Whole-Cell Mechanosensitive Currents in Rat Ventricular Myocytes Activated by Direct Stimulation

Mechanosensitive channels may have a significant role in the development of cardiac arrhythmia following infarction, but the data on mechanical responses at the cellular level are limited. Mechanosensitivity is a ubiquitous property of cells, and although the structure of bacteriological mechanosensitive ion channels is becoming known by cloning, the structure and force transduction pathway in eukaryotes remains elusive. Isolated adult rat ventricular myocytes were voltage clamped and stimulated with a mechanical probe. The probe was set in sinusoidal motion (either in, or normal to, the plane of the cell membrane), and then slowly lowered onto the cell. The sinusoidal frequency was held constant at 1 Hz but the stimulation amplitude was increased and the probe gradually lowered until a mechanically sensitive whole cell current was seen, which usually followed several minutes of stimulation. The whole cell mechanosensitive current in rat cells had two components: (i) a brief large inward current spike current; (ii) a more sustained smaller inward current. The presence of the initial sharp inward current suggests that some structure within the cell either relaxes or is broken, exposing the mechanosensitive element(s) to stress. Metabolic changes induced by continued stress prior to the mechanosensitive response may weaken the elements that break producing the spike, or simple stress-induced fracture of the cytoskeleton itself may occur.     

Mechanosensitive Channels in the Cell Body of Chlamydomonas

Mechanosensitive channels appear ubiquitous but they have not been well characterized in cells directly responding to mechanical stimuli. Here, we identified tension-sensitive channel currents on the cell body of Chlamydomonas, a protist that shows a marked behavioral response to mechanical stimulation. When a negative pressure was applied to the cell body with a patch clamp electrode, single-ion-channel currents of 2.4 pA in amplitude were observed. The currents were inhibited by 10 μm gadolinium, a general blocker of mechanosensitive channels. The currents were most likely due to Ca²⁺ influxes because the current was absent in Ca²⁺-free solutions and the reversal potential was 98 mV positive to the resting potential. The distribution of channel-open times conformed to a single exponential component and that of closed times to two exponential components. This mechanosensitive channel was similar to the one found in the flagella in the following respects: both channels were inhibited by Gd³⁺ at 10 μm but not at 1 μm; both passed Ca²⁺ and Ba²⁺; their kinetic parameters for channel opening were similar. These observations raise the possibility that identical mechanosensitive channels may function both in the behavioral control through the mechanoreception by the flagella and in the regulation of cellular physiology in response to mechanical perturbation on the cell body. Received: 13 May 1998/Revised: 2 September 1998     

Mechanically Activated Currents in Chick Heart Cells

As predicted from stretch-induced changes of rate and rhythm in the heart, acutely isolated embryonic chick heart cells exhibit whole-cell mechanosensitive currents. These currents were evoked by pressing on cells with a fire polished micropipette and measured through a perforated patch using a second pipette. The currents were carried by Na⁺ and K⁺ but not Cl⁻, and were independent of external Ca²⁺. The currents had linear I/V curves reversing at −16 mV and were completely blocked by Gd³⁺≥ 30 μm and Grammostola spatulata venom at a dilution of 1:1000. Approximately 20% of cells showed time dependent inactivation. In contrast to direct mechanical stimulation, hypotonic volume stress produced an increase in conductance for anions rather than cations—the two stimuli are not equivalent. The cells had two types of stretch-activated ion channels (SACs): a 21 pS nonspecific cation-selective reversing at −2 mV and a 90 pS K⁺ selective reversing at −70 mV in normal saline. The activity of SACs was strongly correlated with the presence of whole-cell currents. Both the whole-cell currents and SACs were blocked by Gd³⁺ and by Grammostola spatulata spider venom. Mechanical stimulation of spontaneously active cells increased the beating rate and this effect was blocked by Gd³⁺. We conclude that physiologically active mechanosensitive currents arise from stretch activated ion channels. Received: 8 April 1996/Revised: 8 August 1996     

