Single channel analysis of the inward rectifier K current in the rabbit ventricular cells

The inward rectifier K channel in rabbit ventricular cells was studied by the patch-clamp method. Single channel currents were recorded in giga-sealed cell-attached patches with 150 mM K+ in the pipette. The slope conductance in the membrane potential range from -140 to -40 mV was 46.6 +/- 6.7 pS (mean +/- S.D., n = 16), and was reduced by decreasing [K+] in the pipette (20 or 50 mM). The channel was blocked by an application of Cs+ or Ba2+ (0.04-1 mM) in the pipette. Outwardly directed current, recorded with 50 mM K+ in the pipette, revealed the inward rectification of the single channel current. The probability of the channel being open was 0.33 +/- 0.05 (n = 10) at the resting potential (RP=-81.7 +/- 1.7 mV, n = 16) with 150 mM K+ in the pipette, and it decreased with hyperpolarization. The mean open time of the channel was 178 +/- 25 msec (n = 6) at RP. The closed time of the channel seemed to have two exponential components, with time constants of 11.0 +/- 2.0 msec and 1.92 +/- 0.52 sec (n = 6) at RP. The slower time constant was increased with hyperpolarization. The averaged patch current recorded upon hyperpolarizing pulses demonstrated a time-dependent current decay as expected from the single channel kinetics. The results indicated that the inward rectifier K+ current has time- and voltage-dependent kinetics.     

Antagonistic modulation of a hyperpolarization-activated Cl(-) current in Aplysia sensory neurons by SCP(B) and FMRFamide

Whole cell voltage-clamp recordings from Aplysia mechanosensory neurons obtained from the pleural ganglion were used to investigate the actions on membrane currents of the neuropeptides SCP(B) and FMRFamide. At the start of whole cell recording, SCP(B) typically evoked an inward current at a holding potential of -40 mV, due to the cAMP-mediated closure of the S-type K+ channel, whereas FMRFamide evoked an outward current, due to the opening of the S-type K+ channels mediated by 12-lipoxygenase metabolites of arachidonic acid. However, after several minutes of whole cell recording with a high concentration of chloride in the whole cell patch pipette solution, the responses to SCP(B) and FMRF-amide at -40 mV were inverted; SCP(B) evoked an outward current, whereas FMRFamide and YGGFMRFamide evoked inward currents. Ion substitution experiments and reversal potential measurements revealed that these responses were due to the opposing regulation of a Cl(-) current, whose magnitude was greatly enhanced by dialysis with the high Cl(-) - containing pipette solution. SCP(B) inhibited this Cl(-) current through production of cAMP and activation of PKA. YGGFMRFamide activated this Cl(-) current by stimulating a cGMP-activated phosphodiesterase that hydrolyzed cAMP. Thus a cAMP-dependent Cl(-) current undergoes antagonistic modulation by two neuropeptides in Aplysia sensory neurons.     

Sodium-activated potassium current in guinea pig gastric myocytes

This study was designed to identify and characterize Na+-activated K+ current (I(K(Na))) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+([K+]degrees). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68+/-2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steady-state current produced under [K+]degrees (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+- and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+-selective, since reversal potentials (E(rev)) of various concentrations of [K+]degrees-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 microM), a blocker of I(K(Na)), completely and reversibly inhibited this current. The characteristics of the current coincide with those of I(K(Na)) of other cells. Our results indicate the presence of I(K(Na)) in guinea pig gastric myocytes.     

