Mechanisms of coronary artery depolarization by uridine triphosphate

We sought to define the basic mechanisms by which pyrimidine nucleotides constrict rat coronary resistance arteries. Uridine triphosphate (UTP) caused a dose-dependent constriction in coronary arteries stripped of endothelium. UTP also depolarized and increased cytosolic Ca2+ in coronary smooth muscle cells. Nisoldipine, an antagonist of voltage-operated Ca2+ channels, blocked the rise in cytosolic Ca2+ and reduced UTP-induced vasoconstriction by approximately 75% which suggests a prominent role for depolarization in this constrictor response. The ionic basis of UTP-induced depolarization was subsequently explored in coronary smooth muscle cells using whole-cell patch-clamp electrophysiology. In the absence of K+ and with CsCl in the pipette, UTP (40 microM) activated a sustained inwardly rectifying current (-0.66 +/- 0.10 pA/pF at -60 mV). A 100 mM reduction in bath Na+ shifted the reversal potential of this current (from -2 +/- 1 to -28 +/- 4 mV) and reduced the magnitude (from -2.26 +/- 0.61 to -0.51 +/- 0.11 pA/pF). In addition to activating a depolarizing cation current, UTP inhibited hyperpolarizing outward currents. Specifically, UTP inhibited ATP-sensitive and voltage-dependent K+ currents yet had no effect on inwardly rectifying and Ca2+-activated K+ channels. This study indicates that electromechanical coupling is integral to pyrimidine-induced constriction in coronary resistance arteries.     

Effect of acetylcholine on postjunctional membrane permeability in eel electroplaque

Acetylcholine (ACh) was applied iontophoretically to the innervated face of isolated eel electroplaques while the membrane potential was being recorded intracellularly. At the resting potential (about -85 mV) application of the drug produced depolarizations (ACh potentials) of 20 mV or more which became smaller when the membrane was depolarized and reversed in polarity at about zero membrane potential. The reversal potential shifted in the negative direction when external Na+ was partially replaced by glucosamine. Increasing external K+ caused a shift of reversal potential in the positive direction. It was concluded that ACh increased the permeability of the postjunctional membrane to both ions. Replacement of Cl- by propionate had no effect on the reversal potential. In Na+-free solution containing glucosamine the reversal potential was positive to the resting potential, suggesting that ACh increased the permeability to glucosamine. Addition of Ca++ resulted in a still more positive reversal potential, indicating an increased permeability to Ca++ as well. Analysis of the results indicated that the increases in permeability of the postjunctional membrane to K+, Na+, Ca++, and glucosamine were in the ratios of approximately 1.0:0.9:0.7:0.2, respectively. With these permeability ratios, all of the observed shifts in reversal potential with changes in external ionic composition were predicted accurately by the constant field equation.     

Ionic currents and ion channels of lobster olfactory receptor neurons

The role of the soma of spiny lobster olfactory receptor cells in generating odor-evoked electrical signals was investigated by studying the ion channels and macroscopic currents of the soma. Four ionic currents; a tetrodotoxin-sensitive Na+ current, a Ca++ current, a Ca(++)-activated K+ current, and a delayed rectifier K+ current, were isolated by application of specific blocking agents. The Na+ and Ca++ currents began to activate at -40 to -30 mV, while the K+ currents began to activate at -30 to -20 mV. The size of the Na+ current was related to the presence of a remnant of a neurite, presumably an axon, and not to the size of the soma. No voltage-dependent inward currents were observed at potentials below those activating the Na+ current, suggesting that receptor potentials spread passively through the soma to generate action potentials in the axon of this cell. Steady-state inactivation of the Na+ current was half-maximal at -40 mV. Recovery from inactivation was a single exponential function that was half-maximal at 1.7 ms at room temperature. The K+ currents were much larger than the inward currents and probably underlie the outward rectification observed in this cell. The delayed rectifier K+ current was reduced by GTP-gamma-S and AIF-4, agents which activate GTP-binding proteins. The channels described were a 215-pS Ca(++)-activated K+ channel, a 9.7-pS delayed rectifier K+ channel, and a 35-pS voltage-independent Cl- channel. The Cl- channel provides a constant leak conductance that may be important in stabilizing the membrane potential of the cell.     