Mechanically Induced Calcium Movements in Astrocytes, Bovine Aortic Endothelial Cells and C6 Glioma Cells

Forces applied to resting primary astrocytes, bovine aortic endothelial cells and C6 glioma cells with collagen-coated magnetite particles produce a fast transient change of intracellular Ca²⁺. It peaks in the micromolar range as measured by Fura-2. This mechanical response adapts within seconds so that repeated stimulation causes smaller responses requiring >10 min for recovery. When cytoplasmic Ca²⁺ is high after treating with ATP, cyclopiazonic acid and thapsigargin, stimulation causes a transient decrease in Ca²⁺. In these three cell types, no influx of ions is required for Ca²⁺ elevation showing the response is not caused by activation of plasmalemmal mechanosensitive channels. Approximately half the cells tested showed similar behavior, while the other half, such as fibroblasts, required extracellular Ca²⁺. The Ca²⁺ response is not temperature sensitive suggesting the possible involvement of intracellular mechanosensitive channels. We tested a number of second messenger reagents and were only able to block the response in BAECs, but not C6 glioma cells, with Xestospongin C, a blocker of IP₃-activated channels. Despite the lack of a causal involvement of plasmalemmal mechanosensitive channels, mechanical stimulation immediately activates a persistent Mn²⁺ influx pathway. This Mn²⁺ pathway may be mechanosensitive channels, Ca²⁺-activated cation channels or depletion-activated Ca²⁺ channels. Received: 7 July 1999/Revised: 12 November 1999     

Partners in Protection: Interdependence of Cytoskeleton and Plasma Membrane in Adaptations to Applied Forces

In mechanically active environments mammalian cells must cope with potentially injurious forces to survive, but the most proximal mechanosensors are largely unknown. How mechanoprotective responses to applied forces are generated and regulated is still a mystery. We consider recent evidence that suggests cellular mechanoprotective adaptations involve a coordinated remodeling of the cell membrane and the associated cytoskeleton. The plasma membrane ``protects' the cytoskeleton by maintenance of intracellular ionic balance and can modulate force-induced cytoskeletal rearrangements by stretch-activated (e.g., Ca²⁺) ion channels and mechanosensitive enzymes (e.g., Phospholipase A₂ and Phospholipase C). Conversely, the cytoskeleton protects the plasma membrane by providing structural support, reinforcement of the cortical framework at sites of force application, modulation of mechanosensitive ion channels and by potentially contributing to the membrane resealing process after mechanical rupture. We suggest that the plasma membrane and the cytoskeleton are partners in the cytoprotective response to physical forces. Received: 8 September 1999/Revised: 15 December 1999     

Involvement of Methionine Residues in the Fast Inactivation Mechanism of the Sodium Current from Toad Skeletal Muscle Fibers

The role of methionine residues on the fast inactivation of the sodium channel from toad skeletal muscle fibers was studied with the mild oxidant chloramine-T (CT). Isolated segments of fibers were voltage clamped in a triple Vaseline? gap chamber. Sodium current was isolated by replacing potassium ions by tetramethylammonium ions in the external and internal solutions. Externally applied chloramine-CT was found to render noninactivating a large fraction of sodium channels and to slow down the fast inactivation mechanism of the remainder fraction of inactivatable channels. The action of CT appeared to proceed first by slowing and then removing the fast inactivation mechanism. The voltage dependence of the steady-state inactivation of the inactivatable CT-treated currents was shifted +10 mV. CT also had a blocking effect on the sodium current, but was without effect on the activation mechanism. The effects of CT were time and concentration dependent and irreversible. The use of high CT concentrations and/or long exposure times was found to be deleterious to the fiber. This side effect precluded the complete removal of fast inactivation. The effects of CT on the fast inactivation of the sodium current can be explained assuming that at least two methionine residues are critically involved in the mechanism underlying this process. Received: 10 November 1998/Revised: 4 January 1999     

