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)     

Membrane properties of isolated mudpuppy taste cells

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.     

E-series prostaglandins activate cAMP-mediated potassium currents in follicle-enclosed Xenopus oocyte

Receptor-mediated responses to prostaglandins E1 and E2 are shown by electrophysiological methods in follicle-enclosed oocytes of Xenopus laevis. In voltage-clamped oocytes, prostaglandins E1 and E2 elicited an outward hyperpolarizing current. This outward membrane current was caused by an increase in K+ conductance. The prostaglandin-induced current was augmented by adenylate cyclase activator, forskolin, and by phosphodiesterase inhibitor, theophylline, indicating that adenosine 3', 5'-cyclic monophosphate (cAMP) is involved in activating the K+ current. The prostaglandin responses were either abolished or greatly reduced by removing follicular cells with collagenase, suggesting that prostaglandin receptors reside in the follicular cells.     

Vasoactive intestinal peptide-stimulated Cl- secretion: activation of cAMP-dependent K+ channels

Vasoactive intestinal peptide (VIP) stimulates active Cl- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K+ conductance. To demonstrate the role of this K- conductance, we measured short-circuit current (I(SC)) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I(SC) from 3 +/- 0.4 microA/cm2 to 75 +/- 11 microA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 microM), reduced I(SC) by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K+ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K+ current (131 +/- 26 pA, n = 15) when membrane potential was held at the Cl- equilibrium potential (E(Cl-) = -2 mV), and activated inward current (179 +/- 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (E(K+) = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nM), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba2+ , 5 mM), but not 293B, blocked this K+ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K + current flow to demonstrate the channels that underlie this K+ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca2+ concentration, [Ca2 +]i. Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K+ current (185 +/- 56 pA, n = 8) without altering [Ca2+]i. These results demonstrate that VIP activates a basolateral membrane K+ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.     

Epithelium and/or theca are required for ATP-elicited K+ current in follicle-enclosed Xenopus oocytes

The Xenopus follicular cell membrane is endowed with ATP-sensitive K+ channels, which are operated by various transmitters. These generate the ionic response named IK,cAMP via a mechanism that involves intracellular cAMP synthesis. It is known that opening these K+ channels favors oocyte maturation. Follicle stimulation by adenosine (Ado) or ATP consistently generates a strong IK,cAMP via activation of P1 and P3 purinergic receptors; however, ATP can also inhibit IK,cAMP, apparently acting on a third receptor type. Here, we show that IK,cAMP might be elicited by ATP released within the follicle, and that current activation by ATP was entirely dependent on the presence of epithelial and/or theca layers. Morphological studies confirmed that removal of epithelium/theca in these follicles (e.t.r.) was complete, and activation of fast Cl- (Fin) currents by ATP in e.t.r. follicles confirmed that communication between oocyte and follicular cells remained unchanged. Thus, dependence on epithelium/theca was specific for ATP-elicited K+ current. Using UTP and betagamma-MeATP as specific purinergic agents for IK,cAMP inhibition and activation, respectively, it was found that inhibition of IK,cAMP elicited by ATP or UTP was robustly present in e.t.r. follicles but was absent or strongly decreased in whole follicles (w.f.). Accordingly, this indicated that in w.f., epithelium and/or theca downregulated the IK,cAMP inhibition evoked by ATP, and that this control mechanism was absent in e.t.r. follicles. We suggest that this notable action on follicular cells involves one or both of two mechanisms, a paracrine transmitter released from epithelial and/or theca layers and action of ecto-ATPases.     

