Effects of tetraethylammonium on potassium currents in a molluscan neurons

The effects of tetraethylammonium (TEA) on the delayed K+ current and on the Ca2+-activated K+ current of the Aplysia pacemaker neurons R-15 and L-6 were studied. The delayed outward K+ current was measured in Ca2+-free ASW containing tetrodotoxin (TTX), using brief depolarizing clamp pulses. External TEA blocks the delayed K+ current reversibly in a dose-dependent manner. The experimental results are well fitted with a Michaelis-Menten expression, assuming a one-to-one reaction between TEA and a receptor site, with an apparent dissociation constant of 6.0 mM. The block depends on membrane voltage and is reduced at positive membrane potentials. The Ca2+-activated K+ current was measured in Ca2+-free artificial seawater (ASW) containing TTX, using internal Ca2+ ion injection to directly activate the K+ conductance. External TEA and a number of other quaternary ammonium ions block the Ca2+-activated K+ current reversibly in a dose-dependent manner. TEA is the most effective blocker, with an apparent dissociation constant, for a one-to-one reaction with a receptor site, of 0.4 mM. The block decreases with depolarization. The Ca2+-activated K+ current was also measured after intracellular iontophoretic TEA injection. Internal TEA blocks the Ca2+-activated K+ current (but the block is only apparent at positive membrane potentials), is increased by depolarization, and is irreversible. The effects of external and internal TEA can be seen in measurements of the total outward K+ current at different membrane potentials in normal ASW.     

A Fast Transient Outward Monovalent Current in Rat Saphenous Myocytes Passing Through Ca2+ Channels

Transient outward currents in rat saphenous arterial myocytes were studied using the perforated configuration of the patch-clamp method. When myocytes were bathed in a Na-gluconate solution containing TEA to block large-conductance Ca2+-activated K+ (BK) currents, depolarizing pulses positive to +20 mV from a holding potential of -100 mV induced fast transient outward currents. The activation and inactivation time constants of the current were voltage dependent, and at +40 mV were 3.6 +/- 0.8 ms and 23.9 +/- 6.4 ms (n = 4), respectively. The steady-state inactivation of the transient outward current was steeply voltage dependent (z = 1.7), with 50% of the current inactivated at -55 mV. The current was insensitive to the A-type K+ channel blocker 4-AP (1-5 mM), and was modulated by external Ca, decreasing to approximately 0.85 of control values upon raising Ca2+ from 1 to 10 mM, and increasing approximately 3-fold upon lowering it to 0.1 mM. Transient outward currents were also recorded following replacement of internal K+ with either Na+ or Cs+, raising the possibility that the current was carried by monovalent ions passing through voltage-gated Ca2+ channels. This hypothesis was supported by the finding that the transient outward current had the same inactivation rate as the inward Ba2+ current, and that both currents were effectively blocked by the L-type Ca2+ channel blocker, nifedipine and enhanced by the agonist BAYK8644.     

Diversity of K+ channels in circular smooth muscle of opossum lower esophageal sphincter

We previously demonstrated that a balance of K+ and Ca2+-activated Cl- channel activity maintained the basal tone of circular smooth muscle of opossum lower esophageal sphincter (LES). In the current studies, the contribution of major K+ channels to the LES basal tone was investigated in circular smooth muscle of opossum LES in vitro. K+ channel activity was recorded in dispersed single cells at room temperature using patch-clamp recordings. Whole-cell patch-clamp recordings displayed an outward current beginning to activate at -60 mV by step test pulses lasting 400 ms (-120 mV to +100 mV) with increments of 20 mV from holding potential of -80 mV ([K+]I = 150 mM, [K+]o = 2.5 mM). However, no inward rectification was observed. The outward current peaked within 50 ms and showed little or no inactivation. It was significantly decreased by bath application of nifedipine, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and iberiotoxin (IBTN). Further combination of TEA with 4-AP, nifedipine with 4-AP, and IBTN with TEA, or vice versa, blocked more than 90% of the outward current. Ca2+-sensitive single channels were recorded at asymetrical K+ gradients in cell-attached patch-clamp configurations (100.8+/-3.2 pS, n = 8). Open probability of the single channels recorded in inside-out patch-clamp configurations were greatly decreased by bath application of IBTN (100 nM) (Vh = -14.4+/-4.8 mV in control vs. 27.3+/-0.1 mV, n = 3, P < 0.05). These data suggest that large conductance Ca2+-activated K+ and delayed rectifier K+ channels contribute to the membrane potential, and thereby regulate the basal tone of opossum LES circular smooth muscle.     