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     

Voltage-independent Adaptation of Mechanosensitive Channels in Escherichia coli Protoplasts

Mechanosensitive (MS) ion channels, with 560 pS conductance, opened transiently by rapid application of suction pulses to patches of E. coli protoplast membrane. The adaptation phase of the response was voltage-independent. Application of strong suction pulses, which were sufficient to cause saturation of the MS current, did not abolish the adaptation. Multiple-pulse experimental protocols revealed that once MS channels had fully adapted, they could be reactivated by a second suction pulse of similar amplitude, providing the time between pulses was long enough and suction had been released between pulses. Limited proteolysis (0.2 mg/ml pronase applied to the cytoplasmic side of the membrane patch) reduced the number of open channels without affecting the adaptation. Exposing patches to higher levels of pronase (1 mg/ml) removed responsiveness of the channel to suction and abolished adaptation consistent with disruption of the tension transmission mechanism responsible for activating the MS channel. Based on these data we discuss a mechanism for mechanosensitivity mediated by a cytoplasmic domain of the MS channel molecule or associated protein. Received: 29 January 1998/Revised: 16 April 1998     

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     

External nickel blocks human Kv1.5 channels stably expressed in CHO cells

We have investigated the actions of Nickel (Ni²⁺) on a human cardiac potassium channel (hKv1.5), the main component of human atrial ultra-rapid delayed rectifier current, stably expressed in Chinese hamster ovary cell line using the whole-cell voltage-clamp technique. External Ni²⁺ reversibly decreased the amplitude of the current in a concentration-dependent manner. The concentration for half-maximum inhibition of the current at +50 mV was 568 μm. The activation, deactivation, reactivation kinetics of the current were not affected by Ni²⁺. Block was not voltage-dependent but frequency-dependent block was apparent. The extent of channel block during the first pulse increased when the duration of exposure to Ni²⁺, prior to channel activation, was prolonged indicating that Ni²⁺ interacted with hKv1.5 in the closed state. The percentage of current remaining in presence of Ni²⁺ decreased steeply over the range of steady-state channel inactivation, consistent with an enhanced block with increased inactivation. This suggests that Ni²⁺ preferentially blocks nonconducting hKv1.5 channels, either in the resting or inactivated state in a concentration-dependent manner. The data indicate that the mechanisms of hKv1.5 channel inhibition by Ni²⁺ are distinct from those of other K⁺ channels. Received: 12 October 2000/Revised: 14 May 2001     

N-type Inactivation in the Mammalian Shaker K+ Channel Kv1.4

Mammalian voltage-gated K⁺ channels are oligomeric proteins, some of which may be composed in vivo of subunits derived from several similar genes. We have studied N-type inactivation in the rapidly inactivating Kv1.4 channel and, in specific, heteromultimers of this gene product with Kv1.5 noninactivating subunits. Heteromultimeric channels were analyzed for the stoichiometry of Kv1.4:Kv1.5 subunits by observing shifts in the midpoints of steady-state availability from that of homomultimeric channels. This analysis was employed to examine inactivation of heteromultimeric channels expressed in Xenopus oocytes using two model systems: by expression of a Kv1.4–Kv1.5 tandem fusion construct and by coexpression of native Kv1.4 and Kv1.5 channels across a wide relative concentration range of microinjected mRNA. Additionally, inactivation was examined in coexpression experiments of N-terminal deletion mutants of Kv1.4. We found that (i) a single inactivating subunit conferred inactivation in all hetero-multimers studied; (ii) the rate of inactivation could not be distinguished in channels containing two inactivating subunits from those containing one inactivating subunit; and (iii) large deletions in the linker region between the N-terminal inactivation region and the first membrane-spanning domain had no effect on the rate of inactivation. These data confirm the importance of the proximal N-terminal region in the inactivation of mammalian Kv1.4 channels, and suggest that the inactivation particle remains in close proximity to the permeation pathway even when the channel is in the open state. Received: 24 August 1995/Revised: 7 February 1996     

