Channels involved in transient currents unmasked by removal of extracellular calcium in cardiac cells

In cardiac cells that lack macroscopic transient outward K(+) currents (I(to)), the removal of extracellular Ca(2+) can unmask "I(to)-like" currents. With the use of pig ventricular myocytes and the whole cell patch-clamp technique, we examined the possibility that cation efflux via L-type Ca(2+) channels underlies these currents. Removal of extracellular Ca(2+) and extracellular Mg(2+) induced time-independent currents at all potentials and time-dependent currents at potentials greater than -50 mV. Either K(+) or Cs(+) could carry the time-dependent currents, with reversal potential of +8 mV with internal K(+) and +34 mV with Cs(+). Activation and inactivation were voltage dependent [Boltzmann distributions with potential of half-maximal value (V(1/2)) = -24 mV and slope = -9 mV for activation; V(1/2) = -58 mV and slope = 13 mV for inactivation]. The time-dependent currents were resistant to 4-aminopyridine and to DIDS but blocked by nifedipine at high concentrations (IC(50) = 2 microM) as well as by verapamil and diltiazem. They could be increased by BAY K-8644 or by isoproterenol. We conclude that the I(to)-like currents are due to monovalent cation flow through L-type Ca(2+) channels, which in pig myocytes show low sensitivity to nifedipine.     

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.     

Identification and characterization of the inward K+ channel in the plasma membrane of Brassica pollen protoplasts.

Patch clamp techniques have been used to identify and characterize the whole-cell currents carried by inward K+ channels in isolated matured pollen protoplasts of Brassica chinensis var. chinensis. The whole-cell inward currents in the isolated pollen protoplasts were activated at hyperpolarized membrane potentials more negative than -100 mV. The magnitudes of the whole-cell inward currents were strongly dependent on the external K+ concentration, and were highly selective for K+ over other monovalent cations. The inward currents were not observed when external K+ was replaced with the same concentration of Cs+ or Na+. The addition of 1 mM or 10 mM Ba2+ in external solutions resulted in 30% or 80% inhibition of the inward currents at -180 mV, respectively. These results demonstrated that the inward K+ currents mainly account for the recorded whole-cell inward currents in Brassica pollen protoplasts. Increase of cytoplasmic Ca2+ concentrations from 10 nM to 30 microM or even 5 mM did not affect the inward K+ currents. Decrease of external Ca2+ concentrations from 10 mM to 1 mM inhibited the inward K+ currents by 25%, while the increase of external Ca2+ from 10 mM to 50 mM almost completely blocked the inward K+ currents. Physiological importance of K+ transport into pollen and its possible regulatory mechanisms are also discussed.     

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 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)     

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.     

Characterization of K+ currents in rat malignant lymphocytes (Nb2 Cells)

Membrane K+ currents of malignant lymphocytes (Nb2 cells) were studied with the whole-cell patch-clamp method. Upon depolarization, K+ currents activate with a delay and follow a sigmoid time course, resembling other delayed rectifier K+ currents present in nerve and muscle cells. The activation time constant of these currents is voltage dependent, increasing from 1 msec at +90 mV to approximately 37 msec at -30 mV. The fractional number of open channels has a sigmoid voltage dependence with a midpoint near -25 mV. Deactivation of K+ currents in Nb2 cells is voltage dependent and follows a simple exponential time course. Time constant of this process increases from 5 msec at -115 mV to almost 80 msec at -40 mV. The relative permeability of K+ channels to different monovalent cations follows the sequence: K+ (1) greater than Rb+ (0.75) greater than NH4+ (0.11) greater than Cs+ (0.07) greater than Na+ (0.05). Inactivation of K+ currents is a biexponential process with time constants of approximately 600 and 7,000 msec. Inactivation of K+ currents in Nb2 cells is not a voltage-dependent process. The steady-state inactivation curve of K+ currents has a midpoint near -40 mV. Following a 500-msec voltage pulse, inactivation of K+ currents recovers with a simple exponential process with a time constant of 9 sec. Short duration (approximately 50 msec) voltage-clamp pulses do not induce significant inactivation of these currents. K+ currents in malignant lymphocytes do not display the phenomenon of cumulative inactivation as described for other delayed rectifier-type K+ channels. Application of a train of voltage pulses to positive potentials at different frequencies induces a moderate decrease in peak outward currents. The use of substances (N-bromoacetamide, trypsin, chloramine-T, and papain) that remove the inactivation of Na+ and K+ currents in other cells are not effective in removing the inactivation of K+ currents present in this lymphoma cell line. Significant differences were found between the characteristics of K+ currents in this malignant cell line and those present in normal lymphocytes. Possible physiological implications for these differences and for the role of K+ currents in the proliferation of normal and malignant lymphocytes are discussed.     

