Physiological and structural properties of colchicine-treated chick skeletal muscle cells grown in tissue culture.

Chick muscle cells grown in tissue-culture medium containing colchicine developed into rounded cells called “myosacs.” Electron micrographs of myosacs were similar to untreated myofibers except that microtubules were absent and the contractile material was disorganized. Most of the electrophysiological characteristics of myosacs were similar to those of untreated myofibers. Thus, both cellular types had similar resting potentials, nonlinear current voltage curves, three types of action potentials (Na⁺, Ca²⁺, and Cl^? spikes), and acetylcholine sensitivity with “hot spots.” Both demonstrated contraction with electrical or chemical (acetylcholine, caffeine) stimuli. The one significant difference was that myosacs, unlike myofibers, were always isopotential throughout their intracellular space.     

Sodium-dependent plateau potentials in electrocytes of the electric fish <Emphasis Type="Italic">Gymnotus carapo</Emphasis>

The weakly electric fish Gymnotus carapo emits a triphasic electric organ discharge generated by muscle-derived electrocytes, which is modified by environmental and physiological factors. Two electrode current clamp recordings in an in vitro preparation showed that Gymnotus electrocytes fired repetitively and responded with plateau potentials when depolarized. This electrophysiological behavior has never been observed in electrocytes from related species. Two types of plateaus with different thresholds and amplitudes were evoked by depolarization when Na⁺-dependent currents were isolated in a K⁺- and Ca²⁺-free solution containing TEA and 4-AP. Two electrode voltage clamp recordings revealed a classical fast activating–inactivating Na⁺ current and two persistent Na⁺-dependent currents with voltage-dependencies consistent with the action potential (AP) and the two plateaus observed under current clamp, respectively. The three currents, the APs and the plateaus were reduced by TTX, and were absent in Na⁺-free solution. The different Na⁺-dependent currents in Gymnotus electrocytes may be targets for the modifications of the electric organ discharge mediated by environmental and physiological factors.     

A three-state model for inactivation of sodium permeability

The inactivation of Na⁺ permeability in single myelinated motor nerve fibres of Rana esculenta was investigated under voltage and current clamp conditions at 20°C in Ringer's solution and under blocked K⁺ currents. Development of inactivation and its recovery was described by two potential-dependent time constants: The smaller time constant followed the usual bell-shaped function of membrane potential, whereas the larger one was monotone-increasing with more negative potentials. Several three-state models for inactivation were investigated. The experiments could best be approximated by a model with two open and one closed state for inactivation following: open ? closed ? open. Rate constants were determined for all transitions shown from the voltage clamp experiments. The action potentials computed by means of the proposed model were in good agreement with those measured, both in Ringer's solution and under blocked K⁺ current conditions.     

Persistent inward currents in cultured Retzius cells of the medicinal leech

Current-clamp studies of cultured leech Retzius cells revealed inward rectification in the form of slow voltage sags in response to membrane hyperpolarization. Sag responses were eliminated in Na⁺-free saline and blocked by Cs⁺, but not Ba²⁺. Voltage clamp experiments revealed a Cs⁺-sensitive inward current activated by hyperpolarization negative to −70 mV. Cs⁺ decreased the frequency of spontaneous impulses in Retzius cells of intact ganglia. Plateau potentials were evoked in Retzius cells following block of Ca²⁺ influx with Ni²⁺ and suppression of K⁺ currents with internal tetraethylammonium. Plateau potentials continued to be expressed with Li⁺ as the charge carrier, but were eliminated when Na⁺ was replaced with N-methyl-d-glucamine. A persistent Na⁺ current with similar pharmacology that activated positive to −40 mV and reached its peak amplitude near −5 mV was identified in voltage-clamp experiments. Inactivation of the persistent Na⁺ current was slow and incomplete. The current was revealed by slow voltage ramps and persisted for the duration of 5-s voltage steps. Persistent Na⁺ current may underlie Na⁺-dependent bursting recorded in neurons of intact ganglia exposed to Ca²⁺-channel blockers. Accepted: 22 September 1998     

Appearance and maturation of voltage-dependent conductances in solitary spiking cells during retinal regeneration in the adult newt

Summary Electrical membrane properties of solitary spiking cells during newt (Cynops pyrrhogaster) retinal regeneration were studied with whole-cell patch-clamp methods in comparison with those in the normal retina.The membrane currents of normal spiking cells consisted of 5 components: inward Na⁺ and Ca⁺⁺ currents and 3 outward K⁺ currents of tetraethylammonium (TEA)-sensitive, 4-aminopyridine (4-AP)-sensitive, and Ca⁺⁺-activated varieties. The resting potential was about -40mV. The activation voltage for Na⁺ and Ca⁺⁺ currents was about -30 and -17 mV, respectively. The maximum Na⁺ and Ca⁺⁺ currents were about 1057 and 179 pA, respectively.In regenerating retinae after 19–20 days of surgery, solitary cells with depigmented cytoplasm showed slowrising action potentials of long duration. The ionic dependence of this activity displayed two voltage-dependent components: slow inward Na⁺ and TEA-sensitive outward K⁺ currents. The maximum inward current (about 156 pA) was much smaller than that of the control. There was no indication of an inward Ca⁺⁺ current.During subsequent regeneration, the inward Ca⁺⁺ current appeared in most spiking cells, and the magnitude of the inward Na⁺, Ca⁺⁺, and outward K⁺ currents all increased. By 30 days of regeneration, the electrical activities of spiking cells became identical to those in the normal retina. No significant difference in the resting potential and the activation voltage for Na⁺ and Ca⁺⁺ currents was found during the regenerating period examined.     

Growth hormone-releasing factor reduces voltage-gated Ca2+ channel current in Rat GH3 cells

Summary The action of GRF on GH₃ cell membrane was examined by patch electrode techniques. Under current clamp with patch elecrtrode, spontaneous action potentials were partially to totally eliminated by application of GRF. In the case of partial elimination, the duration of remaining spontaneous action potentials was prolonged and the amplitude of afterhyperpolarization was decreased. The evoked actiion potential in the cells which did not show spontaneous action potentials was also eliminated by GRF. In order to examine what channels were affected by GRF, voltage-clamp analysis was performed. It was revealed that voltage-gated Ca²⁺ channel current and Ca²⁺-induced K⁺ channels current were decreased by GRF, while voltage-gated Na⁺ channel and delayed K⁺ channel current was considered to be a consequence of he decrease of voltage-gated Ca²⁺ channels current. Therefore it is likely that the effect of GRF on GH₃ cells was due to the block of voltage-gated Ca²⁺ channels. The elimination of action potential under current clamp corresponded to the block of voltage-gated Ca²⁺ channels and the prolongation of action potential could be explained by the decrease of Ca²⁺-induced K⁺ channel current. The amplitude decrease of afterhyperpolarization could also be explained by the reduction of Ca²⁺-induced K⁺ channel current. Thus the results under current clamp well coincide with the results under voltage clamp. Hormone secretion from GH₃ cells was not stimulated by GRF. However, the finding that GRF solely blocked voltage-gated Ca²⁺ channel suggested the specific action of GRF on GH₃ cell membranes.     

Patch-clamp recordings of spiking and nonspiking interneurons from rabbit olfactory bulb slices: Membrane properties and ionic currents

Summary Physiological and morphological properties of rabbit, Oryctolagus cuniculus, olfactory bulb interneurons were characterized by using a thin slice preparation in combination with patch-clamp measurements and Lucifer Yellow fills. Two types of interneurons, periglomerular (PG) and juxtaglomerular (JG) cells, were unequivocally distinguished in the glomerular layer. Their properties were compared to those of mitral cells. PG cells closely resembled previously described periglomerular cells in their morphology. During current clamp recording these neurons were characterized by their lack of action potentials upon depolarization. Consistent with these results no Na⁺ currents could be elicited in voltage clamp experiments. Two types of outward K⁺ currents were distinguished: one which inactivated and one which did not. From their morphology JG cells appear to be either short axon cells or external tufted cells. JG cells always responded with a single, TTX-blockable action potential in response to maintained current injection. Two types of membrane currents were identified in JG cells during voltage clamp: a fast, inactivating Na⁺ current that was fully activated at — 80 mV, and a sustained outward current that shared some properties with a delayed rectifier K⁺ current. The particular relationship between the voltage dependence of the Na⁺ and K⁺ currents appeared to preclude repetitive spike activity.Abbreviations JG juxtraglomerular interneuron - LOT lateral olfactory tract - M/T mitral/tufted (cells) - PG periglomerular - SA short axon     

Calcium-dependent K current in plasma membranes of dermal cells of developing bean cotyledons

In developing seeds of bean (Phaseolus vulgaris L.), phloem‐imported assimilates (largely sucrose and potassium) are released from coats to seed apoplasm and subsequently retrieved by the dermal cell complexes of cotyledons. To investigate the mechanisms of K⁺ uptake by the cotyledons, protoplasts of dermal cell complexes were isolated and whole‐cell currents across their plasma membranes were measured with the patch‐clamp technique. A weakly rectified cation current displaying a voltage‐dependent blockade by external Ca²⁺ and acidic pH, dominated the conductance of the protoplasts. The P haseolus v ulgaris Cotyledon Dermal‐cell pH and Calcium‐dependent Cation Conductance (Pv‐CD‐pHCaCC) was highly selective for K⁺ over Ca²⁺ and Cl^–. For K⁺ current through Pv‐CD‐pHCaCC a sigmoid shaped current–voltage (I–V) curve was observed with negative conductance at voltages between ?200 and ?140 mV. This negative K⁺ conductance was Ca²⁺ dependent. With other univalent cations (Na⁺, Rb⁺, NH₄⁺) the currents were smaller and were not Ca²⁺ dependent. Reversal potentials remained constant when external K⁺ was substituted with these cations, suggesting that Pv‐CD‐pHCaCC channels were non‐selective. The Pv‐CD‐pHCaCC would provide a pathway for K⁺ and other univalent cation influx into developing cotyledons. These cation influxes could be co‐ordinated with sucrose influx via pH and Ca²⁺dependence.     

Sodium inward currents through calcium channels in mealworm muscle fibers

The contribution of Na⁺ ions to the nonsynaptic electrogenesis was studied in the larval muscle fibers of mealworm, Tenebrio molitor, using currentclamp and voltage-clamp techniques. Na-dependent graded responses were generated by depolarizing current stimuli in Ca²⁺-free solutions. These responses were insensitive to tetrodotoxin and were blocked by Co²⁺. Large inward-going currents were elicited by step depolarizations in Ca²⁺-free solutions under voltage-clamp conditions. The inward currents were totally eliminated by removal of Na⁺ from the bathing solution. These results indicate that the calcium channel of mealworm muscle is permeable to Na⁺.     

Inhibition of Ca2+-dependent Cl− channels from secretory epithelial cells by low internal pH

The actions of intracellular pH (pH _i ) on Ca²⁺dependent Cl^? channels were studied in secretory epithelial cells derived from human colon carcinoma (T84) and in isolated rat parotid acinar cells. Channel currents were measured with the whole cell voltage clamp technique with pipette solutions of different pH. Ca²⁺dependent Cl^? channels were activated by superfusing ionomycin to increase the intracellular calcium concentration ([Ca²⁺] _i ) or by using pipette solutions with buffered Ca²⁺ levels. Large currents were activated in T84 and parotid cells by both methods with pH _i levels of 7.3 or 8.3. Little or no Cl^? channel current was activated with pH _i at 6.4. We used on-cell patch clamp methods to investigate the actions of low pH _i on single Cl^? channel current amplitude in T84 cells. Lowering the pH _i had little or no effect on the current amplitude of a 8 pS Cl^? channel, but did reduce channel activity. These results suggest that cytosolic acidification may be able to modulate stimulus-secretion coupling in fluid-secreting epithelia by inhibiting the activation of Ca²⁺-activated Cl^? channels.     

L-type Ca<Superscript>2+</Superscript> channel and Ca<Superscript>2+</Superscript>-permeable nonselective cation channel as a Ca<Superscript>2+</Superscript> conducting pathway in myocytes isolated from the cricket lateral oviduct

The Ca²⁺-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs²⁺-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca²⁺ with Ba²⁺ slowed the current decay. Increasing the external Ca²⁺ or Ba²⁺ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na⁺ in the absence of extracellular Ca²⁺. Current carried by Na⁺ (I _Na) was almost completely blocked by the dihydropyridine Ca²⁺ channel antagonist, nifedipine, suggesting that the I _Na is through voltage-dependent L-type Ca²⁺ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs⁺-aspartate and 140 mM Na⁺-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na⁺ was replaced with an equimolar amount of K⁺ or Cs⁺, or 50 mM Ca²⁺ or Ba²⁺. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd³⁺ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca²⁺-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca²⁺ channel and stretch-activated Ca²⁺-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I _Ca Ca²⁺ current - I _Na Na⁺ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier     

The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport

Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na⁺, Cl⁻, and K⁺ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na⁺-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca²⁺ (but not other cations) to functionally replace Na⁺ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca²⁺ and Na⁺ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca²⁺ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na⁺-binding sites.     

Ca2+-activated chloride channel activity during Ca2+ alternans in ventricular myocytes

Cardiac alternans, defined beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is a high risk indicator for cardiac arrhythmias. We investigated mechanisms of cardiac alternans in single rabbit ventricular myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique. A strong correlation between beat-to-beat alternations of AP morphology and CaT alternans was observed. During CaT alternans application of voltage clamp protocols in form of pre-recorded APs revealed a prominent Ca²⁺-dependent membrane current consisting of a large outward component coinciding with AP phases 1 and 2, followed by an inward current during AP repolarization. Approximately 85% of the initial outward current was blocked by Cl^? channel blocker DIDS or lowering external Cl^? concentration identifying it as a Ca²⁺-activated Cl^? current (I_CaCC). The data suggest that I_CaCC plays a critical role in shaping beat-to-beat alternations in AP morphology during alternans.     

Dynamics of chloride and potassium currents during the action potential in Chara studied with action potential clamp

TheCl^– and K⁺ currents underlying the action potential (AP) in the giant alga Chara were directly recorded with the action potential clamp method. An electrically triggered action potential was recorded and repetitively replayed as command voltage to the same cell under voltage clamp. The resulting clamp current was close to zero. Only the initial rectangular current used for stimulation was approximately reproduced by the clamp circuit. Inhibition of Cl^– channels with niflumic acid or ethacrynic acid and of K⁺ channels with Ba²⁺ evoked characteristic compensation currents because the amplifier had to add the selectively inhibited currents. Integration of the compensation currents revealed a mean flux through Cl^– and K⁺ channels of 3.3 10^–6 and 2.1 10^–6 mole M^–2 AP^–1 respectively. The dynamics of CI^– and K⁺ channel activation/inactivation were obtained by converting the relevant clamp currents to ionic permeabilities using the Goldman-Hodgkin-Katz current equation. During the AP the Cl^– permeability reaches a peak 370 ms, on average, after termination of the stimulating pulse. The following inactivation proceeds 3.6 times slower than the activation. The increase in K⁺ permeability lags behind the rise in Cl^– permeability, reaching a peak approximately 2 s after the latter.     

Control of ionic currents in guard cell vacuoles by cytosolic and luminal calcium

Activity of vacuolar ion channels can be regulated by the cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt). Using the whole-vacuole mode of patch-clamp with Vicia faba guard cell vacuoles, three distinct cation currents were apparent that were differentially regulated by [Ca²⁺]_cyt. At ‘zero’ to 100 nM [Ca²⁺]_cyt, instantaneous currents typical of Fast Vacuolar (FV) channels were activated. A 10 fold KCl gradient directed out of the vacuole increased FV currents (up to fivefold) at negative potentials compared with the currents in symmetrical KCl. At [Ca²⁺]_cyt higher than 100 nM, instantaneous currents became smaller and voltage-independent (non-rectifying) and were typical of Vacuolar K,+-selective (VK) channels. These currents were less sensitive to a KCl gradient than were the FV currents, being stimulated less than twofold at negative potentials. Reversal potentials measured in the presence of a KCl gradient indicated a high K⁺ permeability of both FV and VK currents. At [Ca²⁺]_cyt higher than 600 nM time-dependent currents elicited by positive potentials were typical of Slow Vacuolar (SV) channel activation. When the Ca²⁺ mole fraction in the cytosolic or luminal solution was varied the reversal potential of SV currents (determined by tail current analysis) passed through maximum or minimum values. The resultant calculated apparent permeability ratios varied with ionic conditions but indicated high Ca²⁺ and K⁺ permeabilities. If a Cl^? permeability was assumed then the apparent P_Ca was lower. However, substitution of Cl^? by the larger (impermeant) anion gluconate had no effect on the reversal potential of SV tail currents in the presence of Ca²⁺ and a K⁺ gradient, demonstrating that the assumption of Cl^? permeability of the SV channel is invalid. Single-channel SV currents also decreased with increasing cytosolic Ca²⁺ mole fraction. These data indicate that the SV channel is highly cation selective, shows characteristics typical of a multi-ion pore and derives ion selectivity by Ca²⁺ binding. The SV channel currents could also be Mg²⁺-activated and were demonstrated to be Mg²⁺-permeable in the absence of Ca²⁺. The apparent permeability ratio (P_Mg:P_K) also varied under different ionic conditions. The results indicate not only that FV, VK and SV channels are all present in a single cell type, but also that each is differentially regulated by [Ca²⁺]_cyt. The respective roles of these channels in vacuolar ion release are discussed, and possible conditions are presented in which these channels could be activated by disparate signalling pathways during stomatal closure.     

The k/na selectivity of a cation channel in the plasma membrane of root cells does not differ in salt-tolerant and salt-sensitive wheat species

The characteristics of cation outward rectifier channels were studied in protoplasts from wheat root (Triticum aestivum L. and Triticum turgidum L.) cells using the patch clamp technique. The cation outward rectifier channels were voltage-dependent with a single channel conductance of 32 ± 1 picosiemens in 100 millimolar KCl. Whole-cell currents were dominated by the activity of the cation outward rectifiers. The time- and voltage-dependence of these currents was accounted for by the summed behavior of individual channels recorded from outside-out detached patches. The K⁺/Na⁺ permeability ratio of these channels was measured in a salt-sensitive and salt-tolerant genotype of wheat that differ in rates of Na⁺ accumulation, using a voltage ramp protocol on protoplasts in the whole-cell configuration. Permeability ratios were calculated from shifts in reversal potentials following ion substitutions. There were no significant differences in the K⁺/Na⁺ permeability ratios of these channels in root cells from either of the two genotypes tested. The permeability ratio for K⁺/Cl⁻ was greater than 50:1. The K⁺/Na⁺ permeability ratio averaged 30:1, which is two to four times more selective than the same type of channel in guard cells and suspension culture cells. Lowering the Ca²⁺ concentration in the bath solution to 0.1 millimolar in the presence of 100 millimolar Na⁺ had no significant effect on the K⁺/Na⁺ permeability ratios of the channel. It seems unlikely that the mechanism of salt tolerance in wheat is based on differences in the K⁺/Na⁺ selectivity of these channels.     

Effects of sodium chloride on tobacco plants

Abstract The effect of salinity on the growth and ion concentrations in a number of tobacco cultivars is described. Sodium chloride, at a concentration of 200 mol m^?3, hardly affected the fresh weight, but significantly reduced the dry weight. The difference in the response of fresh and dry weights to salt was due to a change in succulence (water per unit leaf area); the latter increased with increasing leaf Na⁺ and Cl^? concentration. Under saline conditions, increasing the external Na⁺: Ca^? ratio by decreasing the Ca²⁺ concentration increased the accumulation of Na⁺ and Cl^? into the leaf tissue.     

The ionic components of the current pulses generated by developing fucoid eggs.

Using a newly developed, extracellular vibrating electrode, we studied the ionic composition of the current pulses which traverse the developing Pelvetia embryo. External Na⁺, Mg²⁺, or SO₄^2?, are not needed for the first 20 min of pulsing. In fact, lowering external Na⁺ or Mg²⁺ (or K⁺) actually stimulates pulsing. Since tracer studies show that Ca²⁺ entry is speeded by Na⁺, Mg²⁺, or K⁺ reduction, these findings suggest that Ca²⁺ entry triggers pulsing. A sevenfold reduction in external Cl^? raises pulse amplitudes by 60%. Moreover, Cl^? is the only major ion with an equilibrium potential near the pulse reversal potential. These facts suggest that Cl^? efflux carries much of the “inward” current. We propose a model for pulsing in which increased Ca²⁺ within the growing tip opens Cl^? channels. The resulting Cl^? efflux slightly depolarizes the membrane and thus drives a balancing amount of K⁺ out. Thus, the pulses release KCl and serve to relieve excess turgor pressure. By letting Ca²⁺ into the growing tip, they should also strengthen the transcytoplasmic electrical field which is postulated to pull growth components toward this tip.     

Electrophysiological evidence suggests a defective Ca2+ control mechanism in a newParamecium mutant

Summary A new mutant ofParamecium tetraurelia, k-shyA, was characterized behaviorally and electrophysiologically. The mutant cell exhibited prolonged backward swimming episodes in response to depolarizing conditions. Electrophysiological comparison of k-shyA with wild type cells under voltage clamp revealed that the properties of three Ca²⁺-regulated currents were altered in the mutant. (i) The voltage-dependent Ca²⁺ current recovered from Ca²⁺-dependent inactivation two- to 10-fold more slowly than wild type. Ca²⁺ current amplitudes were also reduced in the mutant, but could be restored by EGTA injection. (ii) The decay of the Ca²⁺-dependent K⁺ tail current was slower in the mutant. (iii) The decay of the Ca²⁺-dependent Na⁺ tail current was also slower in the mutant. All other membrane properties studied, including the resting membrane potential and resistance and the voltage-sensitive K⁺ currents, were normal in k-shyA. Considered together, these observations are consistent with a defect in the ability of k-shyA to reduce the free intracellular Ca²⁺ concentration following stimulation. The possible targets of the genetic lesion and alternative explanations are discussed. The k-shy mutants may provide a useful tool for molecular and physiological analyses of the regulation of Ca²⁺ metabolism inParamecium.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca²⁺ channels are important structural determinants for the passage of Ca²⁺ across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a_1S subunit of the skeletal L-type channel (Ca_v1.1) to lysine virtually eliminates passage of Ca²⁺ during step depolarizations. In this study, we examined the ability of this mutant Ca_v1.1 channel (SkEIIIK) to conduct inward Na⁺ current. When 150 mM Na⁺ was present as the sole monovalent cation in the bath solution, dysgenic (Ca_v1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na⁺. Ca²⁺ block of SkEIIIK-mediated Na⁺ current was revealed by the substantial enhancement of Na⁺ current amplitude after reduction of Ca²⁺ in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na⁺ currents through the mutant Ca_v1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na⁺ channel when Na⁺ is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca²⁺ permeability mediated by Ca_V channels on physiological processes that extend beyond channel gating and permeability.