Enhanced Closed-state Inactivation in a Mutant Shaker K+ Channel

Many mutations that shift the voltage dependence of activation in Shaker channels cause a parallel shift of inactivation. The I2 mutation (L382I in the Shaker B sequence) is an exception, causing a 45 mV activation shift with only a 9 mV shift of inactivation midpoint relative to the wildtype (WT) channel. We compare the behavior of WT and I2 Shaker 29-4 channels in macropatch recordings from Xenopus oocytes. The behavior of WT channels can be described by both simple and detailed kinetic models which assume that inactivation proceeds only from the open state. The behavior of I2 channels requires that they inactivate from closed states as well, a property characteristic of voltage-gated sodium channels. A detailed ``multiple-state inactivation' model is presented that describes both activation and inactivation of I2 channels. The results are consistent with the view that residue L382 is associated with the receptor for the inactivation particles in Shaker channels. Received: 16 December 1996/Revised: 5 February 1997     

Effects of NS1608 on MaxiK Channels in Smooth Muscle Cells from Urinary Bladder

Using the patch-clamp technique, we have characterized membrane currents in single detrusor smooth muscle cells from rat and human urinary bladder. From the voltage- and Ca²⁺-dependence of the current as well as the single channel conductance we conclude that rat and human urinary bladder smooth muscle cells express MaxiK channels. In smooth muscle cells from rat urinary bladder we tested the action of NS1608 on current through these MaxiK channels. Application of 10 μm NS1608 increased the amplitude of the current and this increase could be explained by a shift in the activation voltage of the MaxiK channels ∼100 mV towards more negative potentials. Charybdotoxin as well as paxilline, well known blockers of MaxiK channels, were able to reduce current through MaxiK channels in our cell preparation. In addition, application of 10 μm NS1608 hyperpolarized the membrane potential of the investigated cells. This hyperpolarization could be antagonized by the application of paxilline. We conclude that application of NS1608 results in the opening of MaxiK channels under physiological conditions that leads to a hyperpolarization of the cells. This hyperpolarization in turn could relax urinary bladder smooth muscle cells. MaxiK channels in these cells could therefore play a role in directly controlling muscle tone by regulating the membrane potential. This opens up the possibility of MaxiK channels being targets for the treatment of urge incontinence. Received: 19 July/Revised: 20 September 1999     

Effect of Calcium and Calcium Channel Blockers on Transient Outward Current of F76 and D1 Neuronal Soma Membranes in the Subesophageal Ganglia of Helix aspersa

Twin-electrode voltage-clamp techniques were used to study the effect of calcium and calcium channel blockers on the transient outward current in isolated F76 and D1 neurones of Helix aspersa subesophageal ganglia in vitro (soma only preparation with no cell processes). On lowering extracellular Ca²⁺ concentration from 10 to 2 mm or removing extracellular calcium from the bathing medium, the threshold for this current shifted in a negative direction by 11.5 and 20 mV, respectively. On the other hand, increasing the extracellular Ca²⁺ concentration from 10 to 20 and to 40 mm shifted the steady-state inactivation curves in positive directions on the voltage axis by 7 and 15 mV, respectively. Upon application of calcium channel blockers, Co²⁺, La³⁺, Ni²⁺ and Cd²⁺, transient potassium current amplitude was reduced in a voltage-dependent manner, being more effective at voltages close to the threshold. The current was elicited even at a holding potential of −34 mV. The specific calcium channel blockers, amiloride and nifedipine did not shift the activation and steady-state inactivation curves and did not reduce the transient outward current amplitude. It was concluded that the transient outward current is not dependent on intracellular Ca²⁺ but that it is modulated by Ca²⁺ and di- and trivalent ions extracellularly. The effects of these ions are very unlikely to be due to a surface charge effect because the addition of La³⁺ (200 μm) completely reverses the shift in a hyperpolarizing direction when the extracellular Ca²⁺ concentration was reduced from 10 to 1 mm and additionally shifts the kinetics further still in a depolarizing direction. The responses seen here are consistent with a specific effect of di- and trivalent ions on the transient outward current channels leading to a modification of gating. Received: 30 March 1999/Revised: 5 October 1999     