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)     

Voltage-gated potassium channels in brown fat cells

We studied the membrane currents of isolated cultured brown fat cells from neonatal rats using whole-cell and single-channel voltage-clamp recording. All brown fat cells that were recorded from had voltage-gated K currents as their predominant membrane current. No inward currents were seen in these experiments. The K currents of brown fat cells resemble the delayed rectifier currents of nerve and muscle cells. The channels were highly selective for K+, showing a 58-mV change in reversal potential for a 10-fold change in the external [K+]. Their selectivity was typical for K channels, with relative permeabilities of K+ greater than Rb+ greater than NH+4 much greater than Cs+, Na+. The K currents in brown adipocytes activated with a sigmoidal delay after depolarizations to membrane potentials positive to -50 mV. Activation was half maximal at a potential of -28 mV and did not require the presence of significant concentrations of internal calcium. Maximal voltage-activated K conductance averaged 20 nS in high external K+ solutions. The K currents inactivated slowly with sustained depolarization with time constants for the inactivation process on the order of hundreds of milliseconds to tens of seconds. The K channels had an average single-channel conductance of 9 pS and a channel density of approximately 1,000 channels/cell. The K current was blocked by tetraethylammonium or 4-aminopyridine with half maximal block occurring at concentrations of 1-2 mM for either blocker. K currents were unaffected by two blockers of Ca2+-activated K channels, charybdotoxin and apamin. Bath-applied norepinephrine did not affect the K currents or other membrane currents under our experimental conditions. These properties of the K channels indicate that they could produce an increase in the K+ permeability of the brown fat cell membrane during the depolarization that accompanies norepinephrine-stimulated thermogenesis, but that they do not contribute directly to the norepinephrine-induced depolarization.     

Expression of a Cs(+)-resistant guard cell K+ channel confers Cs(+)-resistant, light-induced stomatal opening in transgenic arabidopsis.

Inward-rectifying K+ (K+in) channels in the guard cell plasma membrane have been suggested to function as a major pathway for K+ influx into guard cells during stomatal opening. When K+in channels were blocked with external Cs+ in wild-type Arabidopsis guard cells, light-induced stomatal opening was reduced. Transgenic Arabidopsis plants were generated that expressed a mutant of the guard cell K+in channel, KAT1, which shows enhanced resistance to the Cs+ block. Stomata in these transgenic lines opened in the presence of external Cs+. Patch-clamp experiments with transgenic guard cells showed that inward K+(in) currents were blocked less by Cs+ than were K+ currents in controls. These data provide direct evidence that KAT1 functions as a plasma membrane K+ channel in vivo and that K+in channels constitute an important mechanism for light-induced stomatal opening. In addition, biophysical properties of K+in channels in guard cells indicate that components in addition to KAT1 may contribute to the formation of K+in channels in vivo.     

Microvascular endothelial cells from human omentum lack an inward rectifier K+ current

In most macrovascular endothelial cell (EC) preparations, resting membrane potential is determined by the inwardly rectifying K+ current (I(K1)), whereas in microvascular EC the presence of I(K1) varies markedly. Cultured microvascular EC from small vessels of human omentum were examined by means of the voltage-clamp technique to elucidate the putative role of I(K1) in maintaining resting membrane potential. Macrovascular EC from human iliac artery and bovine aorta served as reference. Human omentum EC showed an outwardly rectifying current-voltage relation. Inward current was hardly sensitive to variations of extracellular [K+] and Ba2+ block suggesting lack of I(K1). However, substitution of extracellular [Na+] and/or [Cl-] affected the current-voltage relation indicating that Na+ and Cl- contribute to basal current. Furthermore, outward current was reduced by tetraethylammonium (10 mM), and cell-attached recordings suggested the presence of a Ca2+-activated K+ current. In contrast to human omentum EC, EC from human iliac artery and bovine aorta possessed inwardly rectifying currents which were sensitive to variations of extracellular [K+] and blocked by Ba2+. Thus, the lack of I(K1) in human omentum EC suggests that resting membrane potential is determined by Na+ and Cl- currents in addition to K+ outward currents.     