Halothane suppresses slow inward currents in hippocampal slices

Single-electrode voltage-clamp experiments were made on CA1 neurons in the presence of tetrodotoxin and K channel blockers. Applications of halothane (1-3% v/v) for 3-10 min caused a similar marked and reversible depression of slow inward currents (probably Ca currents) evoked by depolarizing pulses from a holding potential near -80 or near -40 mV. The peak amplitudes of the inward currents were much reduced, in a concentration-dependent manner, and they decayed more rapidly (half-decay time was shortened by a quarter). In most cases, leak conductances were diminished by halothane, making it unlikely that the suppression of inward currents was primarily caused by enhancement of outward currents. A similar inactivation of Ca currents in presynaptic terminals would explain why halothane depresses synaptic transmission.     

Alterations in outward K(+) currents on removal of external Ca(2+) in human atrial myocytes

External divalent cations are known to play an important role in the function of voltage-gated ion channels. The purpose of this study was to examine the sensitivity of the voltage-gated K(+) currents of human atrial myocytes to external Ca(2+) ions. Myocytes were isolated by collagenase digestion of atrial appendages taken from patients undergoing coronary artery-bypass surgery. Currents were recorded from single isolated myocytes at 37 degrees C using the whole-cell patch-clamp technique. With 0.5 mM external Ca(2+), voltage pulses positive to -20 mV (holding potential = -60 mV) activated outward currents which very rapidly reached a peak (I(peak)) and subsequently inactivated (tau = 7.5 +/- 0.7 msec at +60 mV) to a sustained level, demonstrating the contribution of both rapidly inactivating transient (I(to1)) and non-inactivating sustained (I(so)) outward currents. The I(to1) component of I(peak), but not I(so), showed voltage-dependent inactivation using 100 msec prepulses (V(1/2) = -35.2 +/- 0.5 mV). The K(+) channel blocker, 4-aminopyridine (4-AP, 2 mM), inhibited I(to1) by approximately 76% and reduced I(so) by approximately 33%. Removal of external Ca(2+) had several effects: (i) I(peak) was reduced in a manner consistent with an approximately 13 mV shift to negative voltages in the voltage-dependent inactivation of I(to1). (ii) I(so) was increased over the entire voltage range and this was associated with an increase in a non-inactivating 4-AP-sensitive current. (iii) In 79% cells (11/14), a slowly inactivating component was revealed such that the time-dependent inactivation was described by a double exponential time course (tau(1) = 7.0 +/- 0.7, tau(2) = 90 +/- 21 msec at +60 mV) with no effect on the fast time constant. Removal of external Ca(2+) was associated with an additional component to the voltage-dependent inactivation of I(peak) and I(so) (V(1/2) = -20.5 +/- 1.5 mV). The slowly inactivating component was seen only in the absence of external Ca(2+) ions and was insensitive to 4-AP (2 mM). Experiments with Cs(+)-rich pipette solutions suggested that the Ca(2+)-sensitive currents were carried predominantly by K(+) ions. External Ca(2+) ions are important to voltage-gated K(+) channel function in human atrial myocytes and removal of external Ca(2+) ions affects I(to1) and 4-AP-sensitive I(so) in distinct ways.     

