A long-lasting potentiation of calmodulin-mediated chloride channel activity without a mediation of protein kinase C in Xenopus oocytes injected with rat brain mRNA.

When Xenopus oocytes injected with rat brain poly(A)+RNA were voltage-clamped in a recording solution containing Ca2+, a depolarization pulse induced a transient current, ICl(Ca), which reflects calmodulin-mediated opening of endogenous Cl- channels in response to a Ca2+ influx through Ca2+ channels of brain origin. ICl(Ca) could be repetitively observed with a steady amplitude over 1 h, whereas the response was greatly potentiated for more than 30 min after a brief stimulation of muscarinic or other Ca2(+)-mobilizing receptors. The enhancement of ICl(Ca) was mimicked by an injection of inositol-1,4,5-trisphosphate or by a treatment with A23187, but not affected by treatments that stimulate or inhibit protein kinase C activity. Isolated Ba2+ current flowing through voltage-sensitive Ca2+ channels was not augmented during the facilitation of ICl(Ca). These observations indicate that the endogenous calmodulin/Cl- channel system may memorize an over-threshold increase in the intracellular Ca2+ concentration and potentiate the Ca2(+)-sensitiveness of the Cl- channel. A long-lasting autoregulation of Ca2(+)-dependent ion channel activity is suggested.     

Positive regulation of capacitative Ca2+ entry by intracellular Ca2+ in Xenopus oocytes expressing rat TRP4

We have investigated the role of intracellular Ca2+ in the opening of capacitative Ca2+ entry (CCE) channels formed with rat TRP4 (rTRP4) using Xenopus oocytes. In rTRP4-expressing oocytes pretreated with thapsigargin, perfusion with A23187, a Ca2+ ionophore, significantly potentiated the delayed phase of the CCE-mediated Cl- current response evoked by extracellular perfusion with Ca2+, without affecting the transient phase of CCE response. In control oocytes, the potentiation of delayed CCE response by A23187 was not significant. Using cut-open recording in combination with artificial intracellular perfusion of oocytes, CCE-mediated Cl- response was recorded at controlled cytosolic Ca2+ concentrations. Intracellular perfusion with a Ca2+ free solution containing 10 mM EGTA abolished most of the CCE responses of both non-injected and rTRP4-expressing oocytes. The native CCE response was not fully recovered by subsequent increases in the intracellular Ca2+ concentration up to 300 nM. However, CCE response of the rTRP4-expressing oocytes was restored at an internal Ca2+ concentration of 110 nM. Blockade of endogenous Cl- channels with anion channel blocker isolated Ca2+ current flowing through CCE channels and clarified the difference in the sensitivity to an internal Ca2+ concentration. These findings indicate that recombinant CCE channels formed with rTRP4 are positively regulated by cytosolic Ca2+ at higher sensitivity compared to oocyte-endogenous CCE channels.     

Angiotensin II receptors in Xenopus oocytes.

Electrical recordings were used to study the sensitivity of native Xenopus oocytes to the octapeptide angiotensin II (AII). AII elicited oscillatory currents associated with an increase in membrane conductance to Cl-. Responsiveness to AII varied greatly between oocytes taken from different frogs, and to a lesser extent between oocytes from the same ovary. Oocytes from frogs showing high sensitivity had response thresholds between 0.5-1.0 nM AII, and at a holding potential of -60 mV, responded to 1 microM AII with currents greater than 3 microA. In contrast, oocytes from some frogs gave no response, even to 10 microM AII. A total of 618 oocytes from 79 frogs were tested for sensitivity to AII, and oocytes from 85% of frogs gave detectable electrical responses. Oscillatory Cl- currents elicited by AII were largely independent of extracellular Ca2+, were abolished by chelation of intracellular Ca2+ using EGTA and were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3). In addition to oscillatory Cl- currents, AII also evoked an influx of extracellular Ca2+, giving rise to a transient inward Cl- current on membrane hyperpolarizing steps. These experiments all suggested that AII responses were elicited through activation of an intracellular messenger pathway triggered by hydrolysis of inositolphospholipids, mobilization of intracellular Ca2+ by inositol polyphosphates, and activation of Ca(2+)-gated Cl- channels. The effect of manual or enzymic defolliculation on AII responses was studied in nine separate experiments recording from 70 defolliculated oocytes. Efficacy of defolliculation procedures was assayed using scanning electron microscopy, which confirmed removal of 90 to greater than 98% of follicular cells.(ABSTRACT TRUNCATED AT 250 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.     

alpha-Bungarotoxin-sensitive hippocampal nicotinic receptor channel has a high calcium permeability.

