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Single K+-channel currents under steady-state potential conditions in small hippocampal neurons
Brain Research, 596 (1992) 133-141
133
© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 18261
Single K+-channel currents under steady-state potential conditions in small hippocampal neurons Staffan Johansson and Peter ~rhem Nobel Institute for Neurophysiology, Karolinska Instituter, Stockholm (Sweden) (Accepted 16 June 1992)
Key words: Potassium channel; Patch clamp; Outside-out patch; Hippocampal neuron; Small cell
Small cultured hippocampal neurons from rat embryos were studied with the patch-clamp technique. Single-channel currents from outside-out membrane patches were recorded under steady-state potential conditions. The most frequently found channel types were selective to K + and showed conductances of about 30 and 80 pS in the range - 20 to 0 mV. Two basic kinetic patterns were observed for the 80 pS channels. In one type, the fraction of time spent in open state increased with potential, and in the other type it decreased. For both types of 80 pS channel, the distribution of dwell times in the open state was well described by the sum of two exponentials while three exponentials sometimes were required for dwell times in the closed state. The time constants of the fitted exponentials could vary considerably during an experiment.
INTRODUCTION
Small cerebral neurons are poorly characterized electrophysiologically in spite of their abundance. In a series of investigations we have studied small cultured hippocampal neurons under current-clamp and voltage-clamp conditions. The cells, which had a soma diameter less than 10 izm, most likely correspond to dentate granule cells and small interneurons. They showed several unexpected features: the action potentials depended systematically on stimulus strength t6, the input resistance was remarkably high (about 3 G•; see ref. 16) and several lines of data indicated that individual openings and/or closures of single ion channels could initiate the generation of action potentials 14. It is clear that, in the studied cells, very small currents do cause large changes in membrane potential. It is therefore of particular interest to obtain information on the properties of single ion channels active (i.e. opening and closing) under steady membrane potentials. The present investigation is a singlechannel analysis of outside-out membrane patches under steady-state potential conditions. The currents were studied under relatively physiological conditions with no use of channel blockers. Generally, many types of
current steps were detected. The most frequently found channel types were K + selective and showed conductances of about 30 and 80 pS in the range - 2 0 to 0 mV. The 80 pS channels showed two types of kinetic patterns. MATERIALS AND METHODS
Cell culture conditions The cells were prepared from embryonic (day El8-21) rat hippocampi, using the techniques described by Johansson et al. ~6. The electrophysiological recordings were performed after 4-8 days in culture. Only cells with a soma diameter less than 10/zm were used.
Electrophysiological recordings The currents were recorded from 29 outside-out membrane patches under voltage-clamp conditionsit. Borosilicate glass pipettes (GC 120F or 150TF, Clark Electromedical instruments), with a resistance of 8-25 MfJ when filled with and immersed in the bathing solution (see below), were used. The pipette-cell membrane seal had a resistance higher than 5 GfL The signals were recorded using an EPC-7 electrometer (Listelectronic) and stored on an FM tape-recorder (Racal Store 4DS, bandwidth under the used conditions 0-5 kHz). All experiments were performed at room temperature (21-23°C).
Solutions The bathing solution contained (in mM) NaC! 137, KC! 5.0, CaCi 2 1.0, MgC!2 1.2 and HEPES (N-(2-hydroxyethyl)piperazine-N'2-ethanesulphonic acid) 10, pH 7.4. The recording pipette was filled
Correspondence: S. Johansson, Nobel Institute for Neurophysiology, Karolinska lnstitutet, S-104 01 Stockholm, Sweden. Fax: (46) (8) 34 95 44.
134 with either (lj a solution containing (in mM) KC! 140, NaCI 3.0, MgCI 2 1.2, EG'I'A (ethyleneglycol-bis-(0-aminoethyl ether)N,N, N',N'-tetra-acetic acid; 1.0 and HEPES 10, pH 7.2 or (II) a solution containing K O H / K H 2 P O 4 140, NaCI 3.0, MgCi 2 J.2, EGTA 1.0 and Na2-ATP 3.0, pH 7.2.
D~.a analysis Only currents more than 5 s after a potential change were analyzed. The currents recorded were transferred to a computer using a TL-1 DMA interface (Labmaster) and the pCLAMP software (version 5.5.1; Axon Instruments). For this procedure the currents were low-pass filtered (8-pole Bessel filter) at a frequency that kept all noise peaks in the direction opposite to channel opening below 50% of the single-channel current amplitude ls'lg. The resulting dead times varied between 90/zs and 180 ~s. (Dead time is the duration of a rectangular pulse which allows the pulse, after tittering, to reach 50% of its true amplitude). The digitization rate was higher than 25 times the filter frequency ( - 3 dB) to prevent samplillg errors 19. The durations of open and closed intervals were measured by 50% threshold detections during visual inspection to exclude artifacts and periods with apparent subconductance current levels. All intervals shorter than the dead time were ignoredsJs. The kinetic analysis was performed using recorded current periods with a minimum of current steps from channels not in focus of the analysis. The open and closed dwell-time histograms were fitted with sums of exponential components using the pCLAMP software (based on a least-squares method and a Levenberg-Marquardt fitting algorithm). Intervals less than two times the dead time were excluded to prevent detection of 'phantom components' due tc, the limited time resolution t'27. The number of exponential components after which the fit was not significantly improved by additional components was estimated by eye. The current amplitude histograms were based on all sampled data points during a given time interval and fitted with Gaussian curves using the same software and fitting procedures as mentioned above. Leak currents through the patch and through the seal were subtracted by taking the current peak at lowest current level as zero current.