Membrane currents in taste cells of the rat fungiform papilla. Evidence for two types of Ca currents and inhibition of K currents by saccharin

Taste buds were isolated from the fungiform papilla of the rat tongue and the receptor cells (TRCs) were patch clamped. Seals were obtained on the basolateral membrane of 281 TRCs, protruding from the intact taste buds or isolated by micro-dissection. In whole-cell configuration 72% of the cells had a TTX blockable transient Na inward current (mean peak amplitude 0.74 nA). All cells had outward K currents. Their activation was slower than for the Na current and a slow inactivation was also noticeable. The K currents were blocked by tetraethylammonium, Ba, and 4-aminopyridine, and were absent when the pipette contained Cs instead of K. With 100 mM Ba or 100 mM Ca in the bath, two types of inward current were observed. An L-type Ca current (ICaL) activated at -20 mV had a mean peak amplitude of 440 pA and inactivated very slowly. At 3 mM Ca the activation threshold of ICaL was near -40 mV. A transient T-type current (ICaT) activated at -50 mV had an average peak amplitude of 53 pA and inactivated with a time constant of 36 ms at -30 mV. ICaL was blocked more efficiently by Cd and D600 than ICaT. ICaT was blocked by 0.2 mM Ni and half blocked by 200 microM amiloride. In whole-cell voltage clamp, Na-saccharin caused (in 34% of 55 cells tested) a decrease in outward K currents by 21%, which may be expected to depolarize the TRCs. Also, Na-saccharin caused some taste cells to fire action potentials (on-cell, 7 out of 24 cells; whole-cell, 2 out of 38 cells responding to saccharin) of amplitudes sufficient to activate ICaL. Thus the action potentials will cause Ca inflow, which may trigger release of transmitter.     

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

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

Analysis of the K+ Current Profile of Mature Rat Oligodendrocytes in situ

Previous studies have reported that mature oligodendrocytes (OLGs) in vitro display various voltage-dependent K+ currents while in situ OLGs show only voltage-independent K+ currents. Given this discrepancy and the lack of information on myelinating OLG ion channel expression in situ, we characterized mature OLG currents in myelinating corpus callosum slices from 17 to 36-day old rats. OLGs were recorded in cell-attached and whole-cell patch-clamp configurations, displayed morphology typical of mature OLGs, and stained positive for myelin basic protein. OLGs displayed large voltage-independent currents that decayed during the voltage pulse and small voltage-activated outward currents. The latter were blocked by TEA, activated between -40 and -50 mV, and decayed slowly. The former were composed of large voltage-independent, time-dependent Ba2+ (1 mM)-sensitive currents, and voltage-dependent Cs+ (5 mM) and Ba2+ (100 mM)-sensitive currents that reversed near the K+ equilibrium potential and inactivated at hyperpolarized potentials, identifying them as inwardly rectifying K+ currents. Inwardly rectifying single-channel K+currents could be recorded in the cell-attached configuration. The estimated single-channel slope conductance was 30 pS. The steady-state open probability was voltage-dependent and declined from 0.9 to 0.5 between -80 and -150 mV. Overall, mature OLGs in situ possess time- and also voltage-dependent K+ currents, which may facilitate clearance of K+ released during axonal firing.     

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.     

