pump (Hansen et al., 1981). The inward and outward rectifiers were fitted to exponential functions and combined with a linear background current. The excitation state in perfused cells was modeled by including an inward current, i <sub>excit</sub>, with p.d.-dependence described by a combination of hyperbolic tangent functions. An inward current, i <sub>no-ATP</sub>, with a smaller amplitude, but very similar p.d.-dependence was also included in the simulation of the I/V curves from cells without ATP. This approach avoided I/V curve subtraction. The modeling of the total I/V and G/V characteristics provided more information about the parameters of the ``Two-state' pump model, as well as more quantitative understanding of the interaction of the major transport systems in the plasmalemma in generation of the resting potential under a range of circumstances. ATP had little effect on nonpump currents except the excitation current; depolarization profoundly affected the pump characteristics. Received: 23 January/Revised: 10 October 1995     

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     

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     

Downregulation of Epithelial Sodium Channel (ENaC) by CFTR Co-expressed in Xenopus Oocytes is Independent of Cl− Conductance

Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel (ENaC) have been implicated in the elevated Na⁺ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR depends on the ability of CFTR to conduct Cl⁻ into the cell and is negligible when Cl⁻ flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na⁺ current (I _amil ) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to −60 mV, stimulating CFTR with 1 mm IBMX reduced I _amil by up to 80%, demonstrating that ENaC is inhibited when Cl⁻ is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of I _amil downregulation and the CFTR-mediated plasma membrane Cl⁻ conductance, suggesting a direct correlation. However, I _amil downregulation was not affected when Cl⁻ flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl⁻ reversal potential (67% ± 10% inhibition at −20 mV compared to 79% ± 4% at −60 mV) demonstrating that I _amil downregulation was independent of the amount of current flow through CFTR. Studies with the Ca²⁺-sensitive photoprotein aequorin showed that Ca²⁺ is not involved in I _amil downregulation by CFTR, although Ca²⁺ injection into the cytoplasm did inhibit I _amil . These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca²⁺ and is independent of the direction and magnitude of Cl⁻ transport across the plasma membrane. Received: 15 December 1998/Revised: 5 March 1999     

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

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