RESULTS
Channel types Most patches studied showed current steps to many different levels (Fig. 1), indicating the existence of many channels in the patch or a few channels with several conductance states. For the following quantitative analysis seven patches with only a few step amplitudes at each potential were chosen. Current-potential plots based on the most frequent of these steps formed two groups of curves of different slope. Fig. 2 shows data from three patches. All curves showed some rectification with increased slope conductance at more positive potentials. The slope conductance in the range - 2 0 to 0 mV was about 30 and 80 pS for the two groups respectively. The extrapolated reversal potential for both groups of curves was more negative than - 4 0 mV (clearly more negative than - 6 0 mV for the 80 pS curves). This was independent of whether the pipette solution contained a high (solution I) or low (solution It) CIconcentration. Thus the current steps were most likely due to K+-selective channels.
SPAI Fig. 1. Outward current steps of different amplitude from one patch. Potential + 10 mV. Filtered at 500 Hz.
Kinetics In the following a quantitative analysis of the 80 pS current steps will be presented. The 80 pS steps were systematically more frequent than the 30 pS steps. The latler were consequently much more difficult to study isolated from the 80 pS steps, and were not analyzed in detail. Examples of 30 pS steps are shown in Fig. 3. They showed complex kinetics, with long periods (up to several minutes) of only negligible activity followed by relatively frequent transitions between open and closed states (Fig. 3B). The relatively low frequency with respect to the 80 pS steps is reflected by the current amplitude histograms (Figs. 4C and 6C; an interval
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Potential (mY) Fig. 2. Step current amplitudes vs. patch membrane potentials. Data from three different patches as indicated by symbols.
135 with relatively frequent 30 pS channel events at +7 mV was chosen for Fig. 4C). Two basic kinetic patterns were observed for the 80 pS steps. In one type, observed in 5 of the 7 patches studied in detail, the fraction of time spent in the open state increased with potential. In the other type, observed in 2 patches studied in detail, the fraction of time in open state decreased with increased potential. Kinetic pattern 1. Currents of the first type are shown in Fig. 4A. The potential dependence of the fraction of time in open state is illustrated in Fig. 4B. The distribution of dwell times in open and closed states were analyzed at + 17, + 7 and - 3 mV, at which the current amplitudes were high with respect to baseline noise and gating was frequent. Open-time as well as closedtime distributions, each based on more than 1,000
A
"loou l
SPAI
B
intervals, were in this case well fitted by the sum of two exponentials (Fig. 5). Generally, the open times were well fitted by two exponentials, while closed times sometimes required three exponentials. The shortest time constants were short with respect to bin width and varied significantly if the bin width was changed. These values should therefore be regarded with special caution. Kinetic pattern 2. Currents of the second kinetic type are shown in Fig. 6. The fraction of time in the open state decreased with increased potential (Fig. 6B, see also Fig. 6C). The open and closed dwell-time distributions, each based on 6,000-10,000 intervals, analyzed at -2.5, - 5 and - 10 mV are shown in Fig. 7. Also for this channel, the distributions of open times were well fitted by sums of two exponentials, while three expgnentials had to be used for closed times.
Variability of kinetics As described above, the dwell-time distributions ob tained from a large number of intervals could be well fitted by the sum of exponential components (Figs. 5 and 7). The good fit suggested that these components reflect the underlying states of the channel 7. However, if the procedure was repeated at different times during the course of an experiment, the time constants of the fitted components were not always the same. Fig. 8 shows the dwell-time distributions for the open state of the channel with the type 2 kinetic pattern described above. The measurements were made at three consecutive time intervals, separated by more than 1 , i n , during the experiment. The result was typical, with significantly differing time constants although no systematic trend with respect to time was found. Note, however, that the potential had been changed between the three different time intervals analyzed. DISCUSSION
400tm 10 pA I Fig. 3. Examples of,currents from a 30 pS channel. A: currents recorded at +20 mV. Filtration 500 Hz. B: currents at +40 mV. Consecutive traces. Note the transition of kinetics with more frequent openings in the four last traces. Note also the different scales with respect to A. Filtration 200 Hz.
The present investigation concerned single-channel currents under steady-state potential conditions. Under these conditions, many different types of current steps were observed. The most frequently found steps were caused by K ÷ channels, with main conductances of 30 and 80 pS. Previous studies suggested that individual ion channel events may induce action potentials in the studied cell type (see Introduction; ref. 14). The size of the largest current steps presently detected around the resting potential (about - 5 0 mV, see ref. 16) was about 1 pA (see Fig. 2). It is thus clear that the (roughly) simultaneous closure of less than 10 K ÷ channels, generating an excitatory current of about 10
136 potential activated channel in addition to possible potential insensitive channels. The first of the 80 pS channels described (kinetic pattern 1) showed a potential dependence similar to that of the delayed-rectifier K + channel described on basis of the whole-cell current measurements ~s. Possibly it is the basis for this current. The conductance (80 pS) was higher than those of most single-channel measurements of delayed-rectifier channels in other preparations 9,1°.17,26'29-31 (conductance about 10-20 pS). However, values closer to ours have been reported 4 (about 60 pS). Harris et al. 12 have reported K + channels with conductances of about 30 and 80 pS; the latter, however, being potential independent.
pA, would induce impulses in many cells (see ref. 16; 3-pA currents can induce impulses). A!~hough the current Rowing through a single of the studied channels (I pA) seems less than demanded to elicit an action potential in the majority of the cells, it is possible that, in some cells with particularly high input resistance, it would be sufficient for impulse generation.