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.     

Delayed rectifying and calcium-activated K+ channels and their significance for action potential repolarization in mouse pancreatic beta-cells

The contribution of Ca2(+)-activated and delayed rectifying K+ channels to the voltage-dependent outward current involved in spike repolarization in mouse pancreatic beta-cells (Rorsman, P., and G. Trube. 1986. J. Physiol. 374:531-550) was assessed using patch-clamp techniques. A Ca2(+)-dependent component could be identified by its rapid inactivation and sensitivity to the Ca2+ channel blocker Cd2+. This current showed the same voltage dependence as the voltage-activated (Cd2(+)-sensitive) Ca2+ current and contributed 10-20% to the total beta-cell delayed outward current. The single-channel events underlying the Ca2(+)-activated component were investigated in cell-attached patches. Increase of [Ca2+]i invariably induced a dramatic increase in the open state probability of a Ca2(+)-activated K+ channel. This channel had a single-channel conductance of 70 pS [( K+]o = 5.6 mM). The Ca2(+)-independent outward current (constituting greater than 80% of the total) reflected the activation of an 8 pS [( K+]o = 5.6 mM; [K+]i = 155 mM) K+ channel. This channel was the only type observed to be associated with action potentials in cell-attached patches. It is suggested that in mouse beta-cells spike repolarization results mainly from the opening of the 8-pS delayed rectifying K+ channel.     

Characterization of the Participation of Sodium Channels on the Rise in Na+ Induced by 4-Aminopyridine (4-AP) in Synaptosomes

The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.     

The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. II. Closed state reverse use- dependent block by 4-aminopyridine

Block of the calcium-independent transient outward K+ current, I(to), by 4-aminopyridine (4-AP) was studied in ferret right ventricular myocytes using the whole cell patch clamp technique. 4-AP reduces I(to) through a closed state blocking mechanism displaying "reverse use- dependent" behavior that was inferred from: (a) development of tonic block at hyperpolarized potentials; (b) inhibition of development of tonic block at depolarized potentials; (c) appearance of "crossover phenomena" in which the peak current is delayed in the presence of 4-AP at depolarized potentials; (d) relief of block at depolarized potentials which is concentration dependent and parallels steady-state inactivation for low 4-AP concentrations (V1/2 approximately -10 mV in 0.1 mM 4-AP) and steady-state activation at higher concentrations (V1/2 = +7 mV in 1 mM 4-AP, +15 mV in 10 mM 4-AP); and (e) reassociation of 4- AP at hyperpolarized potentials. No evidence for interaction of 4-AP with either the open or inactivated state of the I(to) channel was obtained from measurements of kinetics of recovery and deactivation in the presence of 0.5-1.0 mM 4-AP. At hyperpolarized potentials (-30 to - 90 mV) 10 mM 4-AP associates slowly (time constants ranging from approximately 800 to 1,300 ms) with the closed states of the channel (apparent Kd approximately 0.2 mM). From -90 to -20 mV the affinity of the I(to) channel for 4-AP appears to be voltage insensitive; however, at depolarized potentials (+20 to +100 mV) 4-AP dissociates with time constants ranging from approximately 350 to 150 ms. Consequently, the properties of 4-AP binding to the I(to) channel undergo a transition in the range of potentials over which channel activation and inactivation occurs (-30 to +20 mV). We propose a closed state model of I(to) channel gating and 4-AP binding kinetics, in which 4-AP binds to three closed states. In this model 4-AP has a progressively lower affinity as the channel approaches the open state, but has no intrinsic voltage dependence of binding.     

Ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis

The ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis were examined by using conventional two-electrode voltage clamp and whole-cell patch clamping methods. Several separable currents were identified. These include: (1) a transient and (2) a steady-state voltage-activated inward current; both are tetrodotoxin (TTX) and saxitoxin (STX) insensitive, partly reduced by decreasing external Ca2+ or Na+ or by addition of 5 mM Co2+, D-600 or verapamil and are totally blocked with 5 mM Cd2+; (3) an early, transient, cation-dependent, outward K+ current (IKCa/Na); (4) a transient, voltage-activated, outward K+ current provisionally identified as IA; (5) a delayed, steady-state, voltage-activated outward K+ current (IK) and (6) a late, transient, outward K+ current which is blocked by Cd2+ and evident only during long voltage pulses. Despite their phylogenic origin, most of these currents are similar to currents identified in many vertebrate smooth and cardiac muscle preparations, and other excitable cells in higher animals.     