Inward current in single smooth muscle cells of the guinea pig taenia coli

Using the tight-seal voltage-clamp method, the ionic currents in the enzymatically dispersed single smooth muscle cells of the guinea pig taenia coli have been studied. In a physiological medium containing 3 mM Ca2+, the cells are gently tapering spindles, averaging 201 (length) x 8 microns (largest diameter in center of cell), with a volume of 5 pl. The average cell capacitance is 50 pF, and the specific membrane capacitance 1.15 microF/cm2. The input impedance of the resting cell is 1-2 G omega. Spatially uniform voltage-control prevails after the first 400 microseconds. There is much overlap of the inward and outward currents, but the inward current can be isolated by applying Cs+ internally to block all potassium currents. The inward current is carried by Ca2+. Activation begins at approximately -30 mV, maximum ICa occurs at +10-+20 mV, and the reversal potential is approximately +75 mV. The Ca2+ channel is permeable to Sr2+ and Ba2+, and to Cs+ moving outwards, but not to Na+ moving inwards. Activation and deactivation are very rapid at approximately 33 degrees C, with time-constants of less than 1 ms. Inactivation has a complex time course, resolvable into three exponential components, with average time constants (at 0 mV) of 7, 45, and 400 ms, which are affected differently by voltage. Steady-state inactivation is half-maximal at -30 mV for all components combined, but -36 mV for the fast component and -26 and -23 mV for the other two components. The presence of multiple forms of Ca2+ channel is inferred from the inactivation characteristics, not from activation properties. Recovery of the fast channel occurs with a time-constant of 72 ms (at +10 mV). Ca2+ influx during an action potential can transfer approximately 9 pC of charge, which could elevate intracellular Ca2+ concentration adequately for various physiological functions.     

Purified IP3 receptor from smooth muscle forms an IP3 gated and heparin sensitive Ca2+ channel in planar bilayers.

The IP3 receptor of aortic smooth muscle, purified to near homogeneity, was incorporated into vesicle derived planar bilayers. The receptor forms channels which are gated by Ins(1,4,5)P3 (0.5 microM) and are permeable to Ca2+ (Ca2+ greater than K+ much greater than Cl-). Channel activation is specific for Ins(1,4,5)P3. Essentially no activation of channel currents was found for Ins(1,3,4)P3 or Ins(1,3,4,5)P4 at 10 microM. Heparin (25 micrograms/ml) blocked induced currents completely at all levels of activity while ATP (50 microM) increased mean current levels 2 to 4 fold. Ins(1,4,5)P3 activated mean currents increased non-linearly with voltage above about -40 mV applied voltage. Mean current levels could be reversibly adjusted by voltage to the single channel level (0 to -50 mV) or to macroscopic levels (-50 to -100 mV) over periods exceeding 1 h. Single channel events are characterized by fast transitions between predominantly non-resolved sublevels. Estimates of maximal single event currents yield a slope conductance of 32 +/- 4 pS (0 to -60 mV, 50 mM CaCl2). Thus, the purified IP3 receptor forms a channel with functional properties characteristic of IP3 triggered Ca2+ release.     

Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells.

We studied monovalent permeability of Ca2+ release-activated Ca2+ channels (ICRAC) in Jurkat T lymphocytes following depletion of calcium stores. When external free Ca2+ ([Ca2+]o) was reduced to micromolar levels in the absence of Mg2+, the inward current transiently decreased and then increased approximately sixfold, accompanied by visibly enhanced current noise. The monovalent currents showed a characteristically slow deactivation (tau = 3.8 and 21.6 s). The extent of Na+ current deactivation correlated with the instantaneous Ca2+ current upon readdition of [Ca2+]o. No conductance increase was seen when [Ca2+]o was reduced before activation of ICRAC. With Na+ outside and Cs+ inside, the current rectified inwardly without apparent reversal below 40 mV. The sequence of conductance determined from the inward current at -80 mV was Na+ > Li+ = K+ > Rb+ >> Cs+. Unitary inward conductance of the Na+ current was 2.6 pS, estimated from the ratios delta sigma2/delta Imean at different voltages. External Ca2+ blocked the Na+ current reversibly with an IC50 value of 4 microM. Na+ currents were also blocked by 3 mM Mg2+ or 10 microM La3+. We conclude that ICRAC channels become permeable to monovalent cations at low levels of external divalent ions. In contrast to voltage-activated Ca2+ channels, the monovalent conductance is highly selective for Na+ over Cs+. Na+ currents through ICRAC channels provide a means to study channel characteristics in an amplified current model.     