Peeled mammalian skeletal muscle fibers. Possible stimulation of Ca2+ release via a transverse tubule-sarcoplasmic reticulum mechanism

Single muscle fibers from rabbit soleus and adductor magnus and from semitendinosus muscles were peeled to remove the sarcolemma and then stimulated to release Ca2+ by (a) caffeine application or (b) ionic depolarization accomplished via substitution of choline chloride for potassium propionate at constant [K+] X [Cl-] in the bathing solution. Each stimulus, ionic or caffeine, elicited an isometric tension transient that appeared to be due to Ca2+ released from the sarcoplasmic reticulum (SR). The peak magnitude of the ionic (Cl- -induced) tension transient increased with increasing Cl- concentration. The application of ouabain to fibers after peeling had no effect on either type of tension transient. However, soaking the fibers in a ouabain solution before peeling blocked the Cl- -induced but not the caffeine-induced tension transient, which suggests that ouabain's site of action is extracellular, perhaps inside transverse tubules (TTs). Treating the peeled fibers with saponin, which should disrupt TTs to a greater extent than SR membrane, greatly reduced or eliminated the Cl- -induced tension transient without significantly altering the caffeine-induced tension transient. These results suggest that the Cl- -induced tension transient is elicited via stimulation of sealed, polarized TTs rather than via ionic depolarization of the SR.     

Acetylcholine responses of identified neurons in Helix pomatia--III. Ionic composition of the depolarizing currents induced by acetylcholine

The ionic composition of the currents underlying the acetylcholine (ACh) depolarizations in the identified neurons B1 and B3 of the buccal ganglia of Helix pomatia was analysed. The equilibrium potential of the ACh responses was -2.8 +/- 0.6 mV (N = 49) and -4.0 +/- 0.7 mV (N = 79; mean +/- SEM) in the neurons B1 and B3, respectively. Replacement of NaCl in the bath solution by sucrose shifted the ACh equilibrium potential into the negative direction. A similar but less pronounced shift occurred when Ca2+ was substituted for Na+. Substitution of Cl- in the bath solution by propionate or an increase of the intracellular Cl- concentration did not affect the ACh equilibrium potential. Changes of K+ concentration in the bath between 1 and 50 mmol/l left the ACh equilibrium potential nearly unaffected when the Na+ concentration was at the control level. With a simultaneous reduction of extracellular Na+ an increase of K+ concentration shifted the ACh equilibrium potential towards more positive potentials. The findings are compatible with calculated K+ permeabilities if a K+ redistribution across the cell membrane is considered. In the neurons B1 and B3, channels operated by ACh are permeable for K+, Na+ and Ca2+, with the relative permeabilities 1.6:1.0:0.1.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. I. A study of the "cell-attached" configuration

A single channel current was studied in the membrane of the immature oocyte of the european frog (Rana esculenta) by using the "patch clamp" technique in the "cell attached" configuration. Single channel activity appeared as short outward currents when membrane potential was made positive inside; full activation required seconds to be complete, no inactivation being appreciable. Deactivation (or current block) upon membrane repolarization was so fast that no inward current could be detected in any case. The reversal potential, estimated by interpolating the I/V diagrams, was -30 mV using standard Ringer as electrode filling solution, and the elementary conductance was 95 pS. Neither reversal potential nor elementary conductance were affected by removal of external Ca2+ (Mg2+ or Ba2+ substitution) or external Cl- (methanesulphonate substitution). The reversal potential moved towards positive potentials by substituting external Na+ with K+, the magnitude of the shifts being consistent with a ratio PK/PNa = 6.4. A distinctive property of the current/voltage relation for this K-current is its anomalous bell-shape, the outward current displaying a maximum at membrane potentials around 75 mV with standard Ringer as electrode filling solution and tending to zero with more positive potentials.     

Cyclopiazonic acid activates a Ca2+-permeable, nonselective cation conductance in porcine and bovine tracheal smooth muscle.

Capacitative Ca2+ entry has been examined in several tissues and, in some, appears to be mediated by nonselective cation channels collectively referred to as "store-operated" cation channels; however, relatively little is known about the electrophysiological properties of these channels in airway smooth muscle. Consequently we examined the electrophysiological characteristics and changes in intracellular Ca2+ concentration associated with a cyclopiazonic acid (CPA)-evoked current in porcine and bovine airway smooth muscle using patch-clamp and Ca2+-fluorescence techniques. In bovine tracheal myocytes, CPA induced an elevation of intracellular Ca2+ that was dependent on extracellular Ca2+ and was insensitive to nifedipine (an L-type voltage-gated Ca2+ channel inhibitor). Using patch-clamp techniques and conditions that block both K+ and Cl- currents, we found that CPA rapidly activated a membrane conductance (I(CPA)) in porcine and bovine tracheal myocytes that exhibits a linear current-voltage relationship with a reversal potential around 0 mV. Replacement of extracellular Na+ resulted in a marked reduction of I(CPA) at physiological membrane potentials (i.e., -60 mV) that was accompanied by a shift in the reversal potential for I(CPA) toward more negative membrane potentials. In addition, I(CPA) was markedly inhibited by 10 microM Gd3+ and La3+ but was largely insensitive to 1 microM nifedipine. We conclude that CPA induces capacitative Ca2+ entry in porcine and bovine tracheal smooth muscle via a Gd3+- and La3+-sensitive, nonselective cation conductance.     

Voltage-activated currents in guinea pig pancreatic alpha 2 cells. Evidence for Ca2+-dependent action potentials

Glucagon-secreting alpha 2 cells were isolated from guinea pig pancreatic islets and used for electrophysiological studies of voltage- activated ionic conductances using the patch-clamp technique. The alpha 2 cells differed from beta cells in producing action potentials in the absence of glucose. The frequency of these potentials increased after addition of 10 mM arginine but remained unaffected in the presence of 5- 20 mM glucose. When studying the conductances underlying the action potentials, we identified a delayed rectifying K+ current, an Na+ current, and a Ca2+ current. The K+ current activated above -20 mV and then increased with the applied voltage. The Na+ current developed at potentials above -50 mV and reached a maximal peak amplitude of 550 pA during depolarizing pulses to -15 mV. The Na+ current inactivated rapidly (tau h approximately 0.7 ms at 0 mV). Half-maximal steady state inactivation was attained at -58 mV, and currents could no longer be elicited after conditioning pulses to potentials above -40 mV. The Ca2+ current first became detectable at -50 mV and reached a maximal amplitude of 90 pA (in extracellular [Ca2+] = 2.6 mM) at about -10 mV. Unlike the Na+ current, it inactivated little or not at all. Membrane potential measurements demonstrated that both the Ca2+ and Na+ currents contribute to the generation of the action potential. Whereas there was an absolute requirement of extracellular Ca2+ for action potentials to be elicited at all, suppression of the much larger Na+ current only reduced the upstroke velocity of the spikes. It is suggested that this behavior reflects the participation of a low-threshold Ca2+ conductance in the pacemaking of alpha 2 cells.     

Functional evidence for Na+-activated K+ channels in circular smooth muscle of the opossum lower esophageal sphincter

Na(+) reduction induces contraction of opossum lower esophageal sphincter (LES) circular smooth muscle strips in vitro; however, the mechanism(s) by which this occurs is unknown. The purpose of the present study was to investigate the electrophysiological effects of low Na(+) on opossum LES circular smooth muscle. In the presence of atropine, quanethidine, nifedipine, and substance P, conventional intracellular electrodes recorded a resting membrane potential (RMP) of -37.5 +/- 0.9 mV (n = 4). Decreasing [Na(+)] from 144.1 to 26.1 mM by substitution of equimolar NaCl with choline Cl depolarized the RMP by 7.1 +/- 1.1 mV. Whole cell patch-clamp recordings revealed outward K(+) currents that began to activate at -60 mV using 400-ms stepped test pulses (-120 to +100 mV) with increments of 20 mV from holding potential of -80 mV. Reduction of [Na(+)] in the bath solution inhibited K(+) currents in a concentration-dependent manner. Single channels with conductance of 49-60 pS were recorded using cell-attached patch-clamp configurations. The channel open probability was significantly decreased by substitution of bath Na(+) with equimolar choline. A 10-fold increase of [K(+)] in the pipette shifted the reversal potential of the single channels to the positive by -50 mV. These data suggest that Na(+)-activated K(+) channels exist in the circular smooth muscle of the opossum LES.     

Identification of a spontaneously active, Na+-permeable channel in guinea pig gallbladder smooth muscle

The action potential in gallbladder smooth muscle (GBSM) is caused by Ca2+ entry through voltage-dependent Ca2+ channels (VDCC), which contributes to the GBSM contractions. Action potential generation in GBSM is critically dependent on the resting membrane potential (about -50 mV), which is approximately 35 mV more positive of the K+ equilibrium potential. We hypothesized that a tonic, depolarizing conductance is present in GBSM and contributes to the regulation of the resting membrane potential and action potential frequency. GBSM cells were isolated from guinea pig gallbladders, and the whole cell patch-camp technique was used to record membrane currents. After eliminating the contribution of VDCC and K+ channels, we identified a novel spontaneously active cation conductance (I(cat)) in GBSM. This I(cat) was mediated predominantly by influx of Na+. Na+ substitution with N-methyl-D-glucamine (NMDG), a large relatively impermeant cation, caused a negative shift in the reversal potential of the ramp current and reduced the amplitude of the inward current at -50 mV by 65%. Membrane potential recordings with intracellular microelectrodes or in current-clamp mode of the patch-clamp technique indicated that the inhibition of I(cat) conductance by NMDG is associated with membrane hyperpolarization and inhibition of action potentials. Extracellular Ca2+, Mg2+, and Gd3+ attenuated the I(cat) in GBSM. Muscarinic stimulation did not activate the I(cat). Our results indicate that, in GBSM, an Na+-permeable channel contributes to the maintenance of the resting membrane potential and action potential generation and therefore plays a critical role in the regulation of GBSM excitability and contractility.     

Extracellular ATP-induced inward current in isolated epithelial cells of the endolymphatic sac.

Using whole-cell patch-clamp technique and Fura-2 fluorescence measurement, the presence of ATP-activated ion channels and its dependence on intracellular Ca2+ concentration ([Ca2+]i) in the epithelial cells of the endolymphatic sac were investigated. In zero current-clamp configuration, the average resting membrane potential was -66.8+/-1.3 mV (n=18). Application of 30 microM ATP to the bath induced a rapid membrane depolarization by 43.1+/-2.4 mV (n=18). In voltage-clamp configuration, ATP-induced inward current at holding potential (VH) of -60 mV was 169.7+/-6.3 pA (n=18). The amplitude of ATP-induced currents increased in sigmoidal fashion over the concentration range between 0.3 and 300 microM with a Hill coefficient (n) of 1.2 and a dissociation constant (Kd) of 11.7 microM. The potency order of purinergic analogues in ATP-induced current, which was 2MeSATP>ATPgammas>/=ATP>alpha, beta-ATP>ADP=AMP>/=adenosine=UTP, was consistent with the properties of the P2Y receptor. The independence of the reversal potential of the ATP-induced current from Cl- concentration suggests that the current is carried by a cation channel. The relative ionic permeability ratio of the channel modulated by ATP for cations was Ca2+>Na+>Li+>Ba2+>Cs+=K+. ATP (10 microM) increased [Ca2+]i in an external Ca2+-free solution to a lesser degree than that in the external solution containing 1.13 mM CaCl2. ATP-induced increase in [Ca2+]i can be mimicked by application of ionomycin in a Ca2+-free solution. These results indicate that ATP increases [Ca2+]i through the P2Y receptor with a subsequent activation of the non-selective cation channel, and that these effects of ATP are dependent on [Ca2+]i and extracellular Ca2+.     