Properties of "creep currents" in single frog atrial cells

Changes in membrane current in response to an elevation of [Na]i were studied in enzymatically dispersed frog atrial cells. Na loading by either intracellular dialysis or exposure to the Na ionophore monensin produces changes in membrane current that resemble the "creep currents" originally observed in cardiac Purkinje fibers during exposure to low-K solutions. Na loading induces a transient outward current during depolarizing voltage-clamp pulses, followed by an inward current in response to repolarization back to the holding potential. In contrast to cardiac Purkinje fibers, Na loading of frog atrial cells induces creep currents without accompanying transient inward currents. Creep currents induced by Na loading are insensitive to K channel antagonists like Cs and 4-aminopyridine; they are not influenced by doses of Ca channel antagonists that abolish iCa, but are sensitive to changes in [Ca]o or [Na]o. A comparison of the time course of development of inward creep currents are not tail currents associated with iCa. Inward creep currents can also be induced by experimental interventions that increase the iCa amplitude. Exposure to isoproterenol enhances the iCa amplitude and induces inward creep currents; both can be attenuated by Ca channel antagonists. Both inward and outward creep currents are blocked by low doses of La, independently of La's ability to block iCa. It is concluded that (a) creep currents are not mediated by voltage-gated Na, Ca, or K channels or by an electrogenic Na,K pump; (b) inward creep currents induced either by Na loading or in response to an increase in the amplitude of iCa are triggered by an elevation of [Ca]i; and (c) creep currents may be generated by either an electrogenic Na/Ca exchange mechanism or by a nonselective cation channel activated by [Ca]i.     

A slowly inactivating potassium current in native oocytes of Xenopus laevis

Membrane currents were recorded in voltage-clamped oocytes of Xenopus laevis in response to voltage steps. We describe results obtained in oocytes obtained from one donor frog, which showed an unusually large outward current upon depolarization. Measurements of reversal potentials of tail currents in solutions of different K+ concentration indicated that this current is carried largely by K+ ions. It was strongly reduced by extracellular application of tetraethylammonium, though not by Ba2+ or 4-aminopyridine. Removal of surrounding follicular cells did not reduce the K+ current, indicating that it arises across the oocyte membrane proper. Activation of the K+ conductance was first detected with depolarization to about -12 mV, increased with a limiting voltage sensitivity of 3 mV for an e-fold change in current, and was half-maximally activated at about +10 mV. The current rose following a single exponential timecourse after depolarization, with a time constant that shortened from about 400 ms at -10 mV to about 15 ms at +80 mV. During prolonged depolarization the current inactivated with a time constant of about 4 s, which did not alter greatly with potential. The K+ current was independent of Ca2+, as it was not altered by addition of 10 mM Mn2+ to the bathing medium, or by intracellular injection of EGTA. Noise analysis of K+ current fluctuations indicated that the current is carried by channels with a unitary conductance of about 20 ps and a mean open lifetime of about 300 ms (at room temperature and potential of +10 to +20 mV).     

Conversion of beating to bursting pacemaker activity: Action of quinidine

External quinidine converts the pacemaker neurone L-11, found in the Aplysia abdominal ganglion, from spontaneously "beating" to "bursting" discharge activity. Quinidine-induced bursting ceased when entry of Ca2+ ions into the cells was blocked in a Ca2+-free, Co2+-containing solution or if internal Ca2+ accumulation was prevented by the injection of EGTA. The analysis of membrane currents from voltage clamp experiments showed that quinidine blocks the Ca2+ inward current in a dose- and time-dependent manner. In addition, the currents were displaced to the left on the voltage axis, causing an increase of the inward current at negative membrane potentials. External quinidine suppresses the Ca2+-activated K+ current induced by intracellular Ca2+ injections and acts to prolong its decay phase. The slowing of the decay phase of the Ca2+-activated K+ current by quinidine was prevented after intracellular injection of EGTA, indicating that Ca2+ removal is impaired by the drug. It is suggested that the increase of Ca2+ inward current at negative potentials and the prolonged activation of the Ca2+-activated K+ current play a major role in causing the bursting discharge behavior in normally beating cells.     