Membrane currents in canine bronchial artery and their regulation by excitatory agonists

The bronchial vasculature plays an important role in airway physiology and pathophysiology. We investigated the ion currents in canine bronchial smooth muscle cells using patch-clamp techniques. Sustained outward K(+) current evoked by step depolarizations was significantly inhibited by tetraethylamonium (1 and 10 mM) or by charybdotoxin (10(-6) M) but was not significantly affected by 4-aminopyridine (1 or 5 mM), suggesting that it was primarily a Ca(2+)-activated K(+) current. Consistent with this, the K(+) current was markedly increased by raising external Ca(2+) to 4 mM but was decreased by nifedipine (10(-6) M) or by removing external Ca(2+). When K(+) currents were blocked (by Cs(+) in the pipette), step depolarizations evoked transient inward currents with characteristics of L-type Ca(2+) current as follows: 1) activation that was voltage dependent (threshold and maximal at -50 and -10 mV, respectively); 2) inactivation that was time dependent and voltage dependent (voltage causing 50% maximal inactivation of -26 +/- 22 mV); and 3) blockade by nifedipine (10(-6) M). The thromboxane mimetic U-46619 (10(-6) M) caused a marked augmentation of outward K(+) current (as did 10 mM caffeine) lasting only 10-20 s; this was followed by significant suppression of the K(+) current lasting several minutes. Phenylephrine (10(-4) M) also suppressed the K(+) current to a similar degree but did not cause the initial transient augmentation. None of these three agonists elicited inward current of any kind. We conclude that bronchial arterial smooth muscle expresses Ca(2+)-dependent K(+) channels and voltage-dependent Ca(2+) channels and that its excitation does not involve activation of Cl(-) channels.     

Fast-deactivating calcium channels in chick sensory neurons

Whole-cell Ca and Ba currents were studied in chick dorsal root ganglion (DRG) cells kept 6-10 in culture. Voltage steps with a 15-microseconds rise time were imposed on the membrane using an improved patch-clamp circuit. Changes in membrane current could be measured 30 microseconds after the initiation of the test pulse. Currents through Ca channels were recorded under conditions that eliminate Na and K currents. Tail currents, associated with Ca channel closing, decayed in two distinct phases that were very well fitted by the sum of two exponentials. The time constants tau f and tau s were near 160 microseconds and 1.5 ms at -80 mV, 20 degrees C. The tail current components, called FD and SD (fast-deactivating and slowly deactivating), are Ca channel currents. They were greatly reduced when Mg2+ replaced all other divalent cations in the bath. The SD component inactivated almost completely as the test pulse duration was increased to 100 ms. It was suppressed when the cell was held at membrane potentials positive to -50 mV and was blocked by 100-200 microM Ni2+. This behavior indicates that the SD component was due to the closing of the low-voltage-activated (LVA) Ca channels previously described in this preparation. The FD component was fully activated with 10-ms test pulses to +20 mV at 20 degrees C, and inactivated to approximately 30% during 500-ms test pulses. It was reduced in amplitude by holding at -40 mV, but was only slightly reduced by micromolar concentrations of Ni2+. Replacement of Ca2+ with Ba2+ increased the FD tail current amplitudes by a factor of approximately 1.5. The deactivation kinetics did not change (a) as channels inactivated during progressively longer pulses or (b) when the degree of activation was varied. Further, tau f was affected neither by changing the holding potential nor by varying the test pulse amplitude. Lowering the temperature from 20 to 10 degrees C decreased tau f by a factor of 2.5. In all cases, the FD component was very well fitted by a single exponential. There was no indication of an additional tail component of significant size. Our findings indicate that the FD component is due to closing of a single class of Ca channels that coexist with the LVA Ca channel type in chick DRG neurons.     

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.