Sea Anemone Toxin (ATX II) Modulation of Heart and Skeletal Muscle Sodium Channel α-Subunits Expressed in tsA201 Cells

We have expressed recombinant α-subunits of hH1 (human heart subtype 1), rSkM1 (rat skeletal muscle subtype 1) and hSkM1 (human skeletal muscle) sodium channels in human embryonic kidney cell line, namely the tsA201 cells and compared the effects of ATX II on these sodium channel subtypes. ATX II slows the inactivation phase of hH1 with little or no effect on activation. At intermediate concentrations of ATX II the time course of inactivation is biexponential due to the mixture of free (fast component, τ^fast _h ) and toxin-bound (slow component, τ^slow _h ) channels. The relative amplitude of τ^slow _h allows an estimate of the IC₅₀ values ∼11 nm. The slowing of inactivation in the presence of ATX II is consistent with destabilization of the inactivated state by toxin binding. Further evidence for this conclusion is: (i) The voltage-dependence of the current decay time constants (τ _h ) is lost or possibly reversed (time constants plateau or increase at more positive voltages in contrast to these of untreated channels). (ii) The single channel mean open times are increased by a factor of two in the presence of ATX II. (iii) The recovery from inactivation is faster in the presence of ATX II. Similar effects of ATX II on rSkM1 channel behavior occur, but only at higher concentrations of toxin (IC₅₀= 51 nm). The slowing of inactivation on hSkM1 is comparable to the one seen with rSkM1. A residual or window current appears in the presence of ATX II that is similar to that observed in channels containing mutations associated with some of the familial periodic paralyses. Received: 5 December 1995/Revised: 1 March 1996     

Serotonergic Agonists Inhibit Calcium-Activated Potassium and Voltage-Dependent Sodium Currents in Rat Taste Receptor Cells

Recently we reported that rat taste receptor cells respond to the neurotransmitter serotonin with an inhibition of a calcium-activated potassium current [17]. In the present study, this observation is confirmed and extended by studying the effects of an array of serotonergic agonists on membrane properties, calcium-activated potassium current, and voltage-dependent sodium current in taste receptor cells using the patch-clamp recording technique in the whole-cell configuration. Serotonergic inhibition of calcium-activated potassium current was mimicked by the agonists N-(3-trifluoromethylphenyl)piperazine and by (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Both produced reversible inhibition of K _Ca as well as significantly increasing the input resistance of the cell. The agonists 1-(1-naphthyl)piperazine and buspirone (both serotonin receptor 1A agonists) were similarly effective in reducing K _Ca . Outward current was unaffected by application of phenylbiguanide, a serotonin receptor 3 agonist, though current was affected by subsequent application of (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Two agonists—N-(3-trifluoromethylphenyl)piperazine and (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene—were also tested on voltage-dependent sodium currents; both were effective and reversible in reducing its magnitude at a variety of applied potentials. These data are consistent with the notion that serotonin effects in rat taste receptor cells are mediated by serotonin 1A receptors, though other receptor subtypes may be additionally expressed. Serotonin may affect the taste cell electrical properties during active stimulation in a paracrine fashion. Received: 10 May 1999/Revised: 27 September 1999     

Photofrin II Sensitized Modifications of Ion Transport Across the Plasma Membrane of an Epithelial Cell Line: II. Analysis at the Level of Membrane Patches

