Identification of M-channels in outside-out patches excised from sympathetic ganglion cells

Neuron,

Vol. 10,639-654,

April,

1993, Copyright

0 1993 by Ceil Press

Identification of M-Channels in Outside-Out Patches Excised from Sympathetic Ganglion Cells Catherine E. Stansfeld, Stephen J. Marsh, Alasdair J. Cibb, and David A. Brown Department of Pharmacology University College London Cower Street London WCIE 6BT England

Summary We have identified the species of K+ channel that underlies the neuronal Mcurrent in rat sympathetic ganglion cells. The channels were kinetically and pharmacologically defined using outside-out and cell-attached patches. They exhibited multiple conductance levels, predominantly 3-9 pS. Their slow gating in response to voltage change in outside-out patches was exhibited only in the presence of AIF,- or GTPyS on the inner membrane surface and when the lower conductance states were dominant. In the absence of AIF,- or CTPTS, the channels exhibited rapid activation and deactivation. We conclude that M-channel gating may be controlled by an associated GTP-binding protein. Introduction Neuronal excitability is under tonic control by sustained activity of voltage-gated ionic conductances. Up or down regulation of the general level of excitability can be induced by neurotransmitter action on these persistent membrane currents. One extensively studied K+ current present in both central and peripheral neurons is the M-current. “M” stands for muscarine, since in many cells the current can be shut down by acetylcholine acting on muscarinic receptors: this increases neuronal excitability and forms an important component of cholinergic excitatory synaptic transmission (Brown and Adams, 1980; Adams and Brown, 1982; Ggihwiler and Brown, 1985; Brown, 1988a, 1988b). However, the mechanism of the G proteinrelated pathway involved in closure of the current has not yet been fully elucidated. Furthermore, until now, even the ion channels themselves have evaded intensive efforts to identify them, though there have been estimates of their conductance from noise analysis in NG108-15 neuroblastoma-glioma cells (3 pS; Neher et al., 1988) and from noise analysis and some preliminary single-channel records in rat and frog superior cervical ganglion cells (I-3 pS; Owen et al., 1990; Marrion et al., 1992). The present report provides evidence that Mchannels can be clearly identified and their activity maintained in outside-out patches; that they can exhibit either slow or fast gating modes, the slow mode being favored by G protein activators; that the channels show prominent multiple conductance levels; and

that there may be an association between the smaller channel conductances and slow gating. Finally, in these isolated patches M-channel activitywas blocked by a direct-acting inhibitor, Ba*+, but not by muscarine. This implies that muscarine-induced inhibition might require a cytoplasmic messenger, a suggestion confirmed by parallel studies on M-channel activity using cell-attached patch electrodes (Selyanko et al., 1992). The identification and characterization of these M-channels represent a significant advance toward elucidating the mechanisms by which the M-current may be modulated. Results The M-Current and Delayed Rectifier as Macroscopic Currents Initial measurements of whole-cell currents were made to relate the kinetics and pharmacology of the macroscopic currents to what was seen in the outsideout patches. In these tissue cultured, adult sympathetic ganglion cells, the M-current was the principal current active in the range -60 to -20 mV, so long as internal free Ca*+ levels were less than 80 nM. The current exhibited exponential (nonsigmoidal) activation kinetics in response to a depolarizing step (Figure IA), with its activation time constant (2) at -20 mV being 165 f 9 ms (n = 34). The change of time constant with voltage over the range -40 to -20 mV was around +60 mV per e-fold change. The current’s deactivation at -50 mV usually had two time constants: rl = 67 f 5 ms and r2 = 277 f 38 ms. The proportions varied considerably, with the slow component providing up to 80% of the total slow relaxation. In five cells of -30 f.rrn diameter, the maximum conductance was 5.3 f 0.9 nS. The delayed rectifier current was recorded in the presence of muscarine (to inhibit M-current), using 50 ms steps from -60 mV. It clearly differed from the M-current in several respects (as noted previously in frog cells [Adamset al., 19821): the activation threshold was -10 mV, i.e., 50 mV positive to M-current; the rising phase of the current was sigmoidal and could be fitted by an exponential gating function raised to the power of 2 or 3, with a time constant of 9 ms at 0 mV. The measured conductance for the delayed rectifier in these cultured cells was 28 & 6 nS (n = 8) at +70 mV. Outside-Out Patches In our early experiments, we used the same pipette solution that we normally used to maintain both the M-current and transduction mechanism for whole-cell M-current. It contained inter alia free Ca*+ buffered to IO-80 nM with EGTA or BAPTA, 2 mM ATP-Mg2+, and 0.5 mM CTP (see Experimental Procedures, solution 1). These records regularly revealed channels of