Presence of a calcium-activated chloride current in mouse ventricular myocytes

The properties of several components of outward K(+) currents, including the pharmacological and kinetics profiles as well as the respective molecular correlates, have been identified in mouse cardiac myocytes. Surprisingly little is known with regard to the Ca(2+)-activated ionic currents. We studied the Ca(2+)-activated transient outward currents in mouse ventricular myocytes. We have identified a 4-aminopyridine (4-AP)- and tetraethyl ammonium-resistant transient outward current that is Ca(2+) dependent. The current is carried by Cl(-) and is critically dependent on Ca(2+) influx via voltage-gated Ca(2+) channels and the sarcoplasmic reticulum Ca(2+) store. The current can be blocked by the anion transport blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Single channel recordings reveal small conductance channels (approximately 1 pS in 140 mM Cl(-)) that can be blocked by anion transport blockers. Ensemble-averaged current faithfully mirrors the transient kinetics observed at the whole level. Niflumic acid (in the presence of 4-AP) leads to prolongation of the early repolarization. Thus this current may contribute to early repolarization of action potentials in mouse ventricular myocytes.     

Action of quinidine on ionic currents of molluscan pacemaker neurons

The effects of quinidine on the fast, the delayed, and the Ca2+- activated K+ outward currents, as well as on Na+ and Ca2+ inward currents, were studied at the soma membrane from neurons of the marine mollusk Aplysia californica. External quinidine blocks these current components but to different degrees. Its main effect is on the voltage- dependent, delayed K+ current, and it resembles the block produced by quaternary ammonium ions (Armstrong, C. M., 1975, Membranes, Lipid Bilayers and Biological Membranes: Dynamic Properties, 3:325-358). The apparent dissociation constant is 28 microM at V = +20 mV. The blocking action is voltage and time dependent and increases during maintained depolarization. The data are consistent with the block occurring approximately 70-80% through the membrane electric field. Internal injection of quinidine has an effect similar to that obtained after external application, but its time course of action is faster. External quinidine may therefore have to pass into or through the membrane to reach a blocking site. The Ca2+-activated K+ current is blocked by external quinidine at concentrations 20-50-fold higher compared with the delayed outward K+ current. In addition, it prolongs the time course of decay of the Ca2+-activated K+ current. Na+ and Ca2+ inward currents are also blocked by external quinidine, but again at higher concentrations. The effects of quinidine on membrane currents can be seen from its effect on action potentials and the conversion of repetitive "beating" discharge activity to "bursting" pacemaker activity.     

Voltage-dependent calcium and calcium-activated potassium currents of a molluscan photoreceptor

Two-microelectrode voltage clamp studies were performed on the somata of Hermissenda Type B photoreceptors that had been isolated by axotomy from all synaptic interaction as well as any impulse-generating (i.e., active) membrane. In the presence of 2-10 mM 4-aminopyridine (4-AP) and 100 mM tetraethylammonium ion (TEA), which eliminated two previously described voltage-dependent potassium currents (IA and the delayed rectifier), a voltage-dependent outward current was apparent in the steady state responses to command voltage steps more positive than -40 mV (absolute). This current increased with increasing external Ca++. The magnitude of the outward current decreased and an inward current became apparent following EGTA injection. Substitution of external Ba++ for Ca++ also made the inward current more apparent. This inward current, which was almost eliminated after being exposed for approximately 5 min to a solution in which external Ca++ was replaced with Cd++, was maximally activated at approximately 0 mV. Elevation of external potassium allowed the calcium (ICa++) and calcium-dependent K+ (IC) currents to be substantially separated. Command pulses to 0 mV elicited maximal ICa++ but no IC because no K+ currents flowed at their new reversal potential (0 mV) in 300 mM K+. At a holding potential of -60 mV, which was now more negative than the potassium equilibrium potential, EK+, in 300 mM K+, IC appeared as an inward tail current after positive command steps. The voltage dependence of ICa++ was demonstrated with positive steps in 100 mM Ba++, 4-AP, and TEA. Other data indicated that in 10 mM Ca++, IC underwent pronounced and prolonged inactivation whereas ICa++ did not. When the photoreceptor was stimulated with a light step (with the membrane potential held at -60 mV), there was also a prolonged inactivation of IC. In elevated external Ca++, ICa++ also showed similar inactivation. These data suggest that IC may undergo prolonged inactivation due to a direct effect of elevated intracellular Ca++, as was previously shown for a voltage-dependent potassium current, IA. These results are discussed in relation to the production of training-induced changes of membrane currents on retention days of associative learning.     