Currents related to the sodium-calcium exchange in squid giant axon

We report the measurement of a Cai-activated membrane current in dialyzed squid axon under membrane potential control with a low-noise voltage clamp. Two additional voltage clamp systems were used to clamp the external guard plates to a value that prevented the establishment of potential differences between the central and lateral compartments of the experimental chamber. This reduced to a minimum the contribution of membrane currents generated at the axon ends to the current measured in the central pool. This latter current was reduced by using internal and external solutions designed to diminish at a maximum membrane currents, while maintaining the conditions for optimal operation of the Na+-Ca2+ exchange. Thus TTX was used to block Na+ channels and prolonged exposure to K+-free media was used to eliminate K+ conductance. The maximum concentration of external sodium was 200 mM. The addition of fixed amounts of free ionic calcium to the internal solution, activated a current whose direction and magnitude depended on the thermodynamic driving forces for calcium and sodium. When the experimental conditions determined an inwardly directed current, this depended on the presence of external sodium, and lithium could not substitute for it. The Cai-activated current, was blocked by external lanthanum and showed a high temperature dependence. In experiments in which the reversal potential was measured for the Cai-activated current, it was found to be strikingly similar to the value calculated according to Er = 3ENa - 2ECa, suggesting that the current is the electrical manifestation of the Na+-Ca2+ exchange operating with an stoichiometry of 3Na+:1Ca2+.     

Membrane ionic currents in Rhodobacter capsulatus. Evidence for electrophoretic transport of K+, Rb+ and NH4+

1. The cytoplasmic membrane ionic current of cells of Rhodobacter capsulatus, washed to lower the endogenous K+ concentration, had a non-linear dependence on the membrane potential measured during photosynthetic illumination. Treatment of the cells with venturicidin, an inhibitor of the H(+)-ATP synthase, increased the membrane potential and decreased the membrane ionic current at values of membrane potential below a threshold. 2. The addition of K+ or Rb+, but not of Na+, led to an increase in the membrane ionic current and a decrease in the membrane potential in either the presence or absence of venturicidin. Approximately 0.4 mM K+ or 2.0 mM Rb+ led to a half-maximal response. At saturating concentrations of K+ and Rb+, the membrane ionic currents were similar. The membrane ionic currents due to K+ and Rb+ were not additive. The K(+)-dependent and Rb(+)-dependent ionic currents had a non-linear relationship with membrane potential: the alkali cations only increased the ionic current when the membrane potential lay above a threshold value. The presence of 1 mM Cs+ did not lead to an increase in the membrane ionic current but it had the effect of inhibiting the membrane ionic current due to either K+ or Rb+. 3. Photosynthetic illumination in the presence of either K+ or Rb+, and weak acids such as acetate, led to a decrease in light-scattering by the cells. This was attributed to the uptake of potassium or rubidium acetate and a corresponding increase in osmotic strength in the cytoplasm. 4. The addition of NH4+ also led to an increase in membrane ionic current and to a decrease in membrane potential (half-maximal at 2.0 mM NH4+). The relationship between the NH4(+)-dependent ionic currents and the membrane potential was similar to that for K+. The NH4(+)-dependent and K(+)-dependent ionic current were not additive. However, illumination in the presence of NH4+ and acetate did not lead to significant light-scattering changes. The NH4(+)-dependent membrane ionic current was inhibited by 1 mM Cs+ but not by 50 microM methylamine. 5. It is proposed that the K(+)-dependent membrane ionic current is catalysed by a low-affinity K(+)-transport system such as that described in Rb. capsulatus [Jasper, P. (1978) J. Bacteriol. 133, 1314-1322]. The possibility is considered that, as well as Rb+, this transport system can also operate with NH4+. However, in our experimental conditions NH4+ uptake is followed by NH3 efflux.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vasoactive intestinal peptide activates Ca2(+)-dependent K+ channels through a cAMP pathway in mouse lacrimal cells

The action of vasoactive intestinal peptide (VIP) on Ca2(+)-dependent K+ currents, in dissociated mouse lacrimal cells, was investigated using patch clamp techniques. In whole cell recordings, VIP (10-100 pM) increased the magnitude of the Ca2(+)-dependent K+ current. In single channel recordings, VIP increased the fraction of time the large charybdotoxin-sensitive Ca2(+)-activated K+ channel spent in the open state. The activity of this channel was also increased by adding forskolin or 8-bromo cAMP to the bath. Additionally, application of either cAMP or catalytic subunit of cAMP-dependent protein kinase directly to the cytoplasmic surface of excised inside out patches reversibly lengthened the time Ca2(+)-activated K+ channels spent in the open state. These data suggest that VIP stimulates Ca2(+)-activated K+ channels by a cAMP-dependent pathway in mouse lacrimal acinar cells.     