Comparative aspects A previous analysis of the whole-cell currents in the same type of cell has shown that the main potentialactivated current that does not show fast inactivation is due to 'delayed rectifier' K + channels mS. Thus, in the present study we expected to find mainly this type of
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Current (pA) Fig. 4. Currents from a 80 pS channel with kinetic pattern 1. A: currents at potentials indicated. Filtered at 800 Hz for illustration. B: relation between fraction of time in open state and potential. Line drawn by eye. C: current amplitude histograms with superimposed sums of 2 ( - 3 mV), 3 ( + 17 mV) and 4 ( + 7 mV) fitted Gaussians. The histogram at + 7 mV was obtained from a period with relatively frequent gating of the 30 pS channel {not used for kinetic analysis).
137 The channels displaying kinetic pattern 2 spent a larger fraction of the time open at more negative potentials. However, they showed few similarities to the inward-rectifier K + channels described for other preparations 3'5'2°'2~'2s. In those cases, only small outward currents were observed with Mg2+-containing solutions on the membrane inside. Also, most conductances reported are much smaller than those of the present cells, even when high K + concentrations have been used. Since no inward-rectifier current was oh-
served in the whole-cell recordings ~5, we have to consider the possibility that the membrane in these patches, although formed from the whole-cell configuration in the conventional way ~, did not form outside-out but inside-out patches. Kinetics The kinetic analysis of the 80 pS current steps revealed two channel types: one with increased fraction of time in the open state with increased potential
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Fig. 5. Dwell-time distributions for the channel in Fig. 4. Open-time (left panel) and closed-time (right panel) distributions at indicated potentials were fitted by the sum of two exponentials (superimposed; time constants as indicated). Dead time 180 #s.
138 (kinetic pattern 1) and one with the reversed potential dependence (kinetic pattern 2). The number of exponential components needed to describe open and closed dwell-time distributions suggests the presence of at least two open and two closed channel states for the first chan~el type, and two open and three closed states for the second type (cf. ref. 7). A further characterization of the pathways between the states has to await an analysis of correlations between dwell-times (see e.g. ref. 6). For comparison, it may be
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noted that two closed states and one open state were sufficient to describe the macroscopic delayed-rectifier currents ~5. However, although precautions were taken
to avoid 'phantom components' (see Materials and Methods), the shortest time constants (except for open times of the channel of the second type) were short with respect to the bin width and should be regarded with caution. Kinetic cariability. The differences in dwell-time distributions measured at different time intervals during an
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Current (pA) Fig. 6. Currents from a 80 pS channel with kinetic pattern 2. A: currents at potentials indicated. Four upper traces filtered at 1 ld-lz, three lower at 800 Hz for illustration. Note the presence of 30 pS channel currents at 0 mV. B: relation between fraction of time in open state and potential. Line drawn by eye. C: current-amplitude histograms for - 20, - 5 and 0 mV with superimposed sums of two Gaussians.
139 experiment shows that the kinetics includes variations on a large time scale. Although the individual distributions suggest the presence of distinct states, new states may be adopted during the recording. The lack of any apparent trend in these variations suggests that they were not caused by a systematic change (such as a
continous deterioration) of the preparation. This is in accordance with observations in a number of different single-channel studies wl:ere changes in kinetic patterns, transitions between different kinetic modes, have been o b s e r v e d 2'13'18'22-25. In several of these studies, excised patches were used, as in the present analysis,
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Fig. 7. Dwell-time distributions for the channel in Fig. 6. Open-time distributions (left panel) at indicated potentials were fitted by the sum of two exponentials (superimposed). Closed-time distributions (right panel) were fitted by the sum of three exponentials. Time constants as indicated. D e a d time 90 p s at - 2 . 5 and 5 mV, 120/zs at - 10 mV.
140
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Time (.8 ms/div) Fig. 8. Open-time distributions for a 80 pS channel with kinetic pattern 2. Three different periods at 0 mV, with time of measurement increasing from left to right, separated by more than 1 rain. Distributions fitted by the sum of two exponentials (superimposed). Dead time 90 p.s.