Elevated potassium shortens action potential duration by altering outward currents in chick dorsal root ganglia neurons

Dissociated embryonic chick dorsal root ganglion (DRG) neurons maintained in culture exhibit a mixed Na+/Ca2+ action potential. The characteristic "shoulder" on the repolarizing phase is due to the relatively prolonged inward Ca2+ current. DRG neurons grown in an elevated K+ medium (25 versus. 5 mM) lack the plateau phase of the action potential. Voltage-clamp analysis showed that this plastic change in action potential duration is not due to the loss of the inward Ca2+ current but is partly due to the appearance of a Ca2(+)-dependent, 4-aminopyridine-(4-AP)-sensitive transient outward current. Faster activation of the purely voltage-dependent delayed rectifier outward current also contributes to the rapid repolarization observed in neurons cultured in elevated K+ medium.     

Voltage- and calcium-activated currents in cultured olfactory receptor neurons of male Mamestra brassicae (Lepidoptera)

Insect olfactory receptor neurons (ORNs) grown in primary cultures were studied using the patch-clamp technique in both conventional and amphotericin B perforated whole-cell configurations under voltage-clamp conditions. After 10-24 days in vitro, ORNs had a mean resting potential of -62 mV and an average input resistance of 3.2 GOmega. Five different voltage-dependent ionic currents were isolated: one Na(+), one Ca(2+) and three K(+) currents. The Na(+) current (35-300 pA) activated between -50 and -30 mV and was sensitive to 1 microM tetrodotoxin (TTX). The sustained Ca(2+) current activated between -30 and -20 mV, reached a maximum amplitude at 0 mV (-4.5 +/- 6.0 pA) that increased when Ba(2+) was added to the bath and was blocked by 1 mM Co(2+). Total outward currents were composed of three K(+) currents: a Ca(2+)-activated K(+) current activated between -40 and -30 mV and reached a maximum amplitude at +40 mV (605 +/- 351 pA); a delayed-rectifier K(+) current activated between -30 and -10 mV, had a mean amplitude of 111 +/- 67 pA at +60 mV and was inhibited by 20 mM tetraethylammonium (TEA); and, finally, more than half of ORNs exhibited an A-like current strongly dependent on the holding potential and inhibited by 5 mM 4-aminopyridine (4-AP). Pheromone stimulation evoked inward current as measured by single channel recordings.     

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)     

Developmental regulation of a Na(+)-activated fast outward K+ current in rat myoblasts.

BACKGROUND/AIMS: Myoblasts undergoing differentiation sequentially express multiple K+ channels, and that ion channel expression varies depending on species and state of development. In this report, we reported a developmental regulation of fast activated and fast inactivated outward current in rat myoblasts. METHODS: The kinetic and pharmacological property of the outward current was investigated by using the patch-clamp technique. RESULTS: The outward current was elicited by a depolarizing step from -100 mV holding potential to +40 mV- +80 mV. The activation properties of this channel changed with days in culture. The outward current was blocked by 4-AP in a concentration dependent manner, with 0.5 mM and 2 mM 4-AP inhibiting the current by 10 +/- 3% and 56 +/- 3%, respectively. When 1 mM tetrodotoxin (TTX) was added to the bath solution or the membrane potential was depolarized to -50 mV, the fast outward current was aborted. Na+ dependent inhibition was observed when Na+ in the bath solution was replaced by Li+. In addition, replacement of K+ in the pipette solution by Cs+ almost completely eliminated the outward current. CONCLUSION: The developmentally regulated outward current recorded in rat myoblasts is a Na+ influx-dependent outward K+ current, which may contribute to myoblast membrane firing of action potential or myoblast fusion.     