The hippocampal nicotinic acetylcholine receptor (nAChR) is a newly identified ligand-gated ion channel that is blocked by the snake toxin alpha-bungarotoxin (alpha-BGT) and that probably contains the alpha 7 nAChR subunit in its structure. Here its ion selectivity was characterized and compared with that of the N-methyl-D-aspartate (NMDA) receptor channel. The reversal potentials (VR) of acetylcholine- and NMDA-activated whole-cell currents were determined under various ionic conditions. Using ion activities and a Goldman-Hodgkin-Katz equation for VR shifts in the presence of Ca2+, permeability ratios were calculated. For the alpha-BGT-sensitive nAChR, PNa/PCs was close to 1 and Cl- did not contribute to the currents. Changing the [Ca2+]0 from 1 to 10 mM, the VRs of the nAChR and NMDA currents were shifted by +5.6 +/- 0.4 and +8.3 +/- 0.4 mV, respectively, and the nAChR current decay was accelerated. These shifts yielded PCa/PCss of 6.1 +/- 0.5 for the nAChR channel and 10.3 +/- 0.7 for the NMDA channel. Thus, the neuronal alpha-BGT-sensitive nAChR is a cation channel considerably selective to Ca2+ and may mediate a fast rise in intracellular Ca2+ that would increase in magnitude with membrane hyperpolarization.     

Inositol trisphosphate isomers, but not inositol 1,3,4,5-tetrakisphosphate, induce calcium influx in Xenopus laevis oocytes

To investigate the mechanisms by which inositol phosphates regulate cytosolic free Ca2+ concentration ([Ca2+]c), we injected Xenopus oocytes with inositol phosphates and measured Ca2+-activated Cl- currents as an assay of [Ca2+]c. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) injection (0.1-10.0 pmol) induced an initial transient Cl- current (I1) followed by a second more prolonged Cl- current (I2). Both currents were Ca2+-dependent, but the source of Ca2+ was different. Release of intracellular Ca2+ stores produced I1, whereas influx of extracellular Ca2+ produced I2; Ca2+-free bathing media and inorganic calcium channel blockers (Mn2+, Co2+) did not alter I1 but completely and reversibly inhibited I2. Injection of the Ins(1,4,5)P3 metabolite, inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (0.2-10.0 pmol) generated a Ca2+-dependent Cl- current with superimposed current oscillations that resulted from release of intracellular Ca2+, not Ca2+ influx. Injection of the Ins(1,3,4,5)P4 metabolite, inositol 1,3,4-trisphosphate (10.0 pmol), or the synthetic inositol trisphosphate isomer, inositol 2,4,5-trisphosphate (1.0-10.0 pmol), mimicked the effect of Ins(1,4,5)P3, stimulating an I1 resulting from release of intracellular Ca2+ and an I2 resulting from influx of extracellular Ca2+. The results indicate that several inositol trisphosphate isomers stimulate both release of intracellular Ca2+ and influx of extracellular Ca2+. Ins(1,3,4,5)P4 also stimulated release of intracellular Ca2+, but it was neither sufficient nor required for Ca2+ influx.     