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B Whole cell

Isolated patch

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Av. n=86

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-40

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. 7.2 PS PS

mV Figure

1. M-Channel

Mean low variance amplitude (pA)

Properties

(A) Current recorded from a whole-cell in response to a depolarizing voltage command from a holding potential of -50 to -20 mV. The voltage protocol is shown below the current record. The pipette contained ATP and GTP (solution 1; described in Experimental Procedures). (B) The M-current recorded from an outside-out patch with AIF, in the patch pipette; the trace was not leak subtracted. The record is an average of 86 current traces, such as those shown below it (D). The patch contained at least 3 channels. n, number of records in average. (C) Single-channel open probability calculated from 8 patches containing up to 3 active channels. Open probability at each potential was based on the mean open probability per number of channels during a consecutive sequence of 8-10 steps (each 1 s) at each potential to avoid the induction of prolonged silent periods. Dotted lines indicate the activation curves based on a maximum open probability of either 0.7 or 1, giving half-activation at -30 mV or -23 mV, respectively. (D) Depolarizing a patch from -50 to -30 mV activated channels of around 7 pS. Dotted lines show shut channel level. Traces were leak subtracted. The voltage protocol is shown below the current traces. The pipette contained solution 4. (E) Semi-log plot showing the relationship to voltage of the decay time constant for deactivating M-channel tail currents. Each point was determined from averaged currents from a single, large outside-out patch (recorded with solution 4 in the patch pipette). (F) The rising phase of an averaged current from an outside-out patch (same as [B]) was fitted by a single exponential function (5 =

M-Channels 641

in Outside-Out

Patches

mainly7 pS that, though clearly numerous and voltage gated in the range appropriate for the M-current, never consistently showed slow gating behavior. In over 100 patches examined, though an M-like relaxation was sometimes evident on depolarizing a patch in the first few seconds following excision, the patch maintained this kinetic behavior for usually less than 30 s before apparently converting to a “rapid” mode of gating, with fast activation and deactivation. In addition, manyofthepatchesexhibitedapparentlyaberrant, noisy K+ channel activity. As an exploratory measure, the patch pipette was supplemented with a G protein activator, AIF4-, and a low proportion of these patches successfully maintained their slow kinetic behavior.These”slow”M-like relaxations were seen with pipette media containing AIFd- (13 of 65 patches), BeF3 (5 of 21 patches), or guanosine 5’-(3-thiotriphosphate) (GTPyS) (5 of 21 patches). Using these solutions (solutions 2,3, and 4; see Experimental Procedures), we have identified a series of channels with multiple conductance states as those having features characteristic of the M-current. We shall describe those features initially and then expand on some interesting, less expected features. In a “physiological” K+ gradient, the conductance of these channels was most commonly 7 pS, with an extrapolated reversal potential close to -100 mV, which is the equilibrium potential for K+ (Figures IG and IH). Most channel conductances ranged from 3 to 9 pS, but channels of up to 15 pS were also seen. In symmetrical K+, the most frequent events were of IO-20 pS. At -50 mV, they were barely activated, but a small depolarization of +I0 or +20 mV for 1 s induced marked activation, with the slow kinetics characteristicof theM-current (Figures IBand lD).Theactivation time constant of the average current at -20 or -30 mV varied greatly, but 18 patches with activation time constants greater than 35 ms had a mean time constant of 123.5 f 13.9 ms (range 45-268 ms). In 6 of these, a second faster time constant could also be fitted-13.2 f 4.0 ms (forming about 30% of the relaxation). In every patch, the current onset was quite unlike that of the delayed rectifier in being clearly exponential, not sigmoidal (Figure IF). Deactivation was voltage sensitive. At -50 mV, the deactivation time constant was 104.7 f 11.3 ms (n = 12) (Figure 2) and at -60 mV, 54.0 f 4.4 ms (n = 3). From a particularly large multichannel patch, average tail currents at potentials between -40 and -70 mV showed an e-fold change in time constant for 30 mV (Figure IE). Measurement of open probability in 8 patches from potentials of -60 to -20 mV gave an estimate of the

"I

f:

-20

178ms

-50 mv 1PAJ

cii

L L 3;L ‘:L 5L 6L

Figure 2. Deactivating Tail Channels on Repolarizing the Membrane from

2i

in an Outside-Out -20 to -50 mV

200 ms

Patch

The upper trace (A) is the average current of 82 records. It was fitted by a single time constant of 178 ms. A series of consecutive individual records which form part of the average are shown in traces l-6 in CC), and a blank trace in which no channels were open is shown in (B). These records clearly show burst behavior of the channels giving rise to the prolonged tail current, and they also show that within-burst gaps are of considerable length. Records have been leak subtracted. The pipette contained solution 3.

