Patch-clamp recording in brain slices with improved slicer technology.

The use of advanced patch-clamp recording techniques in brain slices, such as simultaneous recording from multiple neurons and recording from dendrites or presynaptic terminals, demands slices of the highest quality. In this context the mechanics of the tissue slicer are an important factor. Ideally, a tissue slicer should generate large-amplitude and high-frequency movements of the cutting blade in a horizontal axis, with minimal vibrations in the vertical axis. We developed a vibroslicer that fulfils these in part conflicting requirements. The oscillator is a permanent-magnet-coil-leaf-spring system. Using an auto-resonant mechano-electrical feedback circuit, large horizontal oscillations (up to 3 mm peak-to-peak) with high frequency ( approximately 90 Hz) are generated. To minimize vertical vibrations, an adjustment mechanism was employed that allowed alignment of the cutting edge of the blade with the major axis of the oscillation. A vibroprobe device was used to monitor vertical vibrations during adjustment. The system is based on the shading of the light path between a light-emitting diode (LED) and a photodiode. Vibroprobe monitoring revealed that the vibroslicer, after appropriate adjustment, generated vertical vibrations of <1 microm, significantly less than many commercial tissue slicers. Light- and electron-microscopic analysis of surface layers of slices cut with the vibroslicer showed that cellular elements, dendritic processes and presynaptic terminals are well preserved under these conditions, as required for patch-clamp recording from these structures.

Analysing ion channels with hidden Markov models.

Ion channel current amplitudes (mu) and open probabilities (Po) have been analysed so far by defining a 50% threshold to distinguish between open and closed states of the channels. With this standard method (SM) it is very difficult or even impossible to analyse channels of different size in one membrane patch correctly. A stochastical model, named the hidden Markov model (HMM), separates between observation noise and the stochastic process of opening and closing of ion channels. The HMM allows the independent analysis of mu, Po, and mean dwell times (tau) of different channels in one membrane patch, without defining threshold levels. Using this method errors in the analysis are not summarized like in the SM because all different analysing procedures (e.g. filtering, setting of threshold, fitting processes) are done in one step. Two different K+ channels in excised basolateral membranes of the cortical collecting duct of rat (CCD) were analysed by the SM and the HMM. The mu value of the intermediate-conductance K+ channel (i-K+) was 3.9 +/- 0.1 pA (SM) and 3.8 +/- 0.2 pA (HMM) for 11 observations. The Po value of this channel was 10.2 +/- 4.2% (SM) and 10.1 +/- 4.0% (HMM). The mean tau values were 5.4 +/- 0.6 ms for the open state and 9.6 +/- 2.2 ms and 145 +/- 21 ms for the closed states (SM) and 7.8 +/- 1.1 ms, 7.7 +/- 0.9 ms and 148 +/- 24 ms (HMM), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase in cytosolic Ca2+ regulates exocytosis and Cl- conductance in HT29 cells.

Increases of cytosolic Ca2+, as occur with agonists such as ATP, neurotensin (NT), hypotonic cell swelling and ionomycin, enhance the membrane conductance (GM) and hence the input conductance (GI) of HT29 cells. In the present study we have examined whether these increases in GM are paralleled by exocytosis. To this end the membrane capacitance (CM) of HT29 cells was measured by patch clamp techniques. Two methods to monitor CM were used: a direct method (DM) and a phase tracking method (PTM). With the DM the following results were obtained. NT (10(-8) mol/l, n = 9) increased GM and CM significantly from 2.4 +/- 0.3 nS and 23.5 +/- 3 pF to 32 +/- 8 nS and 27.3 +/- 3.1 pF respectively. ATP (10(-4) mol/l, n = 29) had a very similar effect. GM and CM were increased from 5.7 +/- 1 nS and 36 +/- 4.4 pF to 111 +/- 21 nS and 44 +/- 5.4 pF respectively. Hypotonic cell swelling (160 mosmol/l, n = 18) had a comparable effect: GM and CM were increased from 4.9 +/- 1 nS and 30 +/- 4.1 pF to 46 +/- 10 nS and 37 +/- 4.9 pF respectively. Ionomycin (10(-7) mol/l, n = 4) gave similar results. With the PTM it was possible to monitor the rapid changes in GM and CM, as they were induced by ATP (n = 42) and NT (n = 29), with high time resolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation and possible physiological role of the Ca(2+)-dependent K+ channel of cortical collecting ducts of the rat.