suggesting that the modes did not depend on cytosolic factors. Similar transitions appeared also to occur in the 30 pS-channel currents (Fig. 3B). Acknowledgemolts. We thank Dr. Wilma Friedman, Department of Medical Chemistry !I, Karolinska Institutet, Stockholm, (presently at the Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, New Jersey) for providing the cell cultures. This work was supported by grants from the Swedish Medical Research Council (Project 6552) and Karolinska Institutets Fonder. REFERENCES ! Blatz, A.L. and Magleby, K.L., Correcting single channel data for missed events, Biophys. J., 49 (1986) 967-980. 2 Blatz, A.L. and Magleby, K.L., Quantitative description of three modes of activity of fast chloride channels from rat skeletal muscle, J. Physiol., 378 (1986) 141-174. 3 Burton, F.L. and Hutter, O.F., Sensitivity to flow of intrinsic gating in inwardly rectifying potassium channel from mammalian skeletal muscle, I. Physiol., 424 (1990) 253-261. 4 Clapham, D.E. and De Felice, L.J., Voltage-activated K channels in embryonic chick heart, Biophys. J., 45 (1984) 40-42. 5 Clark, R.B., Nakajima, T,, Giles, W., Kanai, K•, Momose, Y. and Szabo, G., Two distinct types of inwardly rectifying K + channels in bull-frog atrial myocytes, J. Physiol., 424 (1990) 229-251. 6 Colquhoun, D., The interpretation of single channel recordings. in N.B. Standen, P.T.A. Gray and M.J. Whitaker (Eds.), Micro. electrode Techniques, The Company of Biologists, Cambridge, 1987, pp. 105-135. 7 Colquhoun, D. and Hawkes, A.G., The principles of the stochastic interpretation of ion-channel mechanisms. In B• Sakmann and E. Neher (Eds.), Single-Channel Recording, Plenum, New York, 1983, pp. 135-175. 8 Colquhoun, D. and Sigworth, F.J., Fitting and statistical analysis of single-channel records. In B. Sakmann and E. Neher (Eds.), Single-Ctzannel Recording, Plenum, New York, 1983, pp. 191-263. 9 Conti, F. and Neher, C., Single-channel recording of K + currents in squid axons, Nature, 285 (1980) 140-143. 10 Coronado, R., Latorre, R. and Mautner, H., Single potassium channels with delayed rectifier behaviour from lobster axon membranes, Biophys. J., 45 (1984) 289-299. II Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100• 12 Harris, G.L., Henderson, L.P. and Spitzer, N.C., Changes in densities and kinetics of delayed rectifier potassium channels during neuronal differentiation, Neuron, 1 (1988) 739-750.
13 Hess, P., Lansman, JB. and Tsien, R.W., Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists, Nature, 311 (1984)538-544. 14 Johansson, S., Electrophysiology of Small Cultured Hippocampal Neurons. On Graded Action Potentials and Ion Channels, Doctoral thesis from The Nobel Institute for Neurophysiology, Karolinska institutet, Stockholm, 1991. 15 Johansson, S. and Arhem, P., Membrane currents in small cultured rat hippocampal neurons: A voltage-clamp study, J. Physiol., 445 (1992) 141-156. 16 Johansson, S., Friedman, W. and ,~rhem, P., Impulses and resting membrane properties of small cultured rat hippocampal neurons, J. Physiol., 445 (1992) 129-140• 17 Koren, G., Liman, E.R., Logothetis, D.E., NadaI-Ginard, B. and Hess, P., Gating mechanism of a cloned potassium channel expressed in frog oocytes and mammalian cells, Neuron, 2 (1990) 39-51. 18 McManus, O.B. and Magleby, K.L., Kinetic states and modes of single large-conductance calcium-activated potassium channels in cultured rat skeletal muscle, J. Physiol,, 402 (1988) 79-120. 19 McManus, O.B., Blatz, A.L. and Magleby, K.L., Samnling, log binning, fitting, and plotting durations of open and shut intervals from single channels and the effects of noise, Pfliigers Arch., 410 (1987) 530-553. 20 Matsuda, H. and Stanfield, P.R., Single inwardly rectifying potassium channels in cultured muscle cells from rat and mouse, J. Physiol., 414 (1989) 111-124. 21 Matsuda, H., Saigusa, A. and Irisawa, H., Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg 2+, Nature, 325 (1987) 156-159. 22 Nilius, B., Modal gating behavior of cardiac sodium channels in cell-free membrane patches, Biophys. J., 53 (1988) 857-862. 23 Patlak, J.B. and Ortiz, M., Slow currents through single sodium channels of the adult rat heart, J. Gen. Physiol., 86 (1985) 89-104. 24 Patlak, J.B., Gration, K.A.F. and Usherwood, P.N.R., Single glutamate-activated channels in locust muscle, Nature, 278 (1979) 643-645. 25 Plummet, M.R. and Hess, P., Reversible uncoupling of inactivation in N-type calcium channels, Nature, 351 (199D 657-659. 26 Rogawski, M.A., Single voltage-dependent potassium channels in cultured rat hippocampal neurons, J. Neurophysiol., 56 (1986) 481-493. 27 Roux, B. and Sauv6, R., A general solution to the time interval omission problem applied to single channel analysis, Biophys. J., 48 ~.1985) 149-158. 28 Sakmann, B. and Trube, G., Conductance propertie,, of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart, J. Physiol., 347 (1984) 641-657. 29 Smith, P.A., Bokvist, K., Arkhammar, P., Berggren, P.-O. anu Rorsman, P., Delayed rectifying and calcium-activated K + channels and their significance for action potential repolarization in mouse pancreatic//-cells, J. Gen. Physiol., 95 (1990) I041-'~059.
141 30 Standen, N.B., Stanfieid, P.R. and Ward, T.A., Properties of single potassium channels in vesicles formed from the sarcolemma of frog skeletal muscle, J. Physiol., 364 (1985) 339-358.
31 Stiihmer, W., Stocker, M., Sakmann, B., Seeburg, P., Baumann, A., Grupe, A. and Pongs, O., Potassium channels expressed from rat brain cDNA have delayed rectifier properties, FEBS Lett., 242 (1988) 199-206.