In the first part of this study, photofrin II sensitized membrane modifications of OK-cells were investigated at the level of macroscopic membrane currents. In this second part, the inside-out configuration of the patch-clamp technique is applied to analyze the phenomena at the microscopic level. It is shown that the characteristic single channel fluctuations of the electric current disappear after the start of illumination of membrane patches in the presence of photofrin II. This holds for all three types of ion channels investigated: the large-conductance Ca²⁺-dependent K⁺ channel (maxi-K_Ca), a K⁺ channel of small conductance (sK), and a stretch-activated nonselective cation channel (SA-cat). Part of the experiments show a transient activation of the channels (indicated by an increase of the probability in the open-channel state) before the channels are converted into a closed nonconductive state. Inactivation of all three channel types proceeds by a continuous reduction of their open probability, while the single channel conductance values are not affected. The process of photodynamically induced channel inactivation is followed by a pronounced increase of the leak conductance of the plasma membrane. The latter process — after light-induced initiation — is found to continue in the dark. The ionic pathways underlying the leak conductance also allow permeation of Ca²⁺ ions. The resulting Ca²⁺-flux may contribute to the photodynamically induced increase of the intracellular Ca²⁺ concentration observed in various cell lines. Received: 26 May 1998/Revised: 8 September 1998     

Polyamine Triggering of Exocytosis in Paramecium Involves an Extracellular Ca2+/(Polyvalent Cation)-Sensing Receptor, Subplasmalemmal Ca-Store Mobilization and Store-Operated Ca2+-Influx via Unspecific Cation Channels

The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca²⁺] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca²⁺ signals also occur when AED is applied in presence of the fast Ca²⁺ chelator, BAPTA. (ii) Extracellular La³⁺ application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca²⁺] _i transient (while injected La³⁺ causes a sustained fluorescence signal). (iii) Simply increasing [Ca²⁺] _o causes a similar rapid, short-lived [Ca²⁺] _i transient. All these phenomena, (i–iii), are compatible with activation of an extracellular ``Ca<sup>2+</sup>/(polyvalent cation)-sensing receptor' known from some higher eukaryotic systems, where this sensor (responding to Ca<sup>2+</sup>, La<sup>3+</sup> and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores (``alveolar sacs') are physically linked to the cell membrane and they can also be activated by the Ca²⁺ releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca²⁺ signal also in absence of Ca²⁺ _o we largely exclude a ``Ca<sup>2+</sup>-induced Ca<sup>2+</sup> release' (CICR) mechanism. Our finding of increased cortical Ca<sup>2+</sup> signals after store depletion and re-addition of extracellular Ca<sup>2+</sup> can be explained by a ``store-operated Ca²⁺ influx' (SOC), i.e., a Ca²⁺ influx superimposing store activation. AED stimulation in presence of Mn²⁺ _o causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOC-type Ca²⁺ influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca²⁺/polyamine-sensing receptor, (ii) release of Ca²⁺ from subplasmalemmal stores, (iii) and Ca²⁺ influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca²⁺] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca²⁺] microdomains (≤0.5 μm, ≤33 msec) upon stimulation. Received: 30 August 1999/Revised: 1 December 1999     

Electrophysiological Study of Chimeric Sodium Channels from Heart and Skeletal Muscle

The α-subunit cDNAs encoding voltage-sensitive sodium channels of human heart (hH1) and rat skeletal muscle (rSkM1) have been expressed in the tsA201 mammalian cell line, in which inactivation properties appear to be normal in contrast to Xenopus oocytes. A series of rSkM1/hH1 chimeric sodium channels has been evaluated to identify the domains of the α-subunits that are responsible for a set of electrophysiological differences between hH1 and rSkM1, namely, midpoints and slope factors of steady-state activation and inactivation, inactivation kinetics and recovery from inactivation kinetics and their voltage-dependence. The phenotype of chimeric channels in which each hH1 domain was successively introduced into a rSkM1 α-subunit framework confirmed the following conclusions. (i) The D4 and or/C-ter. are responsible for the slow inactivation of hH1 sodium channels. (ii) Concerning the other differences between rSkM1 and hH1: steady-state activation and inactivation, kinetics of recovery from inactivation, the phenotypes are determined probably by more than one domain of the α-subunit. Received: 20 January 1998/Revised: 19 March 1998