Effects of ginsenosides on glycine receptor alpha1 channels expressed in Xenopus oocytes

Ginsenosides, major active ingredients of Panax ginseng, are known to regulate the excitatory ligand-gated ion channel activity. Recent reports showed that ginsenosides attenuate nicotinic acetylcholine and NMDA receptor channel activity. However, it is not known whether ginsenosides also affect the inhibitory ligand-gated ion channel activity. We investigated the effect of ginsenosides on human glycine alpha1 receptor channel activity expressed in Xenopus oocytes using a two-electrode voltage clamp technique. Treatment of ginsenoside Rf enhances glycine-induced inward peak current (IGly) with dose dependent and reversible manner but ginsenoside Rf itself did not elicit membrane currents. The half-stimulatory concentrations (EC50) of ginsenoside Rf was 49.8 +/- 8.9 microM. Glycine receptor antagonist strychnine completely blocked the inward current elicited by glycine plus ginsenoside Rf. Cl- channel blocker 4,4'-disothiocyanostilbene-2,2'-disulfonic acid (DIDS) also blocked the inward current elicited by glycine plus ginsenoside Rf. We also tested the effect of eight individual ginsenosides (i.e., Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, and Ro) in addition to ginsenoside Rf. We found that five of them significantly enhanced the inward current induced by glycine with the following order of potency: Rb1 > Rb2 > Rg2 > or = Rc > Rf > Rg1 > Re. These results indicate that ginsenosides might regulate gylcine receptor expressed in Xenopus oocytes and this regulation might be one of the pharmacological actions of Panax ginseng.     

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.     

NMDA and glycine regulate the affinity of the Mg2+-block site in NR1-1a/NR2A NMDA receptor channels expressed in Xenopus oocytes

NMDA receptors are glutamate-regulated ion channels of critical importance for many neurophysiological and neuropathological processes. Mg2+ blocks the NMDA receptor by binding to the channel pore with an apparent affinity that depends on the membrane potential. We have investigated the effect of NMDA and the required co-agonist glycine on the affinity of the Mg2+ block site in NR1-1a/NR2A NMDA receptors expressed in Xenopus oocytes. We found that NMDA and glycine increase the IC50 value of the Mg2+-block site at pH 7.4 and in the presence of physiological concentration of Ca2+. The increase the IC50 value may correspond to a decrease in Mg2+-block affinity. This effect may result in an increased influx of Ca2+, and this influx may constitute up to a third of the total Ca2+ influx induced by NMDA. At high pH, or at low concentrations of Ca2+, NMDA and glycine have an opposite effect and instead decreased the IC50 value of the Mg2+-block. These results indicate that glutamate and glycine can regulate the affinity of the Mg2+-block site. This effect may have implications for the understanding the role of NMDA receptors both under physiological and pathophysiological conditions.     

T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.     

Cellular mechanisms of carbachol-stimulated Cl- secretion in rat epididymal epithelium

Neurotransmitter-controlled Cl- secretions play an important role in maintenance of the epididymal microenvironment for sperm maturation. This study was carried out to investigate the effect of carbachol (CCH) on the cultured rat epididymal epithelium and the signal transduction mechanisms of this response. In normal K-H solution, CCH added basolaterally elicited a biphasic Isc response consisting of a transient spike followed by a second sustained response. Ca2+ activated Cl- channel blocker disulfonic acid stilbene (DIDS, 300 microM) only inhibited part of the CCH-induced Isc response, while nonselective Cl- channel blocker diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced all, indicating the involvement of different conductance pathways. Both peaks of the CCH-induced Isc response could be significantly inhibited by pretreatment with an adenylate cyclase inhibitor, MDL12330A (50 microM). An increase in intracellular cAMP content upon stimulation of CCH was measured. All of the initial peak and part of the second peak could be inhibited by pretreatment with Ca2+-chelating agent BAPTA/AM (50 microM) and an endoplasmic reticulum Ca2+ pump inhibitor, Thapsigagin (Tg, 1 microM). In a whole-cell patch clamp experiment, CCH induced an inward current in the single cell. Two different profiles of currents were found; the first component current exhibited an outward rectifying I-V relationship in a time and voltage-dependent manner, and the current followed showed a linear I-V relationship. The carbachol-induced current was found to be partially blockable by DIDS and could be completely blocked by DPC. The above results indicate that the CCH-induced Cl- secretion could be mediated by Ca2+ and cAMP-dependent regulatory pathways.     