133
© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 18261
Single K+-channel currents under steady-state potential conditions in small hippocampal neurons Staffan Johansson and Peter ~rhem Nobel Institute for Neurophysiology, Karolinska Instituter, Stockholm (Sweden) (Accepted 16 June 1992)
Key words: Potassium channel; Patch clamp; Outside-out patch; Hippocampal neuron; Small cell
Small cultured hippocampal neurons from rat embryos were studied with the patch-clamp technique. Single-channel currents from outside-out membrane patches were recorded under steady-state potential conditions. The most frequently found channel types were selective to K + and showed conductances of about 30 and 80 pS in the range - 20 to 0 mV. Two basic kinetic patterns were observed for the 80 pS channels. In one type, the fraction of time spent in open state increased with potential, and in the other type it decreased. For both types of 80 pS channel, the distribution of dwell times in the open state was well described by the sum of two exponentials while three exponentials sometimes were required for dwell times in the closed state. The time constants of the fitted exponentials could vary considerably during an experiment.
INTRODUCTION
Small cerebral neurons are poorly characterized electrophysiologically in spite of their abundance. In a series of investigations we have studied small cultured hippocampal neurons under current-clamp and voltage-clamp conditions. The cells, which had a soma diameter less than 10 izm, most likely correspond to dentate granule cells and small interneurons. They showed several unexpected features: the action potentials depended systematically on stimulus strength t6, the input resistance was remarkably high (about 3 G•; see ref. 16) and several lines of data indicated that individual openings and/or closures of single ion channels could initiate the generation of action potentials 14. It is clear that, in the studied cells, very small currents do cause large changes in membrane potential. It is therefore of particular interest to obtain information on the properties of single ion channels active (i.e. opening and closing) under steady membrane potentials. The present investigation is a singlechannel analysis of outside-out membrane patches under steady-state potential conditions. The currents were studied under relatively physiological conditions with no use of channel blockers. Generally, many types of
current steps were detected. The most frequently found channel types were K + selective and showed conductances of about 30 and 80 pS in the range - 2 0 to 0 mV. The 80 pS channels showed two types of kinetic patterns. MATERIALS AND METHODS
Cell culture conditions The cells were prepared from embryonic (day El8-21) rat hippocampi, using the techniques described by Johansson et al. ~6. The electrophysiological recordings were performed after 4-8 days in culture. Only cells with a soma diameter less than 10/zm were used.
Electrophysiological recordings The currents were recorded from 29 outside-out membrane patches under voltage-clamp conditionsit. Borosilicate glass pipettes (GC 120F or 150TF, Clark Electromedical instruments), with a resistance of 8-25 MfJ when filled with and immersed in the bathing solution (see below), were used. The pipette-cell membrane seal had a resistance higher than 5 GfL The signals were recorded using an EPC-7 electrometer (Listelectronic) and stored on an FM tape-recorder (Racal Store 4DS, bandwidth under the used conditions 0-5 kHz). All experiments were performed at room temperature (21-23°C).
Solutions The bathing solution contained (in mM) NaC! 137, KC! 5.0, CaCi 2 1.0, MgC!2 1.2 and HEPES (N-(2-hydroxyethyl)piperazine-N'2-ethanesulphonic acid) 10, pH 7.4. The recording pipette was filled
Correspondence: S. Johansson, Nobel Institute for Neurophysiology, Karolinska lnstitutet, S-104 01 Stockholm, Sweden. Fax: (46) (8) 34 95 44.
134 with either (lj a solution containing (in mM) KC! 140, NaCI 3.0, MgCI 2 1.2, EG'I'A (ethyleneglycol-bis-(0-aminoethyl ether)N,N, N',N'-tetra-acetic acid; 1.0 and HEPES 10, pH 7.2 or (II) a solution containing K O H / K H 2 P O 4 140, NaCI 3.0, MgCi 2 J.2, EGTA 1.0 and Na2-ATP 3.0, pH 7.2.
D~.a analysis Only currents more than 5 s after a potential change were analyzed. The currents recorded were transferred to a computer using a TL-1 DMA interface (Labmaster) and the pCLAMP software (version 5.5.1; Axon Instruments). For this procedure the currents were low-pass filtered (8-pole Bessel filter) at a frequency that kept all noise peaks in the direction opposite to channel opening below 50% of the single-channel current amplitude ls'lg. The resulting dead times varied between 90/zs and 180 ~s. (Dead time is the duration of a rectangular pulse which allows the pulse, after tittering, to reach 50% of its true amplitude). The digitization rate was higher than 25 times the filter frequency ( - 3 dB) to prevent samplillg errors 19. The durations of open and closed intervals were measured by 50% threshold detections during visual inspection to exclude artifacts and periods with apparent subconductance current levels. All intervals shorter than the dead time were ignoredsJs. The kinetic analysis was performed using recorded current periods with a minimum of current steps from channels not in focus of the analysis. The open and closed dwell-time histograms were fitted with sums of exponential components using the pCLAMP software (based on a least-squares method and a Levenberg-Marquardt fitting algorithm). Intervals less than two times the dead time were excluded to prevent detection of 'phantom components' due tc, the limited time resolution t'27. The number of exponential components after which the fit was not significantly improved by additional components was estimated by eye. The current amplitude histograms were based on all sampled data points during a given time interval and fitted with Gaussian curves using the same software and fitting procedures as mentioned above. Leak currents through the patch and through the seal were subtracted by taking the current peak at lowest current level as zero current.
RESULTS
Channel types Most patches studied showed current steps to many different levels (Fig. 1), indicating the existence of many channels in the patch or a few channels with several conductance states. For the following quantitative analysis seven patches with only a few step amplitudes at each potential were chosen. Current-potential plots based on the most frequent of these steps formed two groups of curves of different slope. Fig. 2 shows data from three patches. All curves showed some rectification with increased slope conductance at more positive potentials. The slope conductance in the range - 2 0 to 0 mV was about 30 and 80 pS for the two groups respectively. The extrapolated reversal potential for both groups of curves was more negative than - 4 0 mV (clearly more negative than - 6 0 mV for the 80 pS curves). This was independent of whether the pipette solution contained a high (solution I) or low (solution It) CIconcentration. Thus the current steps were most likely due to K+-selective channels.
SPAI Fig. 1. Outward current steps of different amplitude from one patch. Potential + 10 mV. Filtered at 500 Hz.
Kinetics In the following a quantitative analysis of the 80 pS current steps will be presented. The 80 pS steps were systematically more frequent than the 30 pS steps. The latler were consequently much more difficult to study isolated from the 80 pS steps, and were not analyzed in detail. Examples of 30 pS steps are shown in Fig. 3. They showed complex kinetics, with long periods (up to several minutes) of only negligible activity followed by relatively frequent transitions between open and closed states (Fig. 3B). The relatively low frequency with respect to the 80 pS steps is reflected by the current amplitude histograms (Figs. 4C and 6C; an interval
8 6
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-60
-40
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I
20
40
Potential (mY) Fig. 2. Step current amplitudes vs. patch membrane potentials. Data from three different patches as indicated by symbols.
135 with relatively frequent 30 pS channel events at +7 mV was chosen for Fig. 4C). Two basic kinetic patterns were observed for the 80 pS steps. In one type, observed in 5 of the 7 patches studied in detail, the fraction of time spent in the open state increased with potential. In the other type, observed in 2 patches studied in detail, the fraction of time in open state decreased with increased potential. Kinetic pattern 1. Currents of the first type are shown in Fig. 4A. The potential dependence of the fraction of time in open state is illustrated in Fig. 4B. The distribution of dwell times in open and closed states were analyzed at + 17, + 7 and - 3 mV, at which the current amplitudes were high with respect to baseline noise and gating was frequent. Open-time as well as closedtime distributions, each based on more than 1,000
A
"loou l
SPAI
B
intervals, were in this case well fitted by the sum of two exponentials (Fig. 5). Generally, the open times were well fitted by two exponentials, while closed times sometimes required three exponentials. The shortest time constants were short with respect to bin width and varied significantly if the bin width was changed. These values should therefore be regarded with special caution. Kinetic pattern 2. Currents of the second kinetic type are shown in Fig. 6. The fraction of time in the open state decreased with increased potential (Fig. 6B, see also Fig. 6C). The open and closed dwell-time distributions, each based on 6,000-10,000 intervals, analyzed at -2.5, - 5 and - 10 mV are shown in Fig. 7. Also for this channel, the distributions of open times were well fitted by sums of two exponentials, while three expgnentials had to be used for closed times.
Variability of kinetics As described above, the dwell-time distributions ob tained from a large number of intervals could be well fitted by the sum of exponential components (Figs. 5 and 7). The good fit suggested that these components reflect the underlying states of the channel 7. However, if the procedure was repeated at different times during the course of an experiment, the time constants of the fitted components were not always the same. Fig. 8 shows the dwell-time distributions for the open state of the channel with the type 2 kinetic pattern described above. The measurements were made at three consecutive time intervals, separated by more than 1 , i n , during the experiment. The result was typical, with significantly differing time constants although no systematic trend with respect to time was found. Note, however, that the potential had been changed between the three different time intervals analyzed. DISCUSSION
400tm 10 pA I Fig. 3. Examples of,currents from a 30 pS channel. A: currents recorded at +20 mV. Filtration 500 Hz. B: currents at +40 mV. Consecutive traces. Note the transition of kinetics with more frequent openings in the four last traces. Note also the different scales with respect to A. Filtration 200 Hz.
The present investigation concerned single-channel currents under steady-state potential conditions. Under these conditions, many different types of current steps were observed. The most frequently found steps were caused by K ÷ channels, with main conductances of 30 and 80 pS. Previous studies suggested that individual ion channel events may induce action potentials in the studied cell type (see Introduction; ref. 14). The size of the largest current steps presently detected around the resting potential (about - 5 0 mV, see ref. 16) was about 1 pA (see Fig. 2). It is thus clear that the (roughly) simultaneous closure of less than 10 K ÷ channels, generating an excitatory current of about 10
136 potential activated channel in addition to possible potential insensitive channels. The first of the 80 pS channels described (kinetic pattern 1) showed a potential dependence similar to that of the delayed-rectifier K + channel described on basis of the whole-cell current measurements ~s. Possibly it is the basis for this current. The conductance (80 pS) was higher than those of most single-channel measurements of delayed-rectifier channels in other preparations 9,1°.17,26'29-31 (conductance about 10-20 pS). However, values closer to ours have been reported 4 (about 60 pS). Harris et al. 12 have reported K + channels with conductances of about 30 and 80 pS; the latter, however, being potential independent.
pA, would induce impulses in many cells (see ref. 16; 3-pA currents can induce impulses). A!~hough the current Rowing through a single of the studied channels (I pA) seems less than demanded to elicit an action potential in the majority of the cells, it is possible that, in some cells with particularly high input resistance, it would be sufficient for impulse generation.