External anions and volume-sensitive anion current in guinea-pig ventricular myocytes

The objective of this study was to determine the effects of anion replacement on volume-sensitive anion current in guinea-pig ventricular myocytes. Myocytes in the conventional whole-cell voltage-clamp configuration were superfused and dialysed with Na(+)-, K(+)-, and Ca(2+)-free solution, and exposed to external 75 mM Cl- solution of one-half normal osmolality. Prolonged exposures to hyposmotic solution promoted the development of outwardly-rectifying currents that were inactivated at high positive potentials and reversed in a Cl(-)-dependent manner (50 mV per decade pipette Cl- concentration). Replacement of external Cl- by iodide and aspartate affected the reversal potential (E(rev)) and slope conductance of the volume-sensitive current. Relative permeabilities calculated from changes in E(rev) were 1.49 +/- 0.09, 1.00, and 0.29 +/- 0.04 for iodide, Cl-, and aspartate, respectively; relative slope conductances between E(rev) and E(rev) + 40 mV were 1.21 +/- 0.09, 1.00, and 0.43 +/- 0.07, respectively. Replacement of Cl- also affected the time dependence of the volume-sensitive current; replacement by iodide reversibly enhanced the decay of outward current at positive potentials, whereas replacement by aspartate reduced it. These results are compared with earlier findings on noncardiac time- and voltage-dependent anion current activated by hyposmotic solution.     

Kinetic and steady-state properties of Na+ channel and Ca2+ channel charge movements in ventricular myocytes of embryonic chick heart

Nonlinear or asymmetric charge movement was recorded from single ventricular myocytes cultured from 17-d-old embryonic chick hearts using the whole-cell patch clamp method. The myocytes were exposed to the appropriate intracellular and extracellular solutions designed to block Na+, Ca2+, and K+ ionic currents. The linear components of the capacity and leakage currents during test voltage steps were eliminated by adding summed, hyperpolarizing control step currents. Upon depolarization from negative holding potentials the nonlinear charge movement was composed of two distinct and separable kinetic components. An early rapidly decaying component (decay time constant range: 0.12-0.50 ms) was significant at test potentials positive to -70 mV and displayed saturation above 0 mV (midpoint -35 mV; apparent valence 1.6 e-). The early ON charge was partially immobilized during brief (5 ms) depolarizing test steps and was more completely immobilized by the application of less negative holding potentials. A second slower-decaying component (decay time constant range: 0.88-3.7 ms) was activated at test potentials positive to -60 mV and showed saturation above +20 mV (midpoint -13 mV, apparent valence 1.9 e-). The second component of charge movement was immobilized by long duration (5 s) holding potentials, applied over a more positive voltage range than those that reduced the early component. The voltage dependencies for activation and inactivation of the Na+ and Ca2+ ionic currents were determined for myocytes in which these currents were not blocked. There was a positive correlation between the voltage dependence of activation and inactivation of the Na+ and Ca2+ ionic currents and the activation and immobilization of the fast and slow components of charge movement. These complementary kinetic and steady-state properties lead to the conclusion that the two components of charge movement are associated with the voltage-sensitive conformational changes that precede Na+ and Ca2+ channel openings.     

Serotonin depolarizes the membrane potential in rat mesenteric artery myocytes by decreasing voltage-gated K+ currents

We hypothesized that voltage-gated K+ (Kv) currents regulate the resting membrane potential (Em), and that serotonin (5-HT) causes Em depolarization by reducing Kv currents in rat mesenteric artery smooth muscle cells (MASMCs). The resting Em was about -40 mV in the nystatin-perforated patch configuration, and the inhibition of Kv currents by 4-aminopyridine caused marked Em depolarization. The inhibition of Ca2+-activated K+ (KCa) currents had no effect on Em. 5-HT (1 microM) depolarized Em by approximately 11 mV and reduced the Kv currents to approximately 63% of the control at -20 mV. Similar 5-HT effects were observed with the conventional whole-cell configuration with a weak Ca2+ buffer in the pipette solution, but not with a strong Ca2+ buffer. In the presence of tetraethylammonium (1mM), 5-HT caused Em depolarization similar to the control condition. These results indicate that the resting Em is largely under the regulation of Kv currents in rat MASMCs, and that 5-HT depolarizes Em by reducing Kv currents in a [Ca2+]i-dependent manner.     

A store-operated calcium channel in Drosophila S2 cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ > Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.     

Ionic currents underlying the action potential of Rana pipiens oocytes

Ionic currents in immature, ovulated Rana pipiens oocytes (metaphase I) were studied using the voltage-clamp technique. At this stage of maturity the oocyte can produce action potentials in response to depolarizing current or as an "off response" to hyperpolarizing current. Reducing external Na+ to 1/10 normal (choline substituted) eliminated the action potentials and both the negative-slope region and zero-crossing of the I-V relation. Reducing external Cl- to 1/10 or 1/100 normal (methanesulfonate substituted) lengthened the action potential. The outward current was reduced and a net inward current was revealed. By changing external Na+, Cl-, and K+ concentrations and using blocking agents (SITS, TEA), three voltage- and time-dependent currents were identified, INa, IK and ICl. The Na+ current activated at about 0 mV and reversed at very positive values which decreased during maturation. Inward Na+ current produced the upstroke of the action potential. During each voltage-clamp step the Na+ current activated slowly (seconds) and did not inactivate within many minutes. The Na+ current was not blocked by TTX at micromolar concentrations. The K+ current was present only in the youngest oocytes. Because IK was superimposed on a large leakage current, it appeared to reverse at the resting potential. When leakage currents were subtracted, the reversal potential for IK was more negative than -110 mV in Ringer's solution. IK was outwardly rectifying and strongly activated above -50 mV. The outward K+ current produced an after hyperpolarization at the end of each action potential. IK was blocked completely and reversibly by 20 mM external TEA. The Cl- current activated at about +10 mV and was outwardly rectifying. ICl was blocked completely and reversibly by 400 microM SITS added to the bathing medium. This current helped repolarize the membrane following an action potential in the youngest oocytes and was the only repolarizing current in more mature oocytes that had lost IK. The total leakage current had an apparently linear I-V relation and was separated into two components: a Na+ current (IN) and a smaller component carried by as yet unidentified ions.     

Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander

Voltage-dependent membrane currents of cells dissociated from tongues of larval tiger salamanders (Ambystoma tigrinum) were studied using whole-cell and single-channel patch-clamp techniques. Nongustatory epithelial cells displayed only passive membrane properties. Cells dissociated from taste buds, presumed to be gustatory receptor cells, generated both inward and outward currents in response to depolarizing voltage steps from a holding potential of -60 or -80 mV. Almost all taste cells displayed a transient inward current that activated at -30 mV, reached a peak between 0 and +10 mV and rapidly inactivated. This inward current was blocked by tetrodotoxin (TTX) or by substitution of choline for Na+ in the bath solution, indicating that it was a Na+ current. Approximately 60% of the taste cells also displayed a sustained inward current which activated slowly at about -30 mV and reached a peak at 0 to +10 mV. The amplitude of the slow inward current was larger when Ca2+ was replaced by Ba2+ and it was blocked by bath applied CO2+, indicating it was a Ca2+ current. Delayed outward K+ currents were observed in all taste cells although in about 10% of the cells, they were small and activated only at voltages more depolarized than +10 mV. Normally, K+ currents activated at -40 mV and usually showed some inactivation during a 25-ms voltage step. The inactivating component of outward current was not observed at holding potentials more depolarized -40 mV. The outward currents were blocked by tetraethylammonium chloride (TEA) and BaCl2 in the bath or by substitution of Cs+ for K+ in the pipette solution. Both transient and noninactivating components of outward current were partially suppressed by CO2+, suggesting the presence of a Ca2(+)-activated K+ current component. Single-channel currents were recorded in cell-attached and outside-out patches of taste cell membranes. Two types of K+ channels were partially characterized, one having a mean unitary conductance of 21 pS, and the other, a conductance of 148 pS. These experiments demonstrate that tiger salamander taste cells have a variety of voltage- and ion-dependent currents including Na+ currents, Ca2+ currents and three types of K+ currents. One or more of these conductances may be modulated either directly by taste stimuli or indirectly by stimulus-regulated second messenger systems to give rise to stimulus-activated receptor potentials. Others may play a role in modulation of neurotransmitter release at synapses with taste nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transient Cl- and K+ Currents during the Action Potential in Chara inflata (Effects of External Sorbitol,Cations, and Ion Channel Blockers)

In voltage-clamp experiments, a two-pulse procedure was used to investigate the ionic currents underlying the action potential in Chara inflata. A prepulse hyperpolarized the membrane from a resting potential of about -100 to -200 mV. The prepulse was followed by a second pulse that changed the potential difference (p.d.) to -100 mV and less negative values in steps of 20 mV. This two-pulse procedure induces action potentials that have a reproducible time course, which is essential for any comparative investigation of the action potential. The two-pulse procedure reveals that in the charophyte C. inflata the electric current flowing across the cell membranes during positive voltage-clamp steps from the resting p.d. consists of a leak current flowing from the start of the pulse, followed by a transient inward-going current, Ii, commencing after a delay, and preceding a delayed transient outward current, Io. The characteristics of the current components and their response to various ion channel blockers and ionic treatments suggest that: (a) Ii, which is blocked by the external application of 9-anthracenecarboxylic acid, is carried by Cl- and (b) Io, which is blocked by the external application of the organic anions tetraethylammonium (TEA+) and nonyltriethylammonium, is carried mainly by K+. The magnitude and behavior of these K+ and Cl- currents could be modified by changes in the external concentration of CaCl2, LiCl, or NaCl but not sorbitol. Hence, it is concluded that NaCl-enhanced transient inward Cl- current, Ii, is due to ionic effects of NaCl rather than to its osmotic effects. The modification of the K+ current, Io, either by changing external K+ concentrations or by blocking the current with TEA+, also alters the Cl- currents Ii.     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid ADP-evoked currents in human platelets recorded with the nystatin permeabilized patch technique.

"Whole-cell" patch recordings using nystatin permeabilization were made from single human platelets during application of agonists from a "puffer" pipette. In platelets clamped near the resting potential and bathed in Na+ saline, 40 microM ADP activated a transient inward current within tens of milliseconds. At -73 mV the current lasted between 0.1 and 1 s and had a peak of between 13 and 31 pA in different cells. Ion substitution experiments indicated that the channel is permeable to Na+,K+, and Ba2+ and presumably also to Ca2+, but is not permeable to Cl-. The single channel conductance was 15 pS (near the resting potential) in nominally Ca(2+)-free saline and 11 picosiemens in BaCl2 saline. Thrombin, at 1 unit/ml, did not elicit detectable currents during a 3-s application in platelets bathed in 1 mM Ca2+, Na+ saline. Under the same conditions, in fura-2-loaded cells, thrombin-evoked Ca2+ entry (monitored by Mn2+ quench) was detectable after a delay of 1.4 s. This suggests that early thrombin-evoked Ca2+ entry occurs via small conductance channels, below the resolution of the patch clamp technique, or by an electroneutral pathway. The ADP-evoked channel has the requisite speed of activation to account for the rapid Ca2+ influx observed during stopped-flow studies of agonist-evoked changes in [Ca2+]i.