Novel Cl- currents elicited by follicle stimulating hormone and acetylcholine in follicle-enclosed Xenopus oocytes

Voltage-clamp techniques were used to study the membrane currents elicited by follicle stimulating hormone (FSH) and acetylcholine (ACh) in follicle-enclosed oocytes of Xenopus laevis (follicles). Both agonists caused complex responses that were more evident when the follicles were in hypotonic Ringer solution (HR; 190.4 mosM). In this medium, currents activated by FSH regularly showed three phases whereas currents activated by ACh displayed three to six phases. At a holding potential of -60 mV, FSH, and ACh responses involved combinations of inward and outward currents. Both FSH and ACh responses included a slow smooth inward component that was associated with an increase in membrane conductance, mainly to Cl- (S(in)). This current was strongly dependent on the osmolarity of the external solution: an increase in osmolarity of the HR solution of 18-20 mosM caused a 50% decrease in S(in). In contrast, a fast and transient Cl- current (F(in)) specifically elicited by ACh was not dependent on osmolarity. Both, F(in) and S(in) currents required the presence of follicular cells, since defolliculation using three different methods abolished all the response to FSH and at least four components of the ACh responses. The membrane channels carrying F(in) and oscillatory Cl- currents elicited by stimulation of ACh or serum receptors, were much more permeable to I- and Br- than Cl-, whereas S(in) channels were equally permeable to these anions. Unlike the oscillatory Cl- currents generated in the oocyte itself, S(in) and F(in) currents in follicle-enclosed oocytes were not abolished by chelation of intracellular Ca2+, either with EGTA or BAPTA, which suggests that intracellular Ca2+ does not play a critical role in the activation of these currents. Our experiments show that S(in) and F(in) currents are quite distinct from the previously characterized oscillatory Cl- responses of oocytes. Moreover, the results strongly suggest that the FSH and ACh receptors, the Cl- channels mediating the F(in) and S(in) currents, together with the necessary elements for their activation, are all located in the follicular cells and not in the oocyte. Many aspects of follicular cell physiology in Xenopus laevis, and other species, are regulated by hormones and neurotransmitters, including FSH and ACh. The follicular Cl- currents described in this paper may play an important role in the follicular cell-oocyte development.     

Time course of the initial [Ca2+]i response to extracellular ATP in smooth muscle depends on [Ca2+]e and ATP concentration.

In response to extracellular application of 50 microM ATP, all individual porcine aortic smooth muscle cells respond with rapid rises from basal [Ca2+]i to peak [Ca2+]i within 5 s. The time from stimulus to the peak of the [Ca2+]i response increases with decreasing concentration of ATP. At ATP concentrations of 0.5 microM and below, the time to the [Ca2+]i peak varies more significantly from cell to cell than at higher concentrations, and each cell shows complicated initiation and decay kinetics. For any individual cell, the lag phase before a response decreases with increasing concentration of ATP. An increase in lag time with decreasing ATP concentration is also observed in the absence of extracellular Ca2+, but the lag phase is more pronounced, especially at concentrations of ATP below 0.5 microM. Whole-cell patch-clamp electrophysiology shows that in porcine aortic smooth muscle cells, ATP stimulates an inward current carried mainly by Cl- ion efflux with a time course similar to the [Ca2+]i changes and no detectable current from an ATP-gated cation channel. A simple signal cascade initiation kinetics model, starting with nucleotide receptor activation leading to IP3-mediated Ca2+ release from IP3-sensitive internal stores, fits the data and suggests that the kinetics of the Ca2+ response are dominated by upstream signal cascade components.     

Ion currents and membrane domains in the cleaving Xenopus egg

We used an extracellular vibrating probe to measure ion currents through the cleaving Xenopus laevis egg. Measurements indicate sharp membrane heterogeneities. Current leaves the first cleavage furrow after new, unpigmented membrane is inserted. This outward current may be carried by K+ efflux. No direct involvement of the Na+,K+-ATPase in the generation of this outward current is detected at first cleavage. Inward current enters the old, pigmented membrane; however, it does not enter uniformly. The inward current is largest at the old membrane bordering the new membrane. This suggests a heterogeneous ion channel distribution within the old membrane. Experiments suggest that the inward current may be carried by Na+ influx, Ca2+ influx, and Cl- efflux. No steady currents were detected during grey crescent formation, the surface contraction waves preceding cleavage, or with groove formation at the beginning of cleavage.     

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)     

Voltage-dependent conductances in Limulus ventral photoreceptors

The voltage-dependent conductances of Limulus ventral photoreceptors have been investigated using a voltage-clamp technique. Depolarization in the dark induces inward and outward currents. The inward current is reduced by removing Na+ or Ca2+ and is abolished by removing both ions. These results suggest that both Na+ and Ca2+ carry voltage-dependent inward current. Inward current is insensitive to tetrodotoxin but is blocked by external Ni2+. The outward current has a large transient component that is followed by a smaller maintained component. Intracellular tetraethylammonium preferentially reduces the maintained component, and extracellular 4-amino pyridine preferentially reduces the transient component. Neither component is strongly affected by removal of extracellular Ca2+ or by intracellular injection of EGTA. It is concluded that the photoreceptors contain at least three separate voltage-dependent conductances: 1) a conductance giving rise to inward currents; 2) a delayed rectifier giving rise to maintained outward K+ current; and 3) a rapidly inactivating K+ conductance similar to the A current of molluscan neurons.     

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.     

Ion transport in an immortalized rat submandibular cell line SMG-C6

The immortalized rat submandibular epithelial cell line, SMG-C6, cultured on porous tissue culture supports, forms polarized, tight-junction epithelia facilitating bioelectric characterization in Ussing chambers. The SMG-C6 epithelia generated transepithelial resistances of 956+/-84Omega.cm2 and potential differences (PD) of -16.9 +/- 1.5mV (apical surface negative) with a basal short-circuit current (Isc) of 23.9 +/- 1.7 microA/cm2 (n = 69). P2 nucleotide receptor agonists, ATP or UTP, applied apically or basolaterally induced a transient increase in Isc, followed by a sustained decreased below baseline value. The peak DeltaIsc increase was partly sensitive to Cl- and K+ channel inhibitors, DPC, glibenclamide, and tetraethylammonium (TEA) and was completely abolished following Ca2+ chelation with BAPTA or bilateral substitution of gluconate for Cl-. The major component of basal Isc was sensitive to apical Na+ replacement or amiloride (half-maximal inhibitory concentration 392 nM). Following pretreatment with amiloride, ATP induced a significantly greater Isc; however, the poststimulatory decline was abolished, suggesting an ATP-induced inhibition of amiloride-sensitive Na+ transport. Consistent with the ion transport properties found in Ussing chambers, SMG-C6 cells express the rat epithelial Na+ channel alpha-subunit (alpha-rENaC). Thus, cultured SMG-C6 cells produce tight polarized epithelia on permeable support with stimulated Cl- secretory conductance and an inward Isc accounted for by amiloride-sensitive Na+ absorption.     

A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes.

Activation of T lymphocytes results in an increase in intracellular Ca2+ due in large part to influx of extracellular Ca2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of -90 mV but not from -60 mV. This whole-cell current was insensitive to tetrodotoxin, carried by Ba2+, and blocked by Ni2+. Occupancy of the T lymphocyte antigen receptor increased the current's magnitude. These data suggest that antigen receptor-induced Ca2+ entry in T lymphocytes may be mediated by a voltage-regulated Ca channel.     

Excitation contraction coupling in skeletal muscle: evidence for a role of slow Ca2+ channels using Ca2+ channel activators and inhibitors in the dihydropyridine series

Ca2+ current and tension have been simultaneously recorded from single twitch fibres of the semi-tendinosus of Rana esculenta in a medium containing a physiological Ca2+ concentration (1.8 mM). Under appropriate conditions it can be shown that tension develops in two phases. The first is rapid and reaches its maximum before activation of the inward Ca2+ current. The second phase is slower and with a time course which appears to be correlated with that of the inward current. Nifedipine, a specific Ca2+ channel inhibitor greatly reduced ICa2+ and the slower component of tension. Bay K8644 a Ca2+ channel activator, which has receptors on T-tubule, increased ICa2+ and the slow component of tension. These results indicate that a slow component of skeletal muscle contraction is related to the inward Ca2+ current flowing through dihydropyridine sensitive voltage-dependent Ca2+ channels.