Comparative aspects A previous analysis of the whole-cell currents in the same type of cell has shown that the main potentialactivated current that does not show fast inactivation is due to 'delayed rectifier' K + channels mS. Thus, in the present study we expected to find mainly this type of
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Current (pA) Fig. 4. Currents from a 80 pS channel with kinetic pattern 1. A: currents at potentials indicated. Filtered at 800 Hz for illustration. B: relation between fraction of time in open state and potential. Line drawn by eye. C: current amplitude histograms with superimposed sums of 2 ( - 3 mV), 3 ( + 17 mV) and 4 ( + 7 mV) fitted Gaussians. The histogram at + 7 mV was obtained from a period with relatively frequent gating of the 30 pS channel {not used for kinetic analysis).
137 The channels displaying kinetic pattern 2 spent a larger fraction of the time open at more negative potentials. However, they showed few similarities to the inward-rectifier K + channels described for other preparations 3'5'2°'2~'2s. In those cases, only small outward currents were observed with Mg2+-containing solutions on the membrane inside. Also, most conductances reported are much smaller than those of the present cells, even when high K + concentrations have been used. Since no inward-rectifier current was oh-
served in the whole-cell recordings ~5, we have to consider the possibility that the membrane in these patches, although formed from the whole-cell configuration in the conventional way ~, did not form outside-out but inside-out patches. Kinetics The kinetic analysis of the 80 pS current steps revealed two channel types: one with increased fraction of time in the open state with increased potential
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Fig. 5. Dwell-time distributions for the channel in Fig. 4. Open-time (left panel) and closed-time (right panel) distributions at indicated potentials were fitted by the sum of two exponentials (superimposed; time constants as indicated). Dead time 180 #s.
138 (kinetic pattern 1) and one with the reversed potential dependence (kinetic pattern 2). The number of exponential components needed to describe open and closed dwell-time distributions suggests the presence of at least two open and two closed channel states for the first chan~el type, and two open and three closed states for the second type (cf. ref. 7). A further characterization of the pathways between the states has to await an analysis of correlations between dwell-times (see e.g. ref. 6). For comparison, it may be
A
noted that two closed states and one open state were sufficient to describe the macroscopic delayed-rectifier currents ~5. However, although precautions were taken
to avoid 'phantom components' (see Materials and Methods), the shortest time constants (except for open times of the channel of the second type) were short with respect to the bin width and should be regarded with caution. Kinetic cariability. The differences in dwell-time distributions measured at different time intervals during an
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Current (pA) Fig. 6. Currents from a 80 pS channel with kinetic pattern 2. A: currents at potentials indicated. Four upper traces filtered at 1 ld-lz, three lower at 800 Hz for illustration. Note the presence of 30 pS channel currents at 0 mV. B: relation between fraction of time in open state and potential. Line drawn by eye. C: current-amplitude histograms for - 20, - 5 and 0 mV with superimposed sums of two Gaussians.
139 experiment shows that the kinetics includes variations on a large time scale. Although the individual distributions suggest the presence of distinct states, new states may be adopted during the recording. The lack of any apparent trend in these variations suggests that they were not caused by a systematic change (such as a
continous deterioration) of the preparation. This is in accordance with observations in a number of different single-channel studies wl:ere changes in kinetic patterns, transitions between different kinetic modes, have been o b s e r v e d 2'13'18'22-25. In several of these studies, excised patches were used, as in the present analysis,
closed dwell times
open dwell times
-2.5 mV
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Fig. 7. Dwell-time distributions for the channel in Fig. 6. Open-time distributions (left panel) at indicated potentials were fitted by the sum of two exponentials (superimposed). Closed-time distributions (right panel) were fitted by the sum of three exponentials. Time constants as indicated. D e a d time 90 p s at - 2 . 5 and 5 mV, 120/zs at - 10 mV.
140
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Time (.8 ms/div) Fig. 8. Open-time distributions for a 80 pS channel with kinetic pattern 2. Three different periods at 0 mV, with time of measurement increasing from left to right, separated by more than 1 rain. Distributions fitted by the sum of two exponentials (superimposed). Dead time 90 p.s.