Purinergic activation of anion conductance and osmolyte efflux in cultured rat hippocampal neurons

The majority of mammalian cells demonstrate regulatory volume decrease (RVD) following swelling caused by hyposmotic exposure. A critical signal initiating RVD is activation of nucleotide receptors by ATP. Elevated extracellular ATP in response to cytotoxic cell swelling during pathological conditions also may initiate loss of taurine and other intracellular osmolytes via anion channels. This study characterizes neuronal ATP-activated anion current and explores its role in net loss of amino acid osmolytes. To isolate anion currents, we used CsCl as the major electrolyte in patch electrode and bath solutions and blocked residual cation currents with NiCl(2) and tetraethylammonium. Anion currents were activated by extracellular ATP with a K(m) of 70 microM and increased over fourfold during several minutes of ATP exposure, reaching a maximum after 9.0 min (SD 4.2). The currents were blocked by inhibitors of nucleotide receptors and volume-regulated anion channels (VRAC). Currents showed outward rectification and inactivation at highly depolarizing membrane potentials, characteristics of swelling-activated anion currents. P2X agonists failed to activate the anion current, and an inhibitor of P2X receptors did not block the effect of ATP. Furthermore, current activation was observed with extracellular ADP and 2-(methylthio)adenosine 5'-diphosphate, a P2Y(1) receptor-specific agonist. Much less current activation was observed with extracellular UTP, suggesting the response is mediated predominantly by P2Y(1) receptors. ATP caused a dose-dependent loss of taurine and alanine that could be blocked by inhibitors of VRAC. ATP did not inhibit the taurine uptake transporter. Thus extracellular ATP triggers a loss of intracellular organic osmolytes via activation of anion channels. This mechanism may facilitate neuronal volume homeostasis during cytotoxic edema.     

Fast activation of cardiac Ca++ channel gating charge by the dihydropyridine agonist, BAY K 8644.

Nonlinear charge movement (gating current) was studied by the whole-cell patch clamp method using cultured 17-d-old embryonic chick heart cells. Na+ and Ca++ currents were blocked by the addition of 10 microM TTX and 3 mM CoCl2; Cs+ replaced K+ both intra- and extracellularly. Linear capacitive and leakage currents were subtracted by a P/5 procedure. The small size (15 microns in diameter) and the lack of an organized internal membrane system in these myocytes permits a rapid voltage clamp of the surface membrane. Ca++ channel gating currents were activated positive to -60 mV; the rising phase was not distorted due to the system response time. The addition of BAY K 8644 (10(-6) M) caused a shortening of the time to peak of the Ca++ gating current, and a negative shift in the isochronal Qon vs. Vm curve. Qmax was unchanged by BAY K 8644. The voltage-dependent shift produced by BAY K 8644 is similar to that produced by isoproterenol (Josephson, I.R., and N. Sperelakis. 1990. Biophys. J. 57:305a. [Abstr.]). The results suggest that the binding of BAY K 8466 to one or more of the Ca++ channel subunits alters the kinetics and shifts the voltage dependence of gating. These changes in the gating currents can explain the parallel changes in the macroscopic Ca++ currents.     

Activation of a small-conductance Ca(2+)-dependent K+ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells.

Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.     

Time dependence of the calcium-activated potassium current.

We investigated the dependence of the kinetics of the Ca2+-activated K+ current of the molluscan neuron soma upon membrane potential. The K+ current was activated by intracellular Ca2+ ion injection in neurons with blocked inward Na+ and Ca2+ currents. The difference between currents was measured with brief pulses (less than 100 ms) before and immediately after Ca2+ injection and was used as the Ca2+ activated K+ current at difference membrane potentials. The results in normal (10 mM) and in high (200 nM) external K+ show that the time-course of the Ca2+-activated K+ current depends upon membrane voltage and that the current activates more rapidly with membrane depolarization.