A slow calcium-dependent chloride conductance in clonal anterior pituitary cells

1. Whole cell voltage-clamp recordings were made from GH3 cells, a clonal cell line initially derived from a rat anterior pituitary tumor, using patch electrodes filled with CsCl or N-methylglucamine chloride (NMG Cl). The bathing medium contained tetraethylammonium chloride (TEA; 20 mM) and NaCl (120 mM) or NMG Cl (140 mM). These conditions resulted in substantial blockade of outward currents. 2. Depolarizing voltage steps from a holding potential of -50 mV activated transient (T-type) and sustained (L-type) inward Ca2+ currents. In addition, prolonged depolarization (greater than 1 s) invariably elicited a slowly activating inward current that persisted with maintained depolarization, and deactivated slowly on repolarization, resulting in a prominent inward tail current. 3. This tail current could be recorded under conditions where Ca2+ and Cl- were the only membrane-permeant ions (symmetrical NMG Cl). The tail current nulled near 0 mV with symmetrical Cl- and showed a negative reversal potential with nominally Cl--free internal solution. Ba2+ substituted for Ca2+ as a carrier of inward current, but no tail current was expressed. These observations indicate that Cl- is the charge carrier of the slow inward tail current. 4. The voltage dependence for activation of the slow tail current was U-shaped with a peak at approximately -10 mV. This closely paralleled the voltage dependency of the Ca2+ currents. Recordings with 5 mM internal ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) to buffer intracellular Ca2+ to low nM levels exhibited slow inward tail currents that were of lower peak amplitude than with the usual 1.1 mM EGTA-containing pipette solution, but the kinetics of the currents were similar in both cases. In addition, the slow tail current was eliminated on superfusion with the Ca2+ channel blocker Cd2+ or with Ca2+-free medium. These results demonstrate that the current is dependent on Ca2+ influx; it is, therefore, referred to as ICl(Ca). 5. Activation of ICl(Ca) required depolarization of at least 1 s. More prolonged depolarizations activated progressively greater current, to a maximum with 6-s depolarization. In most cases, the decay of the tail current was described by a single exponential function with time constant approximately 0.8-0.9 s within the potential range -80 to -30 mV. At more depolarized potentials the decay was slower (increasing e-fold/20-mV change in membrane potential). 6. In a high proportion of cells, ICl(Ca) rapidly diminished in amplitude on repeated activation. This "rundown" occurred more rapidly than the rundown of the Ca2+ currents.(ABSTRACT TRUNCATED AT 400 WORDS)     

Voltage-dependent currents in isolated cells of the turtle retinal pigment epithelium

The electrophysiological properties of isolated turtle retinal pigment epithelial cells (RPE cells) were investigated using the whole-cell patch-clamp technique. Most RPE cells exhibited a voltage-dependent outward current activated by depolarization beyond about –43 mV that inactivated during a 500-ms voltage step. Tail current measurements indicated that the conductance underlying this current was potassium selective. This current inactivated with prolonged depolarization and was abolished or reduced by extracellular quinidine, barium, tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Steady-state inactivation of the voltage-dependent outward current revealed a time-independent outwardly rectifying current/voltage relationship in many cells. In addition, many cells had an outward current that activated slowly upon depolarization beyond about +40 mV and appeared to reverse near 0 mV in both 3 mM KCl and 30 mM KCl external solutions. This current was often observed in the presence of potassium channel blockers. Hyperpolarizing pulses commonly evoked inward currents that activated slowly and did not inactivate. These currents were commonly observed when fluoride was absent from the pipette, and only occasionally when fluoride was the major pipette anion. Tail current measurements indicated that this current was somewhat anion selective.These currents may play important roles in the homeostatic and phagocytic functions of RPE cells in their interactions with the neural retina.     

Single-channel L-type Ca2+ currents in chicken embryo semicircular canal type I and type II hair cells

Few data are available concerning single Ca channel properties in inner ear hair cells and particularly none in vestibular type I hair cells. By using the cell-attached configuration of the patch-clamp technique in combination with the semicircular canal crista slice preparation, we determined the elementary properties of voltage-dependent Ca channels in chicken embryo type I and type II hair cells. The pipette solutions included Bay K 8644. With 70 mM Ba(2+) in the patch pipette, Ca channel activity appeared as very brief openings at -60 mV. Ca channel properties were found to be similar in type I and type II hair cells; therefore data were pooled. The mean inward current amplitude was -1.3 +/- 0.1 (SD) pA at - 30 mV (n = 16). The average slope conductance was 21 pS (n = 20). With 5 mM Ba(2+) in the patch pipette, very brief openings were already detectable at -80 mV. The mean inward current amplitude was -0.7 +/- 0.2 pA at -40 mV (n = 9). The average slope conductance was 11 pS (n = 9). The mean open time and the open probability increased significantly with depolarization. Ca channel activity was still present and unaffected when omega-agatoxin IVA (2 microM) and omega-conotoxin GVIA (3.2 microM) were added to the pipette solution. Our results show that types I and II hair cells express L-type Ca channels with similar properties. Moreover, they suggest that in vivo Ca(2+) influx might occur at membrane voltages more negative than -60 mV.     

Effects of lysophosphatidylcholine on resting potassium conductance of isolated guinea pig ventricular cells

We studied the effects of lysophosphatidylcholine (LPC), a toxic metabolite of ischemia, on the inward rectifier potassium channel current in isolated guinea pig ventricular cells. LPC (10-50 microM) added to the external solution decreased the resting membrane potential and occasionally induced repetitive action potential discharges, with or without loss of repolarization. In voltage clamp studies, LPC (20 microM) decreased the conductance at the levels of resting potentials (approximately equal to -80 mV) from 26 +/- 8 nS to 16 +/- 3 nS (mean and SD, n = 4) within 10 min. Prolonged application of LPC (greater than 12 min) produced transient inward currents after depolarizing clamp pulses, thereby suggesting that the LPC elevated intracellular Ca2+ concentrations. The effect of LPC on the single inward rectifier K channel current was examined using the patch clamp technique in a cell-attached mode. LPC decreased the single channel conductance, depending on the concentration (5-100 microM). The slope conductance in the presence of 150 mM K+ in the pipette decreased from 45 +/- 7 pS (control) to 32 +/- 17, 20 +/- 19, and 14 +/- 10 pS for 5, 20 and 100 microM LPC, respectively. LPC induced little change with regard to probability of the channel opening. These results suggest that LPC depolarizes membrane by decreasing single channel conductance of the inward rectifier K channel. This reduction partially contributes to the alleged LPC-induced abnormal automaticities and conduction disturbances in the heart.     

Background conductance attributable to spontaneous opening of muscarinic K+ channels in rabbit sino-atrial node cells.

Single myocytes were dissociated from the rabbit sino-atrial node, and the membrane background conductance produced by spontaneous opening of the muscarinic K+ channels was investigated by recording whole-cell and single channel currents in both normal K+ (5.4 mM) and high-K+ (145 mM) external solutions. Increasing external K+ concentration ([K+]o) from 5.4 to 145 mM induced a large inward shift of the whole-cell current accompanied by considerable current fluctuations at -50 mV. The high-K(+)-induced current was both K+ selective and voltage dependent, which was examined by varying [K+]o. This current was almost completely suppressed by 1-5 mM Ba2+ or 2-10 mM Cs+ and it was partly blocked by 10 microM atropine. In high-K+ (145 mM) solution, 20 nM acetylcholine (ACh) further increased the K+ conductance as well as the current noise. The power density spectrum of the noise was fitted with a sum of two Lorentzian functions. The corner frequencies of both the slow (approximately 5 Hz) and fast (approximately 120 Hz) components were comparable between the noise before and during the ACh application. Internal dialysis with a non-hydrolysable derivative of ATP, 5'-adenylylimido-diphosphate (AMP-PNP) or Mg(2+)-free solution markedly decreased both the amplitude and fluctuations of the high-K(+)-induced current. The relation between the variance of the current fluctuations and the mean current amplitude was linear in every experiment using dialysis of AMP-PNP or Mg(2+)-free internal solution, or using superfusion of ACh. The slopes of these relations gave comparable single channel current amplitudes of -0.7 pA at -50 mV. These results indicate that the spontaneous opening of the muscarinic K+ channels is largely responsible for the high-K(+)-induced current. In the high-K+ solution, the variance-mean relation at -50 mV showed that the muscarinic K+ channel provides an inward current of 3.12 +/- 2.13 pA pF-1 (n = 23), which was about 60% of the total inward background current. In the normal K+ solution, the variance-mean relation at -50 mV indicated that an outward current of 6.0 +/- 2.0 pA (0.33 +/- 0.28 pA pF-1, n = 8) was provided by the K+ channel. The single channel current amplitude was estimated to be 0.06 +/- 0.02 pA (n = 9). Cell-attached recordings in the absence of ACh demonstrated sporadic and brief openings of channels identical to the ACh-induced channels. The power density spectra of the single channel currents exhibited kinetic properties comparable with those of the whole-cell currents.(ABSTRACT TRUNCATED AT 400 WORDS)     

Voltage-dependent, slowly activating K+ current (I Ks) and its augmentation by carbachol in rat pancreatic acini

Acetylcholine-stimulated exocrine secretion of Cl- and water requires the concomitant activation of K+ channels. However, there has not been much investigation of the carbachol- (CCH-) activated K+ channel of rodent pancreatic acini. Here, in a study of rat pancreatic acini, we characterize a voltage-dependent, slowly activating outward current (I(Ks)) that is augmented by CCH. Intact acini were obtained by enzymatic digestion and fast-whole-cell patch-clamp was applied. With symmetrical [Cl-] (32 mmol/l) in the pipette and bath solution, acinar cells had resting membrane voltages of -45+/-0.8 mV (n=97) under current-clamp conditions. CCH (10 micromol/l), which is known to activate Cl- channels via a Ca2+-mediated pathway, sharply depolarized the membrane to -4+/-0.5 mV, which was more negative than E(Cl) (0 mV), and reversed it to -41+/-0.9 mV (n=83) by washout. A clamp voltage of 0 mV activated I(Ks) under control conditions (91+/-8.6 pA, n=83). During CCH application an increase of outward current was observed at 0 mV, and at -50 mV a marked increase of inward Cl current occurred. In the presence of CCH the slow activation of I(Ks) was rarely distinguishable because of interference by the huge Cl- conductance. During CCH washout and decrease of inward current, a persistent augmentation of I(Ks) was revealed (486+/-36.3 pA, n=83). I(Ks) and its augmentation were abolished by substituting K+ in the pipette solution with Cs+. Augmentation of I(Ks) was mimicked by applying ionomycin (0.1 micromol/l), a Ca2+ ionophore. Pharmacological blockers were tested. The chromanol 293B and clotrimazole blocked I(Ks) at micromolar concentrations (IC50=3 micromol/l and 9 micromol/l, respectively) and Ba2+ was a poor blocker (IC50=3 mmol/l). In the presence of CCH (0.2 micromol/l), the membrane was depolarized to around -20 mV and the addition of 293B (10 micromol/l) further depolarized the membrane by 11+/-3 mV (n=5). These data suggest the presence of I(Ks) channels in rat pancreatic acini and their muscarinic activation.     

Activation of Ca2+-dependent Cl- and K+ conductances in rat and mouse parotid acinar cells

Isolated acinar cells from rat and mouse parotid glands were studied with patch-clamp whole-cell current recordings. Acetylcholine (ACh) stimulation caused a transient inward current at a membrane potential of -70 mV, and a sustained outward current at a membrane potential of 0 mV, in quasi physiological Na+, K+ ion gradients, except the zero-Cl- ion gradient condition across the membrane. The reversal potential obtained from the ACh-evoked steady current was about -75 mV, in this ionic condition. When major Cl- ions of both the pipette and the bath solution were replaced, either by glutamate or by sulphate, only a large outward current was observed, at a membrane potential of -60 mV, in the presence of ACh. The addition of Ca2+-ionophore A23187 caused responses similar to those evoked by ACh. The reversal potential of A23187-induced current was close to the K+ equilibrium potential of -90 mV, in a Cl- -free condition. When K+-free NaCl solution was used in the pipette and the bath, A23187 caused only a large inward current, at a membrane potential of -60 mV. The reversal potential of A23187-evoked current was about -15 mV, in a symmetrical K+-free, NaCl condition. These results suggest that the ACh and A23187 activate Cl- as well as K+ conducting pathways via an increase in [Ca2+]i in the parotid acinar cells. The A23187-evoked large K+ current could not be explained solely by a rise in open probability of the channels.     

Reversibility and cation selectivity of the K(+)-Cl(-) cotransport in rat central neurons

The reversibility and cation selectivity of the K(+)-Cl(-) cotransporter (KCC), which normally extrudes Cl(-) out of neurons, was investigated in dissociated lateral superior olive neurons of rats using the gramicidin perforated patch technique. Intracellular Cl(-) activity (alpha[Cl(-)](i)) was maintained well below electrochemical equilibrium as determined from the extracellular Cl(-) activity and the holding potential, where the pipette and external solutions contained 150 mM K(+) ([K(+)](pipette)) and 5 mM K(+) ([K(+)](o)), respectively. Extracellular application of 1 mM furosemide or elevated [K(+)](o) increased alpha[Cl(-)](i). When the pipette solution contained 150 mM Cs(+) ([Cs(+)](pipette)), alpha[Cl(-)](i) increased to a value higher than the passive alpha[Cl(-)](i). An increase of alpha[Cl(-)](i) with the [Cs(+)](pipette) was not due to the simple blockade of net KCC by the intracellular Cs(+) since alpha[Cl(-)](i), with the pipette solution containing 75 mM Cs(+) and 75 mM K(+), reached a value between those obtained using the [K(+)](pipette) and the [Cs(+)](pipette). The higher-than-passive alpha[Cl(-)](i) with the [Cs(+)](pipette) was reduced by 1 mM furosemide, but not by 20 microM bumetanide or Na(+)-free external solution, indicating that the accumulation of [Cl(-)](i) in the [Cs(+)](pipette) was mediated by a KCC operating in a reversed mode rather than by Na(+)-dependent, bumetanide-sensitive mechanisms. Replacement of K(+) in the pipette solution with either Li(+) or Na(+) mimicked the effect of Cs(+) on alpha[Cl(-)](i). On the other hand, Rb(+) mimicked K(+) in the pipette solution. These results indicate that K(+) and Rb(+), but not Cs(+), Li(+), or Na(+), can act as substrates of KCC in LSO neurons.     

Characterization of a hyperpolarization-activated inward current in Cajal-Retzius cells in rat neonatal neocortex

Cajal-Retzius cells are among the first neurons appearing during corticogenesis and play an important role in the establishment of cortical lamination. To characterize the hyperpolarization-activated inward current (I(h)) and to investigate whether I(h) contributes to the relatively positive resting membrane potential (RMP) of these cells, we analyzed the properties of I(h) in visually identified Cajal-Retzius cells in cortical slices from neonatal rats using the whole cell patch-clamp technique. Membrane hyperpolarization to -90 mV activated a prominent inward current that was inhibited by 1 mM Cs(+) and was insensitive to 1 mM Ba(2+). The activation time constant for I(h) was strongly voltage dependent. In Na(+)-free solution, I(h) was reduced, indicating a contribution of Na(+). An analysis of the tail currents revealed a reversal potential of -45.2 mV, corresponding to a permeability coefficient (pNa(+)/pK(+)) of 0. 13. While an increase in the extracellular K(+) concentration ([K(+)](e)) enhances I(h), it was reduced by a [K(+)](e) decrease. This [K(+)](e) dependence could not be explained by an effect on the electromotive force on K(+) but suggested an additional extracellular binding site for K(+) with an apparent dissociation constant of 7.2 mM. Complete Cl(-) substitution by Br(-), I(-), or NO(3)(-) had no significant effect on I(h), whereas a complete Cl(-) substitution by the organic compounds methylsulfate, isethionate, or gluconate reduced I(h) by approximately 40%. The I(h) reduction observed in gluconate could be abolished by the addition of Cl(-). The analysis of the [Cl(-)](e) dependence of I(h) revealed a dissociation constant of 9.8 mM and a Hill-coefficient of 2.5, while the assumption of a gluconate-dependent I(h) reduction required an unreasonably high Hill-coefficient >20. An internal perfusion with the lidocaine derivative lidocaine N-ethyl bromide blocks I(h) within 1 min after establishment of the whole cell configuration. An inhibition of I(h) by 1 mM Cs(+) was without an effect on RMP, action potential amplitude, threshold, width, or afterhyperpolarization. We conclude from these results that Cajal-Retzius cells express a prominent I(h) with characteristic properties that does not contribute to the RMP.     

High Activity K+ Channels in Rat Hippocampal Neurones Maintained in Culture

A channel was identified in cell-attached recordings in rat hippocampal neurones maintained in culture. This channel, which was highly active at the resting membrane potential, was present in most (73 %) patches studied. The channel was characterized by long duration openings and a high open probability (Po, mean value 0.73 at -70 mV) at negative patch potentials with mild voltage dependence over the range -40 to -120 mV. It showed inward rectification. There were up to five active channels in cell-attached recordings in experiments where the cells were bathed in sodium-containing Locke solution. The single channel conductances in cell-attached recordings with 140 or 40 mM K+ in the patch pipette were 26 and 12 pS, respectively. The channel was therefore selective for K+ over Na+. The channel was not permeable to Rb+ ions. The single channel conductance was 24 pS in excised inside-out patches bathed in symmetrical K+ (140 mM) solutions. Examination of the channel kinetics revealed that both the open and closed time distributions could be fitted by the sum of three exponentials, there being no pronounced voltage sensitivity between -60 and -120 mV. The 26 pS K+ channel was insensitive to extracellular TEA, apamin, 4-AP and dequalinium. Neither was it sensitive to intracellular Ca2+. Extracellular Ba2+ was effective in reversibly blocking the channel, the IC50 being 2.0 mM. There was no obvious effect of bath application of the K+ channel opener, lemakalim, or a cAMP analogue. This channel appears to contribute a significant proportion (at least 30 %) of the resting conductance in these neurones.     

Chloride channels in the plasma membrane of a foetal Drosophila cell line, S2

We evaluated the suitability of the S2 foetal Drosophila cell line as an expression system for vertebrate anion channel proteins (e.g. cystic fibrosis transmembrane conductance regulator, CFTR) in patch-clamp studies of the endogenous ion channels. In the inside-out configuration (symmetric 150 mM Cl-) we found most frequently an inwardly rectifying Cl- channel with single-channel conductances (gamma) of 57, 45 and 17 pS at -80, 0 and 80 mV, respectively. Reduction of bath [Cl-] to 40 mM caused a shift in reversal potential (Vrev) to -22.5 mV indicating pronounced Cl- selectivity. In the outside-out configuration ([Cl-]pipette = 40 mM, [Cl-]bath = 150 mM) we observed a Cl- channel with a linear unitary current/voltage (i/V) relation for which gamma was 30 pS. The kinetics were quite slow in both configurations. Cl-selectivity was also observed in whole-cell experiments ([Cl-]pipette = 40 mM) in which a Vrev of -43.8 mV, i.e. close to the Cl- equilibrium potential, demonstrated that the membrane current was dominated by Cl-. We conclude that the important features making S2 cells suitable as an expression system for heterologous expressed anion channel proteins are: small total whole-cell currents (less than 100 pA), single-channel and whole-cell currents that, unlike those of CFTR, cannot be described by the Goldman-Hodgkin-Katz regime, and slow kinetics distinctly different from those of CFTR.     

Effects of increased intracellular Cl- concentration on membrane responses to acetylcholine in the isolated endothelium of guinea pig mesenteric arteries

ACh-induced membrane responses in vascular endothelial cells that have been reported vary between preparations from a sustained hyperpolarization to a transient hyperpolarization followed by a depolarization; the reason for this variation is unknown. Using the perforated whole-cell clamp technique, we investigated ACh-induced membrane currents in freshly isolated endothelial layers having a resting membrane potential of less negative than -10 mV. A group of cells was electrically isolated using a wide-bore micropipette, and their membrane potential was well controlled. ACh activated K(+) and Cl(-) currents simultaneously. The K(+) current was blocked by a combination of charybdotoxin and apamin and appears to result from the opening of IK(Ca) and SK(Ca) channels. The Cl(-) current was partially blocked by tamoxifen, niflumic acid, or DIDS and appears to be produced by Ca(2+)-activated Cl(-) channels. When the pipettes contained 20 mM Cl(-), the ACh-induced K(+) conductance started decreasing during a 1-min application of ACh while the Cl(-) conductance continued, making the ACh-induced hyperpolarization sustained. When the pipettes contained 150 mM Cl(-), both conductances started decreasing during a 1-min application of ACh, making the ACh-induced hyperpolarization small and transient. [Cl(-)](i) is very likely modified by experimental procedures such as the cell isolation and the intracellular dialysis with the pipette solution. Such a variability in [Cl(-)](i) may be one of the reasons for the variations in the ACh-induced membrane response.     

Muscarine induces two distinct current responses in adrenal chromaffin cells of the guinea-pig

Muscarine-induced membrane responses were studied in dissociated chromaffin cells of the guinea-pig adrenal medulla, using the whole-cell version of the patch-clamp technique. Bath application of muscarine (1-10 microM) produced two distinct current responses at a holding potential of -40 mV. One is an inward current associated with an increase in current noise. This current response was sustained during stimulation and had a reversal potential of 4.5 +/- 3.4 mV (n = 6) with a negative slope conductance below about -30 mV in 12.5 mM K(+)-containing perfusate. The other is a transient outward current. This was evoked at membrane potentials more positive than -60 mV and completely suppressed by addition of 2 mM TEA to the bath solution, suggesting a possible involvement of the Ca2(+)-dependent K+ channel. Generation of the outward current response was suppressed for at least 60-90 s following 25 s muscarinic stimulation and was facilitated by activation of the nicotinic receptor. The maximum inward current seemed to be produced by 3 microM, whereas the threshold concentration required for generation of the outward current was somewhere between 3 and 10 microM. The outward current was evoked less often in cells treated with 2% collagenase for 1 h than in those treated with 0.2% for 30 min. The results suggest that guinea-pig chromaffin cells have two muscarinic receptors: one is coupled with a cation nonselective channel and the other may be related to a Ca2(+)-dependent K+ channel.     

ATP-sensitive potassium channels in freshly dissociated adult rat striatal neurons: activation by metabolic inhibitors and the dopaminergic receptor agonist quinpirole

The characteristics of adenosine 5'-triphosphate (ATP)-sensitive K+ channels in acutely isolated striatal neurons from adult rats were examined. Neurons had a resting membrane potential of -53.9+/-1.2 mV (n=66), with evoked or spontaneous action potentials firing at 10+/-0.7 Hz, and large inwards and outwards whole-cell currents. In cell-attached patches with a high [K+] in the pipette, a voltage-independent, ATP-insensitive 16.5+/-1.5 pS channel was observed in 375 out of 452 cells. Bath application of Na+-azide (0.5-2 mM) to 108 neurons revealed another 145.7+/-3.5 pS (LKATP) channel in 65 neurons; this channel was blocked by tolbutamide. The LKATP channel exhibited a high open probability (Po, 0.8+/-0.05) at 0 mV pipette potential. Varying the pipette [K+] shifted the reversal potential of LKATP, showing the channel's K+ selectivity. Cytoplasmic ATP (ATPi) reversibly inhibited LKATP, with an inhibitory constant (Ki) of 0.12 mM. LKATP was sensitive to intracellular Ca2+ but insensitive to iberiotoxin. In 25% of cell-attached patches, the presence of quinpirole in the pipette opened a third type of channel (90.6+/-1.7 pS, termed D2KATP). Sulpiride, a dopamine D2-receptor antagonist, inhibited D2KATP. ATPi reversibly inhibited D2KATP, with a Ki of 0.212 mM. The Na+-azide- or quinpirole-induced current caused a tolbutamide-sensitive membrane hyperpolarization and a marked reduction in action potential frequency. We propose that ATP-sensitive K+ channels play a metabolism-dependent role in striatal neurons.     

Single-channel currents through transient-receptor-potential-like (TRPL) channels

Single-channel current recordings were used to examine the properties and modulation of Drosophila transient-receptor-potential-like (TRPL) channels transiently expressed in HEK and COS cells. Recombinant TRPL channels were constitutively active and characterized by a conductance of 104 pS in on-cell membrane patches with 115 mM Na+ and 2 mM Mg2+ in the pipette solution. In inside-out membrane patches exposed to 115 mM Na+ plus 2 mM Mg2+, 115 mM Na+ plus 10 mM Mg2+, 90 mM Ca2+ and 90 mM Ba2+ on both sides, the single-channel conductances were 72 pS, 36 pS, 48 pS and 46 pS, respectively. The single TRPL channel currents reversed close to 0 mV and displayed a linear voltage dependence between -120 mV and +120 mV. Removal of cations from the pipette and bath solutions abolished inward and outward currents, respectively. Similar currents were not observed in mock-transfected and native cells. The opening probability of TRPL channels increased by depolarizing the membrane and accounted for the outward rectification of whole-cell TRPL currents. In on-cell membrane patches, the TRPL channel activity was enhanced by cell dialysis of 300 microM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) and by a rise of intracellular Ca2+ (>2 microM). Constitutively active TRPL channels depolarized the host cells to -10 mV and the membrane potential was restored by cell dialysis with 10 mM BAPTA. The present results suggest that TRPL forms non-selective cationic channels modulated by intracellular Ca2+ in mammalian cells.     

The properties of the inward rectifier potassium currents in rabbit coronary arterial smooth muscle cells

In freshly-isolated, single, smooth muscle cells of rabbit coronary arteries, an inward rectifier K⁺ current [I _K(IR)] was identified using the whole-cell voltage-clamp technique. The current/voltage (I/V) relationship of I _K(IR) showed strong inward rectification with a very small outward current when the smooth muscle cells were dialyzed with a pipette solution containing Mg²⁺. However, dialyzing the cells with a nominally Mg²⁺-free pipette solution revealed a significant outward current hump in the I/V relation of I _K(IR), suggesting that the strong inward rectification of I _K(IR) is partly due to the inhibitory effects of internal Mg²⁺. I _K(IR) was unaffected by tetraethylammonium (1 mM), 4-aminopyridine (1 mM), or glibenclamide (1 μM), but was inhibited by extracellular Ba²⁺ with a concentration of 0.87 μM eliciting half-maximal inhibition at –120 mV. I _K(IR) induced in rabbit coronary smooth muscle cells declined during very negative hyperpolarizing steps, due largely to a block by external Na⁺. I _K(IR) was inhibited by α₁-adrenergic stimuli. Methoxamine, an α₁-adrenergic agonist, concentration dependently inhibited I _K(IR) in the presence of the β-adrenergic antagonist propranolol. The methoxamine concentration required for half-maximal inhibition was 205 μM. We conclude that inward rectifier K⁺ current is present in rabbit coronary smooth muscle cells and that it shares many properties with the inward rectifier K⁺ current described for other cell types. Received: 2 February 1999 / Accepted: 24 March 1999