suggesting that the modes did not depend on cytosolic factors. Similar transitions appeared also to occur in the 30 pS-channel currents (Fig. 3B). Acknowledgemolts. We thank Dr. Wilma Friedman, Department of Medical Chemistry !I, Karolinska Institutet, Stockholm, (presently at the Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, New Jersey) for providing the cell cultures. This work was supported by grants from the Swedish Medical Research Council (Project 6552) and Karolinska Institutets Fonder. REFERENCES ! Blatz, A.L. and Magleby, K.L., Correcting single channel data for missed events, Biophys. J., 49 (1986) 967-980. 2 Blatz, A.L. and Magleby, K.L., Quantitative description of three modes of activity of fast chloride channels from rat skeletal muscle, J. Physiol., 378 (1986) 141-174. 3 Burton, F.L. and Hutter, O.F., Sensitivity to flow of intrinsic gating in inwardly rectifying potassium channel from mammalian skeletal muscle, I. Physiol., 424 (1990) 253-261. 4 Clapham, D.E. and De Felice, L.J., Voltage-activated K channels in embryonic chick heart, Biophys. J., 45 (1984) 40-42. 5 Clark, R.B., Nakajima, T,, Giles, W., Kanai, K•, Momose, Y. and Szabo, G., Two distinct types of inwardly rectifying K + channels in bull-frog atrial myocytes, J. Physiol., 424 (1990) 229-251. 6 Colquhoun, D., The interpretation of single channel recordings. in N.B. Standen, P.T.A. Gray and M.J. Whitaker (Eds.), Micro. electrode Techniques, The Company of Biologists, Cambridge, 1987, pp. 105-135. 7 Colquhoun, D. and Hawkes, A.G., The principles of the stochastic interpretation of ion-channel mechanisms. In B• Sakmann and E. Neher (Eds.), Single-Channel Recording, Plenum, New York, 1983, pp. 135-175. 8 Colquhoun, D. and Sigworth, F.J., Fitting and statistical analysis of single-channel records. In B. Sakmann and E. Neher (Eds.), Single-Ctzannel Recording, Plenum, New York, 1983, pp. 191-263. 9 Conti, F. and Neher, C., Single-channel recording of K + currents in squid axons, Nature, 285 (1980) 140-143. 10 Coronado, R., Latorre, R. and Mautner, H., Single potassium channels with delayed rectifier behaviour from lobster axon membranes, Biophys. J., 45 (1984) 289-299. II Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100• 12 Harris, G.L., Henderson, L.P. and Spitzer, N.C., Changes in densities and kinetics of delayed rectifier potassium channels during neuronal differentiation, Neuron, 1 (1988) 739-750.
13 Hess, P., Lansman, JB. and Tsien, R.W., Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists, Nature, 311 (1984)538-544. 14 Johansson, S., Electrophysiology of Small Cultured Hippocampal Neurons. On Graded Action Potentials and Ion Channels, Doctoral thesis from The Nobel Institute for Neurophysiology, Karolinska institutet, Stockholm, 1991. 15 Johansson, S. and Arhem, P., Membrane currents in small cultured rat hippocampal neurons: A voltage-clamp study, J. Physiol., 445 (1992) 141-156. 16 Johansson, S., Friedman, W. and ,~rhem, P., Impulses and resting membrane properties of small cultured rat hippocampal neurons, J. Physiol., 445 (1992) 129-140• 17 Koren, G., Liman, E.R., Logothetis, D.E., NadaI-Ginard, B. and Hess, P., Gating mechanism of a cloned potassium channel expressed in frog oocytes and mammalian cells, Neuron, 2 (1990) 39-51. 18 McManus, O.B. and Magleby, K.L., Kinetic states and modes of single large-conductance calcium-activated potassium channels in cultured rat skeletal muscle, J. Physiol,, 402 (1988) 79-120. 19 McManus, O.B., Blatz, A.L. and Magleby, K.L., Samnling, log binning, fitting, and plotting durations of open and shut intervals from single channels and the effects of noise, Pfliigers Arch., 410 (1987) 530-553. 20 Matsuda, H. and Stanfield, P.R., Single inwardly rectifying potassium channels in cultured muscle cells from rat and mouse, J. Physiol., 414 (1989) 111-124. 21 Matsuda, H., Saigusa, A. and Irisawa, H., Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg 2+, Nature, 325 (1987) 156-159. 22 Nilius, B., Modal gating behavior of cardiac sodium channels in cell-free membrane patches, Biophys. J., 53 (1988) 857-862. 23 Patlak, J.B. and Ortiz, M., Slow currents through single sodium channels of the adult rat heart, J. Gen. Physiol., 86 (1985) 89-104. 24 Patlak, J.B., Gration, K.A.F. and Usherwood, P.N.R., Single glutamate-activated channels in locust muscle, Nature, 278 (1979) 643-645. 25 Plummet, M.R. and Hess, P., Reversible uncoupling of inactivation in N-type calcium channels, Nature, 351 (199D 657-659. 26 Rogawski, M.A., Single voltage-dependent potassium channels in cultured rat hippocampal neurons, J. Neurophysiol., 56 (1986) 481-493. 27 Roux, B. and Sauv6, R., A general solution to the time interval omission problem applied to single channel analysis, Biophys. J., 48 ~.1985) 149-158. 28 Sakmann, B. and Trube, G., Conductance propertie,, of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart, J. Physiol., 347 (1984) 641-657. 29 Smith, P.A., Bokvist, K., Arkhammar, P., Berggren, P.-O. anu Rorsman, P., Delayed rectifying and calcium-activated K + channels and their significance for action potential repolarization in mouse pancreatic//-cells, J. Gen. Physiol., 95 (1990) I041-'~059.
141 30 Standen, N.B., Stanfieid, P.R. and Ward, T.A., Properties of single potassium channels in vesicles formed from the sarcolemma of frog skeletal muscle, J. Physiol., 364 (1985) 339-358.
31 Stiihmer, W., Stocker, M., Sakmann, B., Seeburg, P., Baumann, A., Grupe, A. and Pongs, O., Potassium channels expressed from rat brain cDNA have delayed rectifier properties, FEBS Lett., 242 (1988) 199-206.