Serotonin decreases a background current and increases calcium and calcium-activated current in pedal neurons ofHermissenda

The effects of serotonin (5-HT) on membrane potential, membrane resistance, and select ionic currents were examined in large pedal neurons (LP1, LP3) of the mollusk Hermissenda. Calcium (Ca) action potentials were evoked in sodium-free artificial seawater containing tetramethylammonium, tetraethylammonium, and 4-aminopyridine (0-Na, 4-AP, TEA ASW). They failed at stimulation rates greater than 0.5/sec and were blocked by cadmium (Cd). Under voltage clamp the calcium current (ICa) responsible for them also failed with repeated stimulation. Thus, ICa inactivation accounts for refractoriness of the Ca action potential. The addition of 10 microM 5-HT to 0-Na, 4-AP, TEA ASW produced a slight depolarization and increased excitability and input resistance. Under voltage clamp the background current decreased. The voltage-dependent inward, late outward, and outward tail currents, sensitive to Cd, increased. ICa inactivation persisted. Under voltage clamp with Ca influx blocked by Cd, the addition of 10 microM 5-HT decreased the remaining current uniformly over membrane potentials of -10 to -100 mV. Thus, 5-HT reduces a background current that is active within the physiological range of the membrane potential, voltage insensitive, independent of Ca influx, noninactivating, and not blocked by 4-AP or TEA.     

Permeation, selectivity, and blockade of the Ca2+-activated potassium channel of the guinea pig taenia coli myocyte

The permeation properties of the 147-pS Ca2+-activated K+ channel of the taenia coli myocytes are similar to those of the delayed rectifier channel in other excitable membranes. It has a selectivity sequence of K+ 1.0 greater than Rb+ 0.65 greater than NH4+ 0.50. Na+, Cs+, Li+, and TEA+ (tetraethylammonium) are impermeant. Internal Na+ blocks K+ channel in a strongly voltage-dependent manner with an equivalent valence (zd) of 1.20. Blockade by internal Cs+ and TEA+ is less voltage dependent, with d of 0.61 and 0.13, and half-blockage concentrations of 88 and 31 mM, respectively. External TEA+ is about 100 times more effective in blocking the K+ channel. All these findings suggest that the 147-pS Ca2+-activated K+ channel in the taenia myocytes, which functions physiologically like the delayed rectifier, is the single-channel basis of the repolarizing current in an action potential.     

Fluctuations of the Ca2+-activated K+ current in Aplysia neurones

Fluctuations of the Ca2+-activated K+ current were measured in identified Aplysia neurones under voltage clamp conditions. The amplitude of IK,Ca was manipulated by ionophoretic injections of Ca2+. At small amplitudes of Ca2+-activated outward currents the variance of the Ca2+-activated current fluctuations increases linearly with the mean outward current. The single-channel conductance estimated from the variance of the fluctuations and the mean outward current is 11 +/- 3 pS at -30 mV. Power spectra of the Ca2+-activated K+ current can be fitted by the sum of two Lorentzian components with corner frequencies of about 10 Hz and 120 Hz.     

Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.     

Competitive Mg2+ block of a large-conductance, Ca(2+)-activated K+ channel in rat skeletal muscle. Ca2+, Sr2+, and Ni2+ also block

The patch-clamp technique was used to investigate the effect of intracellular Mg2+ (Mgi2+) on the conductance of the large-conductance, Ca(2+)-activated K+ channel in cultured rat skeletal muscle. Measurements of single-channel current amplitudes indicated that Mgi2+ decreased the K+ currents in a concentration-dependent manner. Increasing Mgi2+ from 0 to 5, 10, 20, and 50 mM decreased channel currents by 34%, 44%, 56%, and 73%, respectively, at +50 mV. The magnitude of the Mgi2+ block increased with depolarization. For membrane potentials of -50, +50, and +90 mV, 20 mM Mgi2+ reduced the currents 22%, 56%, and 70%, respectively. Mgi2+ did not change the reversal potential, indicating that Mg2+ does not permeate the channel. The magnitude of the Mgi2+ block decreased as the concentration of K+ was increased. At a membrane potential of +50 mv, 20 mM Mgi2+ reduced the currents 71%, 56%, and 25% for Ki+ of 75, 150, and 500 mM. These effects of Mgi2+, voltage, and K+ were totally reversible. Although the Woodhull blocking model could approximate the voltage and concentration effects of the Mgi2+ block (Kd approximately 30 mM with 150 mM symmetrical K+; electrical distance approximately 0.22 from the inner surface), the Woodhull model could not account for the effects of K+. Double reciprocal plots of 1/single channel current vs. 1/[K+] in the presence and absence of Mgi2+, indicated that the Mgi2+ block is consistent with apparent competitive inhibition between Mgi2+ and Ki+. Cai2+, Nii2+, and Sri2+ were found to have concentration- and voltage-dependent blocking effects similar, but not identical, to those of Mgi2+. These observations suggest the blocking by Mgi2+ of the large-conductance, Ca(2+)-activated K+ channel is mainly nonspecific, competitive with K+, and at least partially electrostatic in nature.