lower region of the activation curve (Figure IC). At more positive potentials, recordings were marred by avery slow inactivation process(see later). Open probability measurements were made early after patch excision, from a series of consecutive sweeps that did not contain a cluster of blanks. Channel open probability did not rise above 0.7. On this basis, the activation curve reached half-maximum at -30 mV, with a slope of 10.8 mV per e-fold change. Pharmacological Identification The macroscopic M-current is susceptibleto blockade by externally applied BaZ+ (Constanti and Brown, 1981). In the present experiments, whole-cell recording determined an IGO of 300 VM for Ba*+ inhibition of macroscopic M-currents (Figure 3B). These were much more sensitive to BaB than the delayed rectifier; the Ko at 0 mV was 5.7 mM (n = 9; Figure 3B). Since no significant change in Ko was detected between 0 and +4O mV, this -2O-fold lower sensitivity of the delayed rectifier to Ba*+ was unlikely to be due to the difference in recording voltage. The channels observed in outside-out patches also showed a high sensitivity to blockade by Ba*+ applied

90 ms), with no evident delay. The current is the average from 120 depolarizing steps from -50 to -20 mV. It was leak subtracted using the average of 26 scaled 10 mV hyperpolarizing steps. n, number of records in average. (G and H) Conductance curves for an M-channel and its substate showing an extrapolated reversal potential close to the theoretical equilibrium potential for K+, - 100 mV. The amplitude histogram for the points at -40 mV is presented in (H) and shows two clear amplitude levels at 0.24 and 0.44 f 0.05 pA. The histogram is the result of mean low variance analysis (Patlak, 1988) of all open channel data points.

Neuron 642

Figure 3. Blockade of Whole-Cell and M-Channels by Baz+

control

Current

(A) M-channel patch is blocked

BalmM

recovery I&*. . ..*............................

. . .. . . .. .. . .

B

-I1PA 100 ms

______-------

activity in an outside-out by external Baz+. Channel openings are upward in the trace. The holding potential was 0 mV. The pipette contained solution 4. (B) Inhibition of M-channel activity in outside-out patches by BaZ+ is indicated by closed circles. Measurements were based on 2 min each of control, test, and recovery periods at steady state at around -30 mV. The proportional suppression of activity for each patch in Ba* was calculated from the mean values of both control and recovery periods. Values were obtained from 28 patches (pipettes contained solutions 3 or 4). The inhibition curve indicates a Ko of 270 uM and a Hill slope of 1.1. The open circles are values obtained from whole-cell records for M-current suppression by Baz+, based on tail current suppression at -50 mV (see text). The dotted line was fitted assuming a Hill slope of 1 and predicts an I& of 300 PM. The open squares represent values obtained from wholecell records and show suppression of the delayed rectifier at 0 mV by Ba* (which, in the presence of 200 uM GP+, blocks Caz+ channels). The dotted line here too is fitted with the assumption of a Hill slope of 1 and predicts an ICw, of 5.7 mM.

..---ij

10-4 10-3 Log barium concentration [M]

10-2

to the external membrane (Figure 3). Ba2+ blockade did not reduce unit conductance, but reduced the proportion of time channels spent in the conducting state. In 28 patches, blockade was assessed at around -30 mV, using the ratio of open time to total time expressed as a percentage of both control and recovery conditions. Two minute periods were studied in each condition. In some cases, reduction of the integrated patch current was used to estimate the proportion of suppression. The inhibition curve shown in Figure 3B has a Hill coefficient of 1.1 and a KD for Ba*+ of 270 PM. This is consistent with the estimate from whole-cell recording for the M-current (308 FM; Figure 38, open circles) and clearly inconsistent with the sensitivity of the whole-cell delayed rectifier (Figure 38, open squares). Inclusion of 2 mM Baz+ in the patch pipette did not obviously affect the activity recorded in outside-out patches (n = 8). Since the activity could still be blocked by1 mMBa2Cappliedexternally,thisconfirms the observations of Robbins et al. (1992) in NC10815 cells that M-channels have a higher sensitivity to external than internal Ba*. The inclusion of Ca*+-activated channels in our observations is unlikely, since the inner membrane of the outside-out patches was exposed to Ca*+ levels

buffered to IO-80 nM with BAPTA or EGTA, and the outer membrane was bathed in 0 Ca*+ and 5-7 mM Mg*‘. Neither channel activity nor conductance levels were affected by the application of 1 mM tetraethylammonium (n = 14), 2mM Qaminopyridine (n = 3), 100 uM d-tubocurarine (n = 31, or 180 nM apamin (n = 6). Delayed Rectifier Delayed rectifier-like activity could be recorded from multichannel patches on stepping the membrane voltage to -10 mV or more positive. Single-channel conductances were over 10 pS, and the voltage sensitivity clearly differed from that observed for the M-channels in that they were barely active at potentials negative to -10 mV, yet showed strong voltagedependent activation on further depolarization. In contrast, M-channels showed the most acute voltagedependent activation in the range -50 to -20 mV. Some patches exhibited only delayed rectifier-type behavior; however, a detailed study of these channels has not yet been completed. Inactivation Rat sympathetic neurons exhibit A-current (Calvan and Sedlmeir,

a rapidly inactivating 1984; Belhrzzi et al.,

M-Channels 643

in Outside-Out

Patches

OmV -50 -100

0.5 PA] 50s

B

8

Figure 4. Demonstration side-Out Patches

of A-Channel

Activity

in Excised

Out-

Pipettes contained solution 2. (A) Two averaged currents are superimposed (no leak subtraction) from a patch that contained multiple Achannels and M-channels, but not delayed rectifier channels. The currents shown were the response to voltage steps (the protocol is shown below the current traces). Stepping to 0 mV from -50 mV elicited M-channels (n = 138), whereas preceding this step by a brief sojourn (100 ms) at the hyperpolarized potential, -100 mV, elicited an additional transient current (n = 102), which is clearly fully inactivated within 100 ms. (B) Normalized conductance versus voltage curves show the mean parameters that fitted the activation (half-maximum, -15.8 mV; slope, 15.4) and inactivation (half-maximum, -95.3 mV; slope, 8.1) curves for the averaged A-current in 3 multichannel patches. Averaged A-current amplitudes were measured from the subtracted transient current using paired data such as that shown in (A). The data points from 3 patches are superimposed. Activation curves were measured from the averaged current response to depolarization to various potentials, following a 100 ms hyperpolarizing prep&e to -120 mV. Inactivation curves were measured from the averaged current responsetodepolarizing steps to 0 mV, following a 100 ms prepulse to various hyperpolarized potentials. Steps were repeated at 5 s intervals, between which a holding potential of -50 mV was maintained.

1985). Channels corresponding to this current were present in a number of patches (Figure 4). They were activated on depolarization from potentials negative to -50 mV (using a 100 ms hyperpolarizing prepulse). Their averaged currents peaked within 3 ms at 0 mV and showed a voltage-insensitive rate inactivation with a time constant of 14.3 f 3.3 ms (n = 7). The mean midpoint of the steady-state inactivation curve was -95 f 6 mV (n = 3), with a slope of 8.1 (Figure 4B). Thus, even though the A-channel conductances were within the M-channel range, they would be fully inactivated by our normal protocol, which used a holding

potential of -50 mV. The channel conductances varied, being 5 pS (3 patches), 10 pS (2 patches), and 16.5 pS (2 patches). Although the M-channels did not show the fast inactivation seen in the A-channels, a very slow, partial inactivation process was nevertheless nearly always present. It was different from that observed for the A-current in that the rate of inactivation was very much slower, and it increased with depolarization. For example, at -30 mV, although declinewas insignificant over a 1 s time scale, inactivation over a 2 min period was usually substantial. At more positive potentials, it clearly intensified, with some patches being detectable in the averaged currents over a 1 s period at -20 mV. Examination of trends in open and closed times indicated that at least two separate processes seemed to be involved in inactivation, inducing decreased open times as welt as prolonged closed times This was not a peculiarityonlyof outside-out patches, since it could be clearly demonstrated in cell-attached patches too (Figure 5), despite the fact that the M-current is noninactivating when recorded in whole-cell mode. Channels in cell-attached patches had recognizably the same activity, though transitions between substates were less obvious. The channels were readily activated on small depolarizations of +I0 or +20 mV from the rest potential (around -60 mV) and could give rise to slow relaxations in averaged currents. In parallel experiments, we have shown the activity of these channels to be suppressed by exogenously applied muscarine (Selyanko et al., 1992). Using high K+ in the cell-attached recording pipette, the same characteristic activity could be observed, the channels being around 20 pS under these symmetrical K+ conditions. The experiments shown in Figure 5 werecarried out in cell-attached patches with 130 mM K+ in therecordingpipette.Theyshow increasedactivity as the patch was depolarized from the resting potential (E,,,J together with longer open times, but a subsequent reduction in rate of activity and open timeswhen depolarization was maintained. In a multichannel patch showing quite marked inactivation, the rate of decline was observed over a 40 s time period (Figure 5B); in this particular patch, the decay time constant was 5.9 s. As seen here, the inactivation for potentials negative to 0 mV was never totally absorbing, with considerable activity remaining at steady state. In most cases, this took the form of a low regular rate of activity, with shorter open times or, alternatively, the single-channel activity fluctuated as it inactivated, reemerged to the active state, and then inactivated again (Figure 5C). Inactivation was readily removed by hyperpolarizing the patch negative to -60 mV for 1 s at -80 mV, or 100 ms at -120 mV. The smaller patches seemed to show this inactivation more intensely than very large ones (patches containing more than 8 channels), and it also increased with the duration of the recording. The possibility that inactivation was controlled by cysteine oxidation (Ruppersbergetal.,l991)wasexaminedinoutside-out

Neuron 644

Figure 5. Inactivation Cell-Attached Patches

of

M-Channels

in

The patch pipettes contained 130 mM K+, giving a K+ reversal potential close to 0 mV. (A) Patch activity observed at steady state. Thevoltages indicatedwere those imposed on the patch, so that 0 = the cell’s resting potential (approximately -60 mV), and -20 indicates a 20 mV depolarization, giving a patch membrane potential of about -40 mV. The first 5 traces are in sequence, the patch potential being depolarized progressively, with 2 min at each potential, but at -30 mV, the initial high rate of activity was not sustained: after 1.5 min at -30 (b), events were shorter, with increased closed times. The sixth trace, at -30 (c), shows restoration of activity at -30 mV, following patch hyperpolarization for 2 s to 20 mV negative to E,,,. The trace shown is taken 5 s after imposing the new depolarized potential (-30 mV) on the patch. (B) A patch containing at least 10 channels, of around 25 pS, was depolarized from a pipette voltage of +20 to -30 mV for 40 s. Four records (filtered at 0.5 kHz) were sampled at0.05 kHzand averaged before fitting a single exponential to the decay phase. Two of the original traces (a and b) are shown from the onset of the voltage step. The lowest panel (c)shows the fitted curve, giving a time constant to steady-state level of 5.9 s. Decay is incomplete. (C) Stability plots over 150 s for measure ments of open times, closed times, and open probability from a third patch with apparently 1 active channel. The voltage is 20 mV depolarized from E,,,. Each bin shows a rolling average of 30 intervals, with an increment of 15 intervals between averages. Unitary activity fluctuated, but when the shut times were long, the open times became noticeably short. The open probability during the active periods was close to 0.5, which might be expected for the M-channel at this voltage.

Interval number x 10e3

patches by the inclusion of either 5 mM glutathione or3 mM chloramine-T in the patch pipette, but neither showed an obvious difference in the form of the slow inactivation. Multichannel Conductances As stated earlier, M-channel activity consisted of a range of low conductance events, mostly 3-9 pS in physiological K+ gradient and most commonly 7 pS. Many patches showed clear multiple conductance levels (e.g., Figure 6). It was found that, although substates were not obvious in all patches, in some cases a very high proportion of channel open time (>50%)

was spent at a subconductance level (Figure 6). All levels could be reached from either open or closed states. Although the dominant conductance level often changed with time or with voltage, it did not change in any consistent direction. Frequently, the stable conductance sublevels were obscured by rapid transitions between substates. The patches selected for analysis were those in which the traces were little, or only periodically, affected by these flickering transitions. The number of identifiable conductance levels varied from patch to patch but was usually between two and four levels. Sometimes these levels were evenly spaced. However, any 1 of the compo-

M-Channels 645

in Outside-Out

Patches

Tim 8 d LKI 6 E 3 IFi 0

Figure

6. M-Channels

in Outside-Out

Patches

Have

Several

Subconductance

0.5

Mean

lot-variance

0

015

amp%de

1.5 @A)

~-1 PA -

Levels

(A) Selected sequences (consecutive but not contiguous) from a 2 min record of channel activity at 0 mV, in which the constant rate of activity gave an open probability of 0.6. However, the amplitude of the channel openings during activity changed several times, initially suggesting the presence of several different channels, yet without a single event superimposition amounting to more than the original 0.9 pA (the dotted line shows the 1 pA level). Given the high rate of activity, it is unlikely that this behavior is due to several independent channels of different conductance; rather, this activity is probably generated by a single channel. The pipette contained solution 3. (B) Open point amplitude histogram from part of the patch record seen in (A). The superimposed Gaussian curves indicate the presence of three main amplitudes: 0.28 f 0.08 PA, 0.61 f 0.14 pA, and 0.94 f 0.1 pA. The driving force being 100 mV, these correspond to 2.8, 6.1, and 9.4 pS. The left-most peak entered at 0 pA shows the baseline noise (SD = 0.071 PA) for reference. (C) The same data as (B), subjected to mean low variance analysis (Patlak, 1988), in which a moving window of 15 data points were averaged to give a mean amplitude, which was included in the distribution if the variance of that mean was less than 1.5 times the baseline variance (measured in [B]). The peaks lie at 0.27, 0.59, and 0.93 pA. (D) All point histogram of 150 s of data from the patch shown in (A). The data were more heavily filtered (filter cutoff frequency, 100 Hz), so as to enhance the peak distributions; hence, they are shifted left compared with (B) and (C). This shows that a high proportion of time was spent at the lowest conducting state.

nents could be enhanced or disappear with time independentlyoftheothers.Thisindicatesthatthevarious conductance levels seen are unlikelyto bethe expression of independently gated pores (Matsuda, 1988): their gating must either be linked or they are the expression of a single pore with different conductance states. Although most of these experiments were done in the presence of 5 mM Mg2+ on the outer membrane surface, reducingthe Mg*to0.5 mM and substituting 2.5 mM Ca*+ did not noticeably alter the conductance levels nor the substate behavior. We have not seen any obvious induction or change of substate behavior for inward currents in symmetrical K+ conditions upon raising external Mg2+ (Matsuda, 1988).

Fast and Slow Cating and a Possible Relationship with Conductance levels A rather interesting phenomenon was that M-channel activation at -30 mV could be “fast”(onset T = 10 ms) or “slow” (onset 7 = 100 ms) without an obviously different pattern of steady-state channel activity. As mentioned earlier, on patch excision, the M-currentlike relaxation evident on depolarizing a multichannel patch in the first few seconds could be maintained in some cases for IO-15 min. But more usually, the pattern swiftly changed to one of rapid gating-the response to depolarization being fast (Figure 7A)with a simultaneous, equivalent change in the tail current at -50 mV. This change seemed to occur without loss of channel activity, nor was there any negative

Neuron 646

Figure 7. Slow M-Channels

PA

I

E

D

Oo“ Open point amplitude (PA)

4

5

6

7

8

91

Predominant channel conductance (PS)

shift in the activation curve. It is important to note that those patches which on isolation lost their slow gating behavior did not regain iteven in thecontinued presence of G protein activators. The fast and slow forms of the channel activity had the same sensitivity to Ba2+, and neither were affected by 1 mM tetraethylammonium. Whereas a wide range of channel conductances were seen, in the outside-out patches, only the conductance states below 9 pS were seen to generate a slow relaxation characteristic of M-current activation. As just described, the averaged onset time constant varied widely, reaching 268 ms at -20 mV, but with a curious feature: the slower time constants occurred in patches in which the mean channel conductance was low (Figures 78, 7C, and 7D). We have analyzed a group of 14 patches containing channels up to 9 pS.

and

Fast

Gating

of

(A) Loss of M-channel kinetics in an outsideout patch. Currents recorded from a multichannel patch during the first 60 s following excision. Repeated depolarizing steps were applied at 5 s intervals. The voltage protocol is shown below the traces. (a) is an average of steps l-4, and (b) is the average of steps 9-12. This was considered an unsuccessful patch in maintaining M-current kinetics. The pipette contained solution 4. Traces were leak subtracted, using averaged 20 mV hyperpolarizing steps. (B and C) Data from two separate outsideout patches. Current traces and their averages and channel activity evoked by depolarizing steps from -50 to -20 mV are shown. (B) is from a slow patch: onset r = 115 ms (sample rate, 1 kHz; filter, 250 Hz). K) is from a fast patch: onset T = 7 ms (sample rate, 4 kHz; filter, 500 Hz for the averaging procedure). Pipettes contained solution 4. Traces were leak subtracted, using scaled hyperpolarizing steps. (D) Open point amplitude histograms (clearly defined channel openings were selected to be without superimposed events). Histograms are superimposed for the 2 patches shown in (B) and (C). The slow patch (shaded histogram) has a fitted curve of 3 Gaussian components with mean values of 0.22, 0.42, and 0.6 PA; SD, 0.06 pA (relative areas, 59%, 38%, and 3%). Mean amplitude, 0.31 PA; mean conductance, 3.9 pS. The scale for this histogram is half of that shown (i.e., maximum 580). The unshaded histogram from the fast patch was fitted with 4 Gaussian components with mean values of 0.38, 0.57, 0.71, and 1.03 * 0.1 pA (relative areas, 29%, 29%, 24%, and 18%). Mean amplitude, 0.63 PA; mean conductance, 7.9 pS. (E) Semi-log plot of onset time constant for average currents against mean channel conductance for 14 outside-out patches. A least squares linear regression was fitted giving r = 0.7062, which is significantly different from 0.

Amplitude histograms were created of all wellresolved openings during repeated 1 s depolarizing steps to between -20 and -30 mV. These patches showed a clear relationship between the dominant conductance state and the onset time constant for the averaged current on patch depolarization; the lower channel conductances are associated with the slower time constants (r = 0.7062) (Figure7E). Given the clarity of the substate levels (Figure 6), it seems most unlikely that the correlation between conductance and onset time constant shown in Figure 7E could be due to the presence of more fast, unresolved closures in the slow patches. Close observation of the records did not reveal signs of increased filtering or slow capacitance changes, which might have indicated an unusually high access resistance. In addition, nocorrelation was found between channel conductance and open times

The the a T, b T,

10 f

7.5

Fast

0.7

Tz 34.0 * 2.1 (78%) 44.3 f 3.8 (73%)b

Patches

3.0 f (4%) 2.0 f 60%)

‘I1

Closed

1.1

0.8

Times

(24%)

23.2 * 3.4 (30%) 19.6 f 8.0

‘c2

>2 ms (ms)

205.6 (16%)

f

38.8

44.3

Mean Closed Times (ms)

Numbers analyzed.

111.5

* 14.6

(24%)

150.0

T3

table compares the kinetic properties of M-channels in patches with slow or fast ensemble current activation. mean relative percent area of each component is shown in parentheses. Four slow and 6 fast patches were present in 2 patches: 6.3 and 10.7 ms (39% and 47%, respectively). present in 3 patches; r2 present in 5 patches.

16.1 f (78%Jb

-a

2

Tl 12

118 *

5.5

in Outside-Out Open Times >2 ms (ms)

Slow

of M-Channels

Activation

Properties Onset Time Constant (ms)

1. Single-Channel

Dominant Conductance (PS)

Table

are mean

Behavior

fms) Duration

30.9 * 13.2 (33%) 23.0 f 4.0 (29%)

Tl

Burst

+ SEM. For distribution

8.2

& 2.8 38.5 f

47.9

T&t

Burst

T2

f

f

time

21.0

17.4

constants,

(67%)

(72%) 142.5

158.1

Neuron 648

Figure

‘Fast ’ A

4 -

a.

B a.

-30 mV

144 36

100 64

16

36 16 4

0

1

10 100 Open time (ms) (log scale)

1000

0

1

10 100 Open time (ms) (log scale)

b.

4

Shut time (ms) (log scale)

Burst

Shut time (ms) (log scale)

Burst length (ms) (log scale)

length (ms) (log scale)

(r = 0.0703) nor between open times (r = 0.1013).

onset

time

constant

and

Single-Channel Kinetic Measurements Measurement of open and closed times was restricted to channel activity evoked by 1 s depolarizing steps from -50 mV. The reasons for this were twofold: to avoid induction of the slow inactivation described earlier and to ensure that the form of gating behavior was known and could be assessed from averaged currents. An important note of caution is that there was an apparent requirement for more than 2 channels to be present in the patch before the slow M-current kinetics were expressed; smaller patches with a single channel invariably had fast activation. (These observations also applied to cell-attached patches, as well as to these outside-out patches.) Possibly, patch formation induced a critical change in membrane-

lOGil

8. Single-Channel

Gating

Comparison of single-channel kinetics in outside-out patches from a slow patch (A) and a fast patch (B), both recorded with solution 4 in the patch pipette. Channels of similar conductance have been chosen for the comparison, (A) being 7.8 pS and (B), 7.9 pS. The leak-subtracted, average currents in response to depolarizing steps from -50 mV to -30 mV or -20 mV are shown above. (A) shows an average of 118 records with an activation T = 96 ms. (B) shows an average of 97 records; the activationr = 7.0ms.A partial inactivationduring the 1 s step is evident. Measurements were made from channel activity evoked during depolarizing stepsof 1 s duration,from -50 mV. Note the log scales for the ordinate and square root scale for the abscissa of these dwell time distributions. The peaks of the curves correspond to the time constant of the exponential distribution (McManus et al., 1987; Sigworth and Sine, 1987). The resolution was 2 ms for both openings and closings. (a) Open time histograms fitted by exponential components (curves). For (A), r = 40.8 ms, and for (B), T, = 17.1 (67%) and ~2 = 56.1 ms (33%) (mean, 30.2 ms). (b) Shut time histograms for (A) and (B), each with 3 components. In (A), the time constants are 2.0 ms (44%), 17.5 ms (25%), and 135.5 ms (31%) (mean, 47.3 ms). In (B), the time constants are 1.4 ms (45%), 16.0 ms (28%), and 130.6 ms (27%) (mean, 40.7 ms). (c) Burst length histograms for (A) and (B). In (A), bursts were defined using a T,,,, of 38.5 ms (see Experimental Procedures); r = 98.5 ms. In (B), using a T,,,r of 38.0 ms, two time constants were present: r, = 27.5 ms (63%) and rZ = 169.6 ms (38%) (mean, 80.7 ms). The mean number of openings per burst for (A) was 2.1 and for (B), 2.2.

associated structures, and hence channels in the larger patches had a better chance to maintain their normal gating. Unitary activity has been studied from 4outside-out patches, each containing at least 3 channels with slow gating kinetics,asdemonstrated from theaveragecurrent response to depolarizing steps: onset time constant was 118 ms between -20 and -30 mV (range: 77-147 ms) (Table 1; Figure 8). In 1 of these patches, the measurements were supported by consistent measurements from channels at steady state at -30 mV. Ignoring openings briefer than 2 ms, the principal open time was well defined at 34.0 ms. In 2 of these patches, a second component (6.3 and 10.7 ms) was present. Shut times had at least 3 components: 3.6,21.9, and 147 ms. The slow kinetic behavior of the macroscopic current may have its basis in long burst durations with long interburst intervals, rather than in long open times, as shown in the single-channel

M-Channels

in Outside-Out

Patches

649

Figure 9. First Latency Fast and Slow Patches

B a.

A a.

A h.

B h.

0

I04

Y?(x) 104

xx)

500

600

00

20

40

nel. For example, patch (A) had 3 channels that were assumed by 3. Patch (B) had 2 channels, and the latency measurements

records of Figure 2. Distributions of burst durations were fitted with 2 exponential components of 30.9 ms and 158.1 ms at -30 mV. Examination of well-isolated channels from 6 fast patchesgave results surprisinglysimilartothosefrom slow patches. At the same potential as that of the slow patches described above, their onset time constant for average currents was 10.0 ms (range 3.9-16.4 ms) (Table 1; Figure 8). Two open time components, 16.1 and 44.3 ms, were present; the faster component was present in 3 patches, but dominant in all three. The mean open time for all 6 patches was 31.8 ms. Three shut time components were present: 2.0, 19.6, and 205.6 ms. Two burst length components were identified: 23.0 and 142.3 ms.

First latency Analysis In view of the multiple closed times, we might expect a delay in the current onsets. However, the leaksubtracted, averaged currents designated slow characteristically showed no evident sigmoidicity in the current onset, though in at least 1 patch, a delay was detectable. For the fast patches, it was more difficult to obtain the resolution required to determine the difference between a single or multicomponent fitted curve. To obtain a clearer measure of the onset kinetics, we have analyzed first event latencies in 5 patches, 1 slow and 4 fast (Figure 9). The onset time constant for the slow patch was 93 ms at -30 mV. The fast patches had average current onset time constants ranging from 6 to 31 ms at either -30 or -20 mV. A noticeable feature of all of the first latency distributions was that when an apparent delay was present, it was brief in comparison with the main onset time constant with negligible effect on the cumulative first

60

80

to be equally were similarly

100

active, and adjusted.

Measurement

for

First latency plots for the activity underlying the two average currents shown in Fig ure 8. They indicate the time from the be ginning of the depolarizing step to the first channel opening, measured over 88 and 188 steps, respectively, for (A) and (B). (A, graph a) and (B, graph a) show first latency histograms. (A) is for the patch shown in Figure 8A, and (B) is for that in Figure 88. Below are plots of the cumulative distribution of times from the same data arbitrarily scaled, indicating the probability that a channelwillopenatagiven time.Thefitted curve for (A, graph b) gives two time constants: r,, 62.5 ms (74%) and Q, 434.1 ms. For (B, graph b), the single time constant was 16.9 ms. The lack of exact accord between these time constants and those measured for the average currents may arise from a degree of inactivation in each case, or from the presence of more than 1 chanthe latency measurements were multiplied

latency curve (Figure the average currents.

9), reflecting

what was seen in

Tests of Muscarine Effects on Outside-Out Patches Application of 10 uM muscarine produced no convincing change in channel activity, either when patch pipettes containing AIF,- were used or with the alternative complement of 2 mM ATP and 0.5 mM GTP. Discussion Identification of M-Channels Several lines of evidence indicate that the channels recorded in these experiments are indeed M-channels: their activation voltage range and voltage sensitivity, their kinetic behavior, and their pharmacology. These features have been described for the whole-cell rat ganglion M-current by Constanti and Brown (1981), Brown et al. (1989), and Owen et al. (1990). The channels were sensitive to membrane voltage in the range -60 to0 mV, with half-maximum activation at -30 mV. They showed slow, exponential but nonsigmoidal activation,asdetermined both by averaged currentsand by analysis of first event latency. The deactivation rate at -50 mV was only a little faster than the rate of activation at -30 mV, whereas deactivation for the delayed rectifier is much faster (Galvan and Sedlmeir, 1984; Belluui and Sacchi, 1991). The lack of effect of 4-aminopyridine but the sensitivity of the channