Characterization of sodium currents in mammalian sensory neurons cultured in serum-free defined medium with and without nerve growth factor

Summary The influence of nerve growth factor (NGF) on Na currents of rat dorsal root ganglia (DRG) was studied in neurons obtained from newborns and cultured for 2–30 hr inserum-free defined medium (SFM). Cell survival for the period studied was 78–87% both with and without NGF. Na currents were detected in all cells cultured for 6–9 hr. They were also detected after 2 hr in culture in 21.5% of the cells cultured without NGF (–NGF cells), and in 91.5% of the cells cultured with NGF (+NGF cells). Current density of the -NGF cells was 2.3 and 2 pA/m² after growth for 2 and 6–9 hr, respectively, compared to 3.0 and 3.9 pA/m² for the +NGF cells. The +NGF cells were separated into fast (F), Intermediate (I) and slow (S) cells, based on the Na current they expressed, while -NGF cells were all of theI type.F, I andS currents differed in their voltage-dependent inactivation (Vh ₅₀=–79, –28 and –20 mV), kinetics of inactivation (tau _h =0.55, 1.3 and 7.75 msec), and TTX sensitivity (K _i=60, 550 and 1100nm). All currents were depressed by [Ca] _o with aKd _Ca of 22, 17 and 8mm forF, I andS currents, respectively. Current density ofF andS currents was 5.5 and 5 pA/m² for theI current. The concentration-dependent curve ofI currentvs. TTX indicated thatI current has two sites: one withF-like and another withS-likeK _i for TTX. Hybridization ofF andS currents yieldI-like currents. Thus, the major effect of NGF on Na currents in SFM is the accleration of Na current acquisition and diversity, reflected in an increase of either theS orF type in a cell.     

Inwardly rectifying whole-cell and single-channel K currents in the murine macrophage cell line J774.1

Summary Inward currents in the murine macrophage-like cell line J774.1 were studied using the whole-cell and cell-attached variations of the patch-clamp technique. When cells were bathed in Na Hanks' (KCl=4.5mm, NaCl=145mm), and the electrode contained Na-free K Hanks' (KCl=145mm) single-channel currents were observed at potentials below –40 mV which showed inward rectification, were K-selective, and were blocked by 2.5mm Ba in the pipette. Single-channel conductance was 29 pS, and was proportional to the square root of [K] _o . Channels manifested complex kinetics, with multiple open and closed states. The steady-state open probability of the channel was voltage dependent, and declined from 0.9 to 0.45 between –40 and –140 mV. When hyperpolarizing voltage pulses were repetitively applied in the cell-attached patch mode, averaged single-channel currents showed inactivation. Inactivation of inwardly rectifying whole-cell current was measured in Na Hanks' and in two types of Na-free Hanks': one with a normal K concentration (4.5mm) and the other containing 145mm K. Inactivation was shown to have Na-dependent and Na-independent components. Properties of single-channel current were found to be sufficient to account for the behavior of the macroscopic current, except that single-channel current showed a greater degree of Na-independent inactivation than whole-cell current.     

Patch-clamp study of isolated taste receptor cells of the frog

Summary Taste discs were dissected from the tongue ofR. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110mm KCl and the cells were superfused with NaCl Ringer's during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was –65.2 mV and the slope resistance 150 to 750 M. Pulse-depolarization from a holding voltage of –80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at –25 mV and was half-maximal at –8 mV. Steady-state inactivation was half-maximal at –67 mV and complete at –50 mV. Peak Na current averaged –0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5mm) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5mm) and 4-aminopyridine (1mm) did not block, but 5mm Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted theI _K(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10mm Ca caused a reversible 5-to 10-mV depolarization in the current-clamp mode. Quinine (0.1mm, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5mm in the external solution or 0.5 m in the pipette) caused reversible depolarization (to –40 to –20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP. Inclusion of ATP--S in the pipette caused slow depolarization to –40 to –20 mV, due to partial blockage of K currents. Subsequently, cAMP was without effect. The remaining K currents were blockable by Ba. These results suggest that cAMP initiates phosphorylation of one set of K channels to a nonconducting conformation.     

Voltage-dependent calcium and potassium conductances in striated muscle fibers from the scorpion,Centruroides sculpturatus

Ionic currents responsible for the action potential in scorpion muscle fibers were characterized using a three-intracellular microelectrode voltage clamp applied at the fiber ends (8–12°C). Large calcium currents (I _Ca) trigger contractile activation in physiological saline (5 mm Ca) but can be studied in the absence of contractile activation in a low Ca saline (2.5 mm). Barium (Ba) ions (1.5–3 mm) support inward current but not contractile activation.Ca conductance kinetics are fast (time constant of 3 msec at 0 mV) and very voltage dependent, with steady-state conductance increasing e-fold in approximately 4 mV. Half-activation occurs at –25 mV. Neither I _Ca nor I _Ba show rapid inactivation, but a slow, voltage-dependent inactivation eliminates I _Ca at voltages positive to –40 mV. Kinetically, scorpion channels are more similar to L-type Ca channels in vertebrate cardiac muscle than to those in skeletal muscle.Outward K currents turn on more slowly and with a longer delay than do Ca currents, and K conductance rises less steeply with voltage (e-fold change in 10 mV; half-maximal level at 0 mV). K channels are blocked by externally applied tetraethylammonium and 3,4 diaminopyridine.This work was supported by a grant from the NIH (NS-17510) to W.F.G. and a NRSA award to T.S. (GM-09921).     

Effects of fatty acids on membrane currents in the squid giant axon

Summary The effects of fatty acids on the ionic currents of the voltage-clamped squid giant axon were investigated using intracellular and extracellular application of the test substances. Fatty acids mainly suppress the Na current but have little effect on the K current. These effects are completely reversed after washing with control solution. The concentrations required to suppress the peak inward current by 50% and Hill number were determined for each fatty acid. ED₅₀ decreased about 1/3 for each increase of one carbon atom. The standard free energy was –3.05 kJ mole^–1 for CH₂. The Hill number was 1.58 for 2-decenoic acid. The suppression effect of the fatty acids depends on the number of carbon atoms in the compounds and their chemical structure. Suppression of the Na current was clearly observed when the number of carbon atoms exceeded eight. When fatty acids of the same chain length were compared, 2-decenoic acid had strong inhibitory activity, but sebacic acid had no effect at all on the Na channel. The currents were fitted to equations similar to those proposed by Hodgkin and Huxley (J. Physiol. (London) 117:500–544, 1952) and the changes in the parameters of these equations in the presence of fatty acids were calculated. The curve of the steady-state activation parameter (m ) for the Na current against membrane potential and the time constant of activation ({ie113-1}) were shifted 20 mV in a depolarizing direction by the application of fatty acids. The time constant for inactivation ({ie113-2}) was almost no change by application of the fatty acids. The time constant for activation ({ie113-3}) of K current was shifted 20 mV in a depolarizing direction by the application of the fatty acids.     

Human Adipose Cells Have Voltage-dependent Potassium Currents

The whole-cell patch-clamp method was used to study the membrane electrical properties of human adipocyte cells obtained by differentiating from precursors of human abdominal and mammary tissues. All differentiated cells exhibited outward currents with sigmoidal activation kinetics. The outward currents showed activation thresholds between –20 to –30 mV and slow inactivation. The ionic channels underlying the macroscopic current were highly selective for K⁺. Their selectivity was for typical K⁺ channels with relative permeabilities of K⁺>NH ₄ ⁺ >Cs⁺>Na⁺. No evidence of any other type of voltage-gated channel was found. The potassium currents (I _KV) were blocked reversibly by tetraethylammonium and barium. The IC ₅₀ value and Hill coefficient of tetraethylammonium inhibition of I _KV were 0.56 mM and 1.17 respectively. These results demonstrate that human adipose cells have voltage-dependent potassium currents.     

Block of single cardiac sodium channels by intracellular magnesium

Currents through single cardiac sodium channels have been measured in inside-out patches from guinea pig ventricular cells. To abolish the fast inactivation, Na channels were modified by DPI 201–106. In symmetrical Na solutions, a diminution of outward sodium currents can be observed that depends on the intracellular magnesium concentration and the membrane potential. Inward currents were not altered by the concentrations of magnesium used (between 0 and 22.5 mmol/1). In Mg free solutions a linear current-voltage relation can also be measured in the range of outward Na currents. At +60 mV (symmetrical Na solutions, single channel conductance 24 pS) a half maximal block of cardiac Na channels by intracellular magnesium was found at 2.1 mmol/l. From the analysis of single channel current-voltage relationships the concentration and voltage-dependent block by intracellular magnesium of cardiac sodium channels could be described as binding of Mg at one site with a K _d value of 5.1 mmol/1 at 0 mV. The site is located at an electrical distance of 0.18 from the inside. Offprint requests to: B. Nilius     

The effects of Al on the calcium currents inHelix neurons

Summary 1. The effects of aluminium (Al) on calcium (Ca) currents were investigated by using the conventional two-electrode voltage clamp technique inHelix pomatia neurons. The peak amplitude, kinetics, and voltage dependence of activation and inactivation of the Ca currents were studied in the presence of 10^–5–10^–3 M AlCl₃, at pH 6.2. Al prolonged the rising phase of the Ca currents and therefore increased the time to peak at each command voltage step used.3. There was no significant influence of Al on the peak amplitude of the Ca currents, but the voltage dependence of the time to peak, activation, and inactivation of the Ca currents shifted to more positive potentials as a consequence of Al treatment.4. The leak currents were not influenced by Al up to 1 mM, which was the maximal dose applied.5. The results support the suggestion that Al may modify the Ca homeostasis and that it exerts a neurotoxic effect, at least in part, by modulation of the Ca current of the neuronal membrane.     

K<Superscript>+</Superscript> Channels in Cardiomyocytes of the Pulmonate Snail <Emphasis Type="Italic">Helix</Emphasis>

We used the patch-clamp technique to identify and characterize the electrophysiological, biophysical, and pharmacological properties of K⁺ channels in enzymatically dissociated ventricular cells of the land pulmonate snail Helix. The family of outward K⁺ currents started to activate at –30 mV and the activation was faster at more depolarized potentials (time constants: at 0 mV 17.4 ± 1.2 ms vs. 2.5 ± 0.1 ms at + 60 mV). The current waveforms were similar to those of the A-type family of voltage-dependent K⁺ currents encoded by Kv4.2 in mammals. Inactivation of the current was relatively fast, i.e., 50.2 ± 1.8% of current was inactivated within 250 ms at + 40 mV. The recovery of K⁺ channels from inactivation was relatively slow with a mean time constant of 1.7 ± 0.2 s. Closer examination of steady-state inactivation kinetics revealed that the voltage dependency of inactivation was U-shaped, exhibiting less inactivation at more depolarized membrane potentials. On the basis of this phenomenon, we suggest that a channel encoded by Kv2.1 similar to that in mammals does exist in land pulmonates of the Helix genus. Outward currents were sensitive to 4-aminopyridine and tetraethylammonium chloride. The last compound was most effective, with an IC₅₀ of 336 ± 142 µmol l^–1. Thus, using distinct pharmacological and biophysical tools we identified different types of voltage-gated K⁺ channels. Present address for S.A.K.: Brigham and Womens Hospital, Cardiovascular Division, Harvard Medical School, 75 Francis St., Thorn 1216, Boston, MA 02115.     

Electrophysiological and Theoretical Analysis of Depolarization-Dependent Outward Currents in the Dendritic Membrane of an Identified Nonspiking Interneuron in Crayfish

Depolarization-dependent outward currents were analyzed using the single-electrode voltage clamp technique in the dendritic membrane of an identified nonspiking interneuron (LDS interneuron) in situ in the terminal abdominal ganglion of crayfish. When the membrane was depolarized by more than 20 mV from the resting potential (65.0 ± 5.7 mV), a transient outward current was observed to be followed by a sustained outward current. Pharmacological experiments revealed that these outward currents were composed of 3 distinct components. A sustained component (I _s) was activated slowly (half rise time > 5 msec) and blocked by 20 mM TEA. A transient component (I _t1) that was activated and inactivated very rapidly (peak time < 2.5 msec, half decay time < 1.2 msec) was also blocked by 20 mM TEA. Another transient component (I _t2) was blocked by 100 M 4AP, activated rapidly (peak time < 10.0 msec) and inactivated slowly (half decay time > 131.8 msec). Two-step pulse experiments have revealed that both sustained and transient components are not inactivated at the resting potential: the half-maximal inactivation was attained at –21.0 mV in I _t1, and –38.0 mV in I _t2. I _s showed no noticeable inactivation. When the membrane was initially held at the resting potential level and clamped to varying potential levels, the half-maximal activation was attained at –36.0 mV in I _s, –31.0 mV in I _t1 and –40.0 mV in I _t2. The activation and inactivation time constants were both voltage dependent. A mathematical model of the LDS interneuron was constructed based on the present electrophysiological records to simulate the dynamic interaction of outward currents during membrane depolarization. The results suggest that those membrane conductances found in this study underlie the outward rectification of the interneuron membrane as well as depolarization-dependent shaping of the excitatory synaptic potential observed in current-clamp experiments.     

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle

We investigated the effects of pressure overload hypertrophy on inward sodium (I _Na) and calcium currents (I _Ca) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that I _Ca density was unchanged whereas I _Na density after stepping from –80 to –30 mV was decreased by 30% (–9.0 ± 1.16 pA pF^–1 in control and –6.31 ± 0.67 pA pF^–1 in hypertrophy, p < 0.05, n= 6). Steady-state activation/inactivation variables of I _Na, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of I _Na was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca²⁺ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in I _Na density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na⁺ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased I _Na density so that net Na⁺ influx remains unaltered. Changes in the fast I _Na do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle. (Mol Cell Biochem 261: 217–226, 2004)     

Patch-clamp studies in human macrophages: Single-channel and whole-cell characterization of two K+ conductances

Summary Human peripheral blood monocytes cultured for varying periods of time were studied using whole-cell and single-channel patch-clamp recording techniques. Whole-cell recordings revealed both an outward K current activating at potentials >20 mV and an inwardly rectifying K current present at potentials negative to –60 mV. Tail currents elicited by voltage steps that activated outward current reversed nearE _K, indicating that the outward current was due to a K conductance. TheI–V curve for the macroscopic outward current was similar to the mean single-channelI–V curve for the large conductance (240 pS in symmetrical K) calcium-activated K channel present in these cells. TEA and charybdotoxin blocked the whole-cell outward current and the single-channel current. Excised and cell-attached single-channel data showed that calcium-activated K channels were absent in freshly isolated monocytes but were present in >85% of patches from macrophages cultured for >7 days. Only 35% of the human macrophages cultured for >7 days exhibited whole-cell inward currents. The inward current was blocked by external barium and increased when [K] _o increased. Inward-rectifying single-channel currents with a conductance of 28 pS were present in cells exhibiting inward whole-cell currents. These single-channel currents are similar to those described in detail in J774.1 cells (L.C. McKinney & E.K. Gallin,J. Membrane Biol. 103:41–53, 1988).     

Voltage-gated chloride currents in cultured canine tracheal epithelial cells

Summary Chloride ions (Cl^–) are concentrated in airway epithelial cells and subsequently secreted into the tracheal lumen by downhill flux through apical Cl^– channels. We have studied Cl^– currents in cultured canine tracheal cells using the whole-cell voltage-clamp technique. Ultrastructural techniques demonstrated that the cells used in the electrophysiological experiments possessed apical membrane specializations known to be present in the intact, transporting cell type. Cultured cells 2–6 days old were characterized by an input resistance of 3.4±0.8 G (n=11) and a capacitance of 63.8±10.8 pF (n=26). A comparison of 3 and 4 day-old cells with 5 and 6 day-old cells showed that the input resistance decreased almost 50%, and the cell capacitance and the inward and outward currents increased concomitantly approximately 200%. Cultured cells 3–4 days old held at –40 mV produced currents of 196±22 pA at 50 mV and –246±27 pA at –90 mV (n=212) with pipette and bath solutions containing primarily 140 KCl and 140 NaCl, respectively. The chloride channel blocker diphenylamine-2-carboxylate (DPC, 100 m) suppressed whole-cell currents by 76.8% at 60 mV; however, currents were unaffected by the stilbenes SITS (1mm) and DNDS (1–30 m). Replacement of K⁺ with Cs⁺ in the pipette solution did not affect the outward current, the current reversal potential, or the input resistance of the cells, indicating that the current was not significantly K⁺ dependent when the intrapipette solution was buffered to a Ca²⁺ concentration of 20nm. The Cl^–/Na⁺ permeability ratio was estimated to be greater than 11 as calculated from reversal potential measurements in the presence of an internal to external NaCl concentration ratio of 12. Current equilibrium permeabilities, relative to Cl^– were: I^– (2.9)NO ₃ ^– (1.1)Br^– (1.1)Cl^– (1.0)F^– (0.93)MeSO ₄ ^– (0.19)gluconate (0.18)aspartate (0.14). Depolarizations to potentials greater than 20 mV elicited a time-dependent component in the outward current in 71% of the cells studied. Currents inactivated with a double exponential time course at the most depolarized voltages. Recovery from inactivation was fast, holding potential-dependent, and followed a double exponential time course. Current amplitude was increased via a cAMP-dependent pathway as has been demonstrated for single Cl-selective channels in cell-attached patches from cultured canine and human tracheal epithelial cells. Forskolin, an activator of adenylate cyclase, produced a 260% increase in the outward current at +50 mV. In summary, cultured canine tracheal cells have a single resting conductance that is Cl^– selective, voltage-dependent, and modulated by a cAMP-dependent mechanism. This preparation appears to be appropriate for analysis of cellular modulation of airway Cl^– channels and Cl^– secretion.     

Ionic mechanisms for the antiarrhythmic action of cinnamophilin in rat heart

We investigated the electrophysiological effect and antiarrhythmic potential of cinnamophilin (Cinn), a thromboxane A₂ antagonist isolated fromCinnamomum philippinense, on rat cardiac tissues. Action potential and ionic currents in single rat ventricular cells were examined by current clamp or voltage clamp in a whole-cell configuration. In 9 episodes of ischemia-reperfusion arrhythmia, 10 µM Cinn converted 6 of them to normal sinus rhythm. Cinn suppressed the maximal rate of rise of the action potential upstroke (V_max) and prolonged the action potential duration at 50% repolarization (APD₅₀). Voltage clamp study showed that the suppression of V_max by Cinn was associated with an inhibition of sodium inward current (I_Na, IC₅₀=10.0 ± 0.4 µM). At 30 µM, V_1/2 for the steady-state inactivation curve of I_Na was shifted from –84.1 ± 0.2 to –93.0 ± 0.5 mV. Cinn also reduced calcium inward current (I_Ca) dose-dependently with an IC₅₀ value of 9.5 ± 0.3 µM. Cinn (10 µM) reduced the I_Ca with a negative shift of V_1/2 for the steady-state inactivation curve of I_Ca from –32.2 ± 0.3 to –50.7 ± 0.4 mV. The prolongation of APD₅₀ was associated with an inhibition of the integral of potassium outward current with IC₅₀ values between 4.8 and 7.1 µM. At 10 µM, Cinn reduced I_Na without a negative shift of its voltage-dependent steady-state inactivation curves. The inhibition of transient outward current (I_to) by Cinn (3–30 µM) was associated with an acceleration of its time constant of inactivation and negative shift of its potential-dependent steady-state inactivation curves. The equilibrium dissociation constant (K_d) of Cinn to inhibit open state I_to channels, as calculated from the time constant of developing block, was 18.3 µM. The time constant of recovery of I_to from inactivation state was unaffected by Cinn. The rate constant for the relief from the depolarization-dependent block of I_to was calculated to be 23.9 ms. As compared with its effect on I_to, Cinn exerted about half the potency to block I_Na and I_Ca. These results indicate that the inhibition of I_Na, I_Ca and I_to may contribute to the antiarrhythmic activity of Cinn against ischemia-reperfusion arrhythmia.     

Expression of low voltage-activated calcium channels inXenopus oocytes

Calcium channels were expressed inXenopus oocytes by means of messenger RNA extracted from the rat thalamo-hypothalamic complex, mRNA(h). Inward barium currents,I _Ba, were recorded in Cl^–-free extracellular solution with 40 mM Ba²⁺ as a charge carrier, using two-microelectrode technique. Depolarizations from a very negative holding potential (V _h=–120 mV) began to activateI _Ba at about –80 mV; this current peaked at –30 to –20 mV and reversed at +50 mV, indicating that I _Ba may be transferred through the low voltage-activated (LVA) calcium channels. The time-dependent inactivation of the current during a prolonged depolarization to –20 mV was quite slow, followed a single exponential decay with a time constant of 1550 msec, and contained a residual component constituting 30% of the maximum amplitude. The current could not be completely inactivated at any holding potential. As expected for LVA current, a steady-state inactivation curve was shifted towards negative potentials. It could be described by the Boltzmann's equation with the half-inactivation potential of –78 mV, slope factor of 15 mV, and residual level of 0.3. ExpressedI _Ba could be blocked by flunarizine (K _d=0.42 µM), nifedipine (K _d=10 µM), and amiloride at a 500 µM concentration. Among the inorganic Ca²⁺ channel blockers, the most potent was La³⁺ (K _d=0.48 µM), while Cd²⁺ and Ni²⁺ were not very selective and almost thousand-fold less effective (K _d=0.52 mM andK _d=0.62 mM, respectively) than La³⁺. Our data show that mRNA(h) induces expression in the oocytes of almost exclusively LVA Ca²⁺ channels with voltage-dependent and pharmacological properties very similar to those observed for T-type Ca²⁺ current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 183–189, May–June, 1995.     

Distinct Ca<Superscript>2+</Superscript>-permeable Cation Currents Are Activated by Internal Ca<Superscript>2+</Superscript>-Store Depletion in RBL-2H3 Cells and Human Salivary Gland Cells,HSG and HSY

Store-operated Ca²⁺ influx, suggested to be mediated via store-operated cation channel (SOC), is present in all cells. The molecular basis of SOC, and possible heterogeneity of these channels, are still a matter of controversy. Here we have compared the properties of SOC currents (I _SOC) in human submandibular glands cells (HSG) and human parotid gland cells (HSY) with I _CRAC (Ca²⁺ release-activated Ca²⁺ current) in RBL cells. Internal Ca²⁺ store-depletion with IP₃ or thapsigargin activated cation channels in all three cell types. 1 μM Gd³⁺ blocked channel activity in all cells. Washout of Gd³⁺ induced partial recovery in HSY and HSG but not RBL cells. 2-APB reversibly inhibited the channels in all cells. I _CRAC in RBL cells displayed strong inward rectification with E _rev(Ca) = >+90 mV and E _rev (Na) = +60 mV. I _SOC in HSG cells showed weaker rectification with E _rev(Ca) = +25 mV and E _rev(Na) = +10 mV. HSY cells displayed a linear current with E _rev = +5 mV, which was similar in Ca²⁺- or Na⁺-containing medium. pCa/pNa was >500, 40, and 4.6 while pCs /pNa was 0.1,1, and 1.3, for RBL, HSG, and HSY cells, respectively. Evidence for anomalous mole fraction behavior of Ca²⁺/Na⁺ permeation was obtained with RBL and HSG cells but not HSY cells. Additionally, channel inactivation with Ca²⁺ + Na⁺ or Na⁺ in the bath was different in the three cell types. In aggregate, these data demonstrate that distinct store-dependent cation currents are stimulated in RBL, HSG, and HSY cells. Importantly, these data suggest a molecular heterogeneity, and possibly cell-specific differences in the function, of these channels.This revised version was published online in June 2005 with a corrected cover date.     

Whole-cell currents in macrophages: I. Human monocyte-derived macrophages

Summary We examined the variability of occurrence and frequency of voltage-dependent whole-cell currents in human peripheral blood monocyte-derived macrophages (HMDM) maintained in culture for up to three weeks. An increase in cell capacitance from an average value of 9 pF on the day of isolation to 117 pF at 14 days accompanied growth and differentiation in culture. The average resting potential was approximately –34 mV for cells beyond two days in culture. Cells exhibited a voltage-and time-dependent outward current upon membrane depolarization above approximately –30 mV, which appeared to be composed of a number of separate currents with variable expression from donor to donor. Three of these currents are carried by K⁺. The frequency of each outward current type was calculated for 974 cells obtained from 36 donors. The HMDMs in these studies exhibited two 4-aminopyridine (4-AP) sensitive, time-dependent outward currents (I _A andI _B ) that could be differentiated on the basis of the presence or absence of steady-state inactivation in the physiological potential range, time course of inactivation during maintained depolarization, as well as threshold of activation. The 4-AP-insensitive outward current activated at approximately 10 mV. One component of the 4-AP insensitive-outward current (I _C ) could be blocked by external TEA and by the exchange of internal Cs⁺ or Na⁺ for K⁺. The probability of observingI _B andI _C appeared to be donor dependent. Following total replacement of internal K⁺ with Cs⁺, two additional currents could be identified (i) a delayed component of outward current (I _D ) remained which could be blocked by low concentrations of external Zn²⁺ (4 m) and was insensitive to anion replacement in the external solution and (ii) a Cl^– current with a reversal potential which shifted in the presence of external anion replacement and which was irreversibly inhibited by the stilbene SITS. The activation of a prominent time-independent inward currents was often observed with increasing hyperpolarization. This inward current was blocked by external Ba²⁺ and corresponded to the inwardly rectifying K⁺ current. Neither inward nor outward current expression appeared dependent on whether cells were differentiated in adherent or suspension culture nor was there demonstrable differential current expression observed upon transition from suspension to adherent form.     

Role of Arginine Residues on the S4 Segment of the <Emphasis Type="Italic">Bacillus halodurans</Emphasis> Na<Superscript>+</Superscript> Channel in Voltage-sensing

The one-domain voltage-gated sodium channel of Bacillus halodurans (NaChBac) is composed of six transmembrane segments (S1–S6) comprising a pore-forming region flanked by segments S5 and S6 and a voltage-sensing element composed of segment S4. To investigate the role of the S4 segment in NaChBac channel activation, we used the cysteine mutagenesis approach where the positive charges of single and multiple arginine (R) residues of the S4 segment were replaced by the neutrally charged amino acid cysteine (C). To determine whether it was the arginine residue itself or its positive charge that was involved in channel activation, arginine to lysine (R to K) mutations were constructed. Wild-type (WT) and mutant NaChBac channels were expressed in tsA201 cells and Na⁺ currents were recorded using the whole-cell configuration of the patch-clamp technique. The current/voltage (I-V) and conductance/voltage (G-V) relationships steady-state inactivation (h _∞) and recovery from inactivation were evaluated to determine the effects of the S4 mutations on the biophysical properties of the NaChBac channel. R to C on the S4 segment resulted in a slowing of both activation and inactivation kinetics. Charge neutralization of arginine residues mostly resulted in a shift toward more positive potentials of G-V and h _∞ curves. The G-V curve shifts were associated with a decrease in slope, which may reflect a decrease in the gating charge involved in channel activation. Single neutralization of R114, R117, or R120 by C resulted in a very slow recovery from inactivation. Double neutralization of R111 and R129 confirmed the role of R111 in activation and suggested that R129 is most probably not part of the voltage sensor. Most of the R to K mutants retained WT-like current kinetics but exhibited an intermediate G-V curve, a steady-state inactivation shifted to more hyperpolarized potentials, and intermediate time constants of recovery from inactivation. This indicates that R, at several positions, plays an important role in channel activation. The data are consistent with the notion that the S4 is most probably the voltage sensor of the NaChBac channel and that both positive charges and the nature of the arginine residues are essential for channel activation.This revised version was published online in June 2005 with a corrected cover date.     

Three classes of potassium channels in large monopolar cells of the blowfly Calliphora vicina

Summary We have used single electrode voltage clamp in the intact animal and whole-cell recording from dissociated cell bodies to investigate the properties of potassium conductances in large monopolar cells (LMCs) of the first optic ganglion of the blowfly Calliphora vicina. Two classes of voltage gated potassium conductances were found: a delayed rectifier current (Kd) with slow inactivation (_inac = 1–3 sec), and an A current (Ka) showing both faster inactivation (_inac = 21 ms) and also more rapid activation. The reversal potential of both currents is ca. -90 mV with 2 mM [K_o] and 140 mM [K_i], and follows the Nernst slope with increasing [K_o]. The voltage operating range of Ka is unusually negative, with the mid point of the steady-state inactivation curve (V₅₀) at- 101 mV. V₅₀ for Kd is - 84 mV. Although no inward currents were detected, for technical reasons their presence cannot be excluded.In inside-out patches from LMC soma membranes the single channels underlying the currents both have a conductance of ca. 20 pS in symmetrical 140 mM K solutions and channel densities may be as high as 10/m². Less frequently, inside-out patches contained a large conductance (110 pS) calcium-activated potassium channel which existed almost exclusively in a rapidly flickering mode. Open probability increased with depolarization and Ca concentrations greater than 40 nM.In whole-cell recordings, dissociated LMC cell bodies fall into two classes with respect to their voltage sensitive currents: 37 % of cells only showed Kd; the remainder (63%) were dominated by Ka with a variable (0–30%) contribution from Kd. In the intact animal, intracellular recordings from LMCs, combined with dye-marking, indicate that cells expressing only Kd are type L3 cells, whilst L1 and L2 express predominantly Ka. Since L1 and L2 have resting potentials of ca. - 40 mV and maximum hyperpolarizations reaching -90 mV only transiently, inactivation of Ka is unlikely to be removed under most physiological conditions. In contrast, L3 cells have a more negative resting potential (–60 mV) and Kd should play a significant role in signal-shaping, in particular contributing to the falling phase of a prominent spike-like transient in response to dimming.Abbreviations Ka A current - Kd delayed rectifier - LMC large monopolar cell - L1-L3 classes thereof - TTX tetrodotoxin