In the luminal membrane of rat cortical collecting duct (CCD) a big Ca(2+)-dependent and a small Ca(2+)-independent K+ channel have been described. Whereas the latter most likely is responsible for the K+ secretion in this nephron segment, the function of the large-conductance K+ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K+ channel was strongly voltage- and calcium-dependent. At 3 mumol/l cytosolic Ca2+ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 mumol/l Ca2+ activity the open probability (Po) of this channel was pH-dependent. At pH 7.0 Po was decreased to 4 +/- 2% (n = 9) and at pH 8.5 it was increased to 425 +/- 52% (n = 9) of the control. At this low Ca2+ activity the Po of the channel was reduced by 1 mmol/l ATP to 8 +/- 4% (n = 6). Cell swelling activated the large-conductance K+ channel (n = 14) and hyperpolarized the membrane potential of the cells by 9 +/- 1 mV (n = 23). Intracellular Ca2+ activity increased after hypotonic stress. This increase depended on the extracellular Ca2+ activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Cation specificity and pharmacological properties of the Ca(2+)-dependent K+ channel of rat cortical collecting ducts.

The luminal membrane of principal cells of rat cortical collecting duct (CCD) is dominated by a K+ conductance. Two different K+ channels are described for this membrane. K+ secretion probably occurs via a small-conductance Ca(2+)-independent channel. The function of the second, large-conductance Ca(2+)-dependent channel is unclear. This study examines properties of this channel to allow a comparison of this K+ channel with the macroscopic K+ conductance of the CCD and with similar K+ channels from other preparations. The channel is poorly active on the cell. It has a conductance of 263 +/- 11 pS (n = 36, symmetrical K+ concentrations) and of 139 +/- 3 pS (n = 91) with 145 mmol/l K+ on one side and 3.6 mmol/l K+ on the other side of the membrane. Its open probability is high after excision (0.71 +/- 0.03, n = 85). The channel flickers rapidly between open and closed states. Its permeability in the cell-free configuration was 7.0 +/- 0.2 x 10(-13) cm3/s (n = 85). It is inhibited by several typical blockers of K+ channels such as Ba2+, tetraethylammonium, quinine, and quinidine and high concentrations of Mg2+. The Ca2+ antagonist verapamil and diltiazem also inhibit this K+ channel. As is typical for the maxi K+ channel, it is inhibited by charybdotoxin but not by apamin. The selectivity of this large-conductance K+ channel demonstrates significant differences between the permeability sequence (pK > pRb > pNH4 > pCs = pLi = pNa = pcholine = 0) and the conductance sequence (gK > gNH4 > gRb > gLi = gcholine > gCs = gNa = 0). The only other cations that are significantly conducted by this channel besides K+ (gK at Vc = infinity is 279 +/- 8 pS, n = 88) re NH+4 (gNH4 = 127 +/- 22 pS, n = 10) and Rb+ (gRb = 36 +/- 5 pS, n = 6). The K+ currents through this channel are reduced by high concentrations of choline+, Cs+, Rb+, and NH+4. These properties and the dependence of this channel on Ca2+ and voltage classify it as a "maxi" K+ channel. A possible physiological function of this channel is discussed in the accompanying paper.

Ion conductances of isolated cortical collecting duct cells.

The study of ion conductances in the intact cortical collecting duct (CCD) with the patch-clamp method is rather difficult. An optimized method to isolate CCD cells from rat kidneys using an in vivo followed by an in vitro enzyme digestion is described. Individual CCD segments were collected after this digestion and incubated in EGTA-buffered medium. This procedure resulted in single cells or cell clusters. These freshly isolated CCD cells were studied with different modifications of the patch-clamp method. Membrane voltages measured in the cell-attached-nystatin configuration were -74 +/- 1 mV (n = 13) and -68 +/- 3 mV (n = 22) in cells isolated from normal and mineralocorticoid-treated rats respectively. These values and those measured with the nystatin-perforated slow-whole-cell configuration (-79 +/- 1 mV, n = 23) are comparable to those measured in principal cells of isolated CCD segments. The cells hyperpolarized after the addition of amiloride and depolarized with the addition of adiuretin to the bath. The amiloride effect was enhanced when cells were isolated from deoxycorticosterone-acetate-treated rats. The cells were strongly depolarized upon elevation of the extracellular K(+)-concentration and did not demonstrate a measurable Cl- conductance. A large-conductance K+ channel (174 pS, n = 5, cell-attached, 145 mmol/l K+ in the pipette; 140 pS, n = 12, cell-free, 3.6 mmol/l K+ in the bath) was seen. It had a very low activity on the cell, but a high open probability when excised into a solution with 1 mmol/l Ca2+ on the cytosolic side.(ABSTRACT TRUNCATED AT 250 WORDS)

Different types of blockers of the intermediate-conductance outwardly rectifying chloride channel in epithelia.

Epithelial chloride channels can be blocked by various inhibitors, which show considerable differences in their molecular structure. In the present patch-clamp study, we compared different blockers of one type of epithelial Cl- channel with respect to their inhibitory potency. We applied the blockers to excised inside-out-or outside-out-oriented membrane patches of cultured HT29 colon carcinoma and respiratory epithelial cells (REC) containing the outwardly rectifying intermediate-conductance (ICOR) chloride channel. Four types of inhibitory compounds were tested: stilbene disulphonate derivatives, indanyloxyacetic acid, amidine, and arylaminobenzoates. The concentrations for half-maximal inhibition (IC50) for the different channel blockers were (mumol/l): 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid 100; 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid 80; indanyloxyacetic acid 9; 4,4'-dinitrostilbene-2,2'-disulphonic acid 8; amidine 8 and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) 0.9. All compounds, when applied to the cytosolic side of the channel, induced a flicker-type block of the ICOR Cl- channel at lower concentrations and a complete channel inhibition at higher concentrations. The inhibitory potency of NPPB was much higher when it was added to the external surface of the channel in outside-out-oriented membrane patches. At 1 mumol/l the inhibition was complete. All blocker effects were fully reversible. The probe with the highest affinity (NPPB) and a closely related compound 5-nitro-2-(3-phenylethylamino)-benzoate (NPEB) were used to construct macromolecular probes by linking these blockers to aminopolyethyleneglycol (PEG) or amino-ethyl-O-dextran (5 kDa).2+ These macromolecular NPPB and NPEB derivatives inhibited the ICOR Cl- channels only from the outside but had no effect on the cytosolic side.(ABSTRACT TRUNCATED AT 250 WORDS)

Bicarbonate permeability of epithelial chloride channels.

Bicarbonate permeability of epithelial chloride channels has been studied using the patch-clamp technique. The experiments were performed in excised inside-out oriented membrane patches from three different cultured cell types: (a) HT29 colon carcinoma cell line, (b) T84 colon carcinoma cell line, and (c) respiratory epithelial cells (REC) in primary culture. In all three preparations we observed outwardly rectifying chloride channels with similar conductances with 145 mmol/l NaCl solution in the pipette and in the bath (Cl- pipette/Cl- bath). When Cl- was replaced by HCO3- in the bath (Cl-/HCO3-) the conductance of the channel at negative clamp voltages was reduced significantly by 40% for HT29 (n = 6), 39% for T84 (n = 7), and 38% for REC (n = 6). Similarly, the zero-current potential (V1 = 0) was shifted from 0 mV (Cl-/Cl-) to negative values (Cl-/HCO3-) revealing permeability ratios PCl/PHCO3 of 2.4 +/- 0.1 for HT29 (n = 6), 2.0 +/- 0.1 for T84 (n = 7), and 1.8 +/- 0.1 for REC (n = 7). With NaHCO3 as the pipette solution and NaCl in the bath, the V1 = 0 was positive and a PCl/PHCO3 value of 2.3 +/- 0.1 was determined for HT29 (n = 5). Replacement of Cl- in the bath by HCO3- reduced V1 = 0 to values close to 0 mV. In another series of experiments, the pipette was filled with 145 mmol/l NaCl and the bath contained 35 mmol/l NaCl to which 35 mmol/l NaHCO3 were added. We found that neither the conductance for the inward current nor V1 = 0 was changed significantly with the addition of NaHCO3 (HT29, n = 6). We conclude that the HCO3- permeability and HCO3- conductance of these channels is about half of that for Cl-.

Antidiuretic hormone acts via V1 receptors on intracellular calcium in the isolated perfused rabbit cortical thick ascending limb.

The effect of antidiuretic hormone [( Arg]vasopressin, ADH) on intracellular calcium activity [Ca2+]i of isolated perfused rabbit cortical thick ascending limb (cTAL) segments was investigated with the calcium fluorescent dye fura-2. The fluorescence emission ratio at 500-530 nm (R) was monitored as a measure of [Ca2+]i after excitation at 335 nm and 380 nm. In addition the transepithelial potential difference (PDte) and transepithelial resistance (Rte) of the tubule were measured simultaneously. After addition of ADH (1-4 nmol/l) to the basolateral side of the cTAL R increased rapidly, but transiently, from 0.84 +/- 0.05 to 1.36 +/- 0.08 (n = 46). Subsequently, within 7-12 min R fell to control values even in the continued presence of ADH. The increase in R evoked by the ADH application corresponded to a rise of [Ca2+]i from a basal level of 155 +/- 23 nmol/l [Ca2+]i up to 429 +/- 53 nmol/l [Ca2+]i at the peak of the transient, as estimated by intra- or extracellular calibration procedures. The electrical parameters (PDte and Rte) of the tubules were not changed by ADH. The ADH-induced Ca2+ transient was dependent on the presence of Ca2+ on the basolateral side, whereas luminal Ca2+ had no effect. d(CH2)5[Tyr(Me)2]2,Arg8vasopressin, a V1 antagonist (Manning compound, 10 nmol/l), blocked the ADH effect on [Ca2+]i completely (n = 5). The V2 agonist 1-desamino-[D-Arg8]vasopressin (10 nmol/l, n = 4), and the cAMP analogues, dibutyryl-cAMP (400 mumol/l, n = 4), 8-(4-chlorophenylthio)-cAMP (100 mumol/l, n = 1) or 8-bromo-cAMP (200 mumol/l, n = 4) had no influence on [Ca2+]i. The ADH-induced [Ca2+]i increase was not sensitive to the calcium-channel blockers nifedipine and verapamil (100 mumol/l, n = 4). We conclude that ADH acts via V1 receptors to increase cytosolic calcium activity transiently in rabbit cortical thick ascending limb segments, possibly by an initial Ca2+ release from intracellular stores and by further Ca2+ influx through Ca2+ channels in the basolateral membrane. These channels are insensitive to L-type Ca2+ channel blockers, e.g. nifedipine and verapamil.

Single cardiac outwardly rectifying K+ channels modulated by protein kinase A and a G-protein.

Elementary K+ currents were recorded at 19 degrees C in cell-attached and in inside-out patches excised from neonatal rat heart myocytes. An outwardly rectifying K+ channel which prevented Na+ ions from permeating could be detected in about 10% of the patches attaining (at 5 mmol/l external K+ and between -20 mV and +20 mV) a unitary conductance of 66 +/- 3.9 pS. K+(outw.-rect.) channels have one open and at least two closed states. Open probability and tau open rose steeply on shifting the membrane potential in the positive direction, thereby tending to saturate. Open probability (at -7 mV) was as low as 3 +/- 1% but increased several-fold on exposing the cytoplasmic surface to Mg-ATP (100 mumol/l) without a concomitant change of tau open. No channel activation occurred in response to ATP in the absence of cytoplasmic Mg++. The cytoplasmic administration of the catalytic subunit of protein kinase A (120-150 mu/ml) or GTP-gamma-S (100 mumol/l) caused a similar channel activation. GDP-beta-S (100 mumol/l) was also tested and found to be ineffective in this respect. This suggests that cardiac K+(outw.-rect.) channels are metabolically modulated by both cAMP-dependent phosphorylation and a G-protein.

Gating in iodate-modified single cardiac Na+ channels.

Elementary Na+ currents were recorded at 19 degrees C during 220-msec lasting step depolarizations in cell-attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the modifying influence of iodate, bromate and glutaraldehyde on single cardiac Na+ channels. Iodate (10 mmol/liter) removed Na+ inactivation and caused repetitive, burst-like channel activity after treating the cytoplasmic channel surface. In contrast to normal Na+ channels under control conditions, iodate-modified Na+ channels attain two conducting states, a short-lasting one with a voltage-independent lifetime close to 1 msec and, likewise tested between -50 and +10 mV, a long-lasting one being apparently exponentially dependent on voltage. Channel modification by bromate (10 mmol/liter) and glutaraldehyde (0.5 mmol/liter) also included the occurrence of two open states. Also, burst duration depended apparently exponentially on voltage and increased when shifting the membrane in the positive direction, but there was no evidence for two bursting states. Chemically modified Na+ channels retain an apparently normal unitary conductance (12.8 +/- 0.5 pS). Of the two substates observed, one of them is remarkable in that it is mostly attained from full-state openings and is very short living in nature; the voltage-independent lifetime was close to 2 msec. Despite removal of inactivation, open probability progressively declined during membrane depolarization. The underlying deactivation process is strongly voltage sensitive but, in contrast to slow Na+ inactivation, responds to a voltage shift in the positive direction with a retardation in kinetics. Chemically modified Na+ channels exhibit a characteristic bursting state much shorter than in DPI-modified Na+ channels, a difference not consistent with the hypothesis of common kinetic properties in noninactivating Na+ channels.

Metabolites of the glycolytic pathway modulate the activity of single cardiac Na+ channels.

Elementary Na+ currents through single cardiac Na+ channels were recorded at 19 degrees C in patch clamp experiments with cultured neonatal rat cardiocytes. The metabolites of the glycolytic pathway, 2,3-diphosphoglycerate and glyceraldehyde phosphate, were identified as a novel class of modulators of Na+ channel activity. In micromolar concentrations (1-10 mumol/liter), their presence at the cytoplasmic membrane face increased the number of sequential openings during depolarization and prolonged the conductive channel state. As found after ensemble averaging, the decay kinetics of reconstructed macroscopic Na+ currents became retarded and slow Na+ inactivation may have been evoked. Both metabolites attenuated the rundown of channel activity that regularly develops after patch excision in the inside-out patch configuration. It is tempting to assume that interference with Na+ inactivation is the mode of action underlying the increase in single-channel activity.

On the mechanism of drug-induced blockade of Na+ currents: interaction of antiarrhythmic compounds with DPI-modified single cardiac Na+ channels.

In patch-clamped membranes from neonatal rat cardiocytes, elementary Na+ currents were recorded at 19 degrees C for study of the inhibitory influence of several antiarrhythmic drugs including lidocaine, diprafenone, propafenone, and prajmalium on DPI-modified cardiac Na+ channels. Diprafenone (20 mumol/l) and lidocaine (300 mumol/l) induced a voltage- and time-dependent block of reconstructed macroscopic sodium current (INa). The drugs depressed the sustained, noninactivating INa component (which reflects the number and open probability of DPI-modified Na+ channels) effectively, in a voltage- and time-dependent fashion. Once opened, DPI-modified Na+ channels are highly drug-sensitive. Antiarrhythmic drugs (propafenone, diprafenone, and, to a lesser extent, lidocaine) provoke a flicker block, that is, the long-lasting openings are chopped into a large number of short and grouped openings. This indicates rapid transitions between a drug-associated, blocked state and a drug-free, conducting state. The latter has a unitary conductance of 12 pS, very similar to the control value in the absence of antiarrhythmic drugs. The decrease in open time of drug-treated DPI-modified Na+ channels is concentration-dependent. Hill coefficients for propafenone of about 1.0 and for prajmalium of about 0.7 were calculated. A blocking rate constant of 6.1 x 10(7) mol-1sec-1 for propafenone, but of 1.5 x 10(7) mol-1sec-1 for prajmalium was obtained at -30 mV. The unblocking rate constant for propafenone was, also at -30 mV, about twice as large as the unblocking rate constant for prajmalium. The open channel block kinetics are essentially voltage-dependent. The affinity of the channel-associated drug receptor increases on membrane depolarization. The blocking rate constant was inversely related to the number of Na+ ions moving through the open channel. It is concluded that the manifestation of this voltage- and Na+-dependent flicker block is intimately related to removal of fast Na+ inactivation.

Versatile supplement device with remote control for the control of patch clamp experiments.

A versatile device for a patch-clamp amplifier is described. This device contains: (i) an acoustic indicator to monitor the input resistance of the patch pipette, which is used in search-mode to indicate the formation of seals; (ii) two pulse generators; and (iii) a staircase generator to produce various pulse and voltage step programs; (iv) a low-pass filter which is used to filter the output of the patch clamp amplifier; and (v) a remote control which is used to control the entire patch clamp experiment. This remote control is used to switch between search-, current clamp-, and voltage clamp-mode, to activate the respective stimulus potential programs, and to control the tape recorder. This electronic device can be easily connected to patch clamp amplifiers.

Predominance of poorly reopening single Na+ channels and lack of slow Na+ inactivation in neonatal cardiocytes.

Elementary Na+ currents through single cardiac Na+ channels were recorded at -50 mV in cell-attached patches from neonatal rat cardiocytes kept at holding potentials between -100 and -120 mV. Na+ channel activity may occur as burst-like, closely-timed repetitive openings with shut times close to 0.5-0.6 msec, indicating that an individual Na+ channel may reopen several times during step depolarization. A systematic quantitative analysis in 19 cell-attached patches showed that reopening may be quite differently pronounced. The majority, namely 16 patches, contained Na+ channels with a low tendency to reopen. This was evidenced from the average value for the mean number of openings per sequence, 2.5. Strikingly different results were obtained in a second group of three patches. Here, a mean number of openings per sequence of 3.42, 3.72, and 5.68 was found. Ensemble averages from the latter group of patches revealed macroscopic Na+ currents with a biexponential decay phase. Reconstructed Na+ currents from patches with poorly reopening Na+ channels were devoid of a slow decay component. This strongly suggests that reopening may be causally related to slow Na+ inactivation. Poorly pronounced reopening and, consequently, the lack of slow Na+ inactivation could be characteristic features of neonatal cardiac Na+ channels.

Differences in open state of NBA-modified cardiac Na+ channels.

Patch clamp recordings from neonatal cardiac Na+ channels treated with N-bromoacetamide (NBA, 5-50 X 10- mol/l) showed modified Na+ channel activity. By chemical removal of inactivation, repetitive openings with an increased life time and burst-like activity occurred. NBA-modified Na+ channels differ in life time and may attain either a slightly (mean open time 3.1 +/- 0.2 ms) or a strongly (mean open time 15.2 +/- 1.4 ms) prolonged open state. This strongly suggests a heterogeneous population of NBA-modified Na+ channels in newborn rat cardiocytes.

Removal of inactivation and blockade of cardiac Na+ channels by DPI 201-106: different voltage-dependencies of the drug actions.

The influence of the novel cardiotonic diphenylpiperazinylindole derivative, the racemic DPI 201-106, on cardiac Na+ channels was studied in conventional microelectrode experiments on papillary muscles of guinea pigs and in patch clamp experiments using inside-out patches excised from cultured neonatal rat cardiocytes. The maximal rate of rise (Vmax) of Na+-dependent action potentials was taken as an estimate for INa. Racemic DPI (3 X 10(-6) mol/l) exerts a dual effect as it removes channel inactivation and may also block cardiac Na+ channels. Both drug actions proved highly voltage-dependent but a given change in membrane potential had a strictly different modulating influence on the two effects. The Vmax depression induced by racemic DPI became attenuated due to hyperpolarization and finally tended to disappear at about -90 mV, while at the same time INa modification became increasingly accentuated. An increase in holding potential caused the non-decaying portion of the macroscopic INa to increase significantly. Resting inactivation remained operative in non-inactivating cardiac Na+ channels and showed a similar voltage-dependence as in normal Na+ channels. The differential voltage-dependencies of both DPI effects strongly suggest the existence of two binding sites for DPI.

Single nonselective cation channels and Ca2+-activated K+ channels in aortic endothelial cells.

In cultured bovine aortic endothelial cells, elementary K+ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150 +/- 10 pS and a small channel with a mean unitary conductance of 12.5 +/- 1.1 pS. The 150-pS channel proved to be voltage- and Ca2+-activatable and seems to be a K+ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca2+ concentration at the cytoplasmic side of the membrane from 10(-7) to 10(-4) M. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced run-down nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K+ and Na+ whose open probability was minimal near -60 mV but increased on membrane hyperpolarization. An increase in internal Ca2+ from 10(-7) to 10(-4) M left the open probability unchanged. Although the K+ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca2+-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na+.

Modification of single cardiac Na+ channels by DPI 201-106.

In inside-out patches from cultured neonatal rat heart cells, single Na+ channel currents were analyzed under the influence of the cardiotonic compound DPI 201-106 (DPI), a putative novel channel modifier. In absence of DPI, normal cardiac single Na+ channels studied at -30 mV have one open state which is rapidly left with a rate constant of 826.5 sec -1 at 20 degrees C during sustained depolarization. Reconstructed macroscopic currents relax completely with 7 to 10 msec. The current decay fits a single exponential. A considerable percentage of openings may occur during relaxation of the macroscopic current. In patches treated with 3 X 10(-6) m DPI in the pipette solution, stepping to -30 mV results in drastically prolonged and usually repetitive openings. This channel activity mostly persists over the whole depolarization (usually 160 msec in duration) but is abruptly terminated on clamping back the patch to the holding potential. Besides these modified events, apparently normal openings occur. The open time distribution of DPI-treated Na+ channels is the sum of two exponentials characterized by time constants of 0.85 msec (which is close to the time constant found in the control patches, 1.21 msec) and 12 msec. Moreover, DPI-modified Na+ channels exhibit a sustained high, time-independent open probability. Similar to normal Na+ channels, is voltage-dependent and increases on shifting the holding potential in the hyperpolarizing direction. These kinetic changes suggest an elimination of Na+ channel inactivation as it may follow from an interaction of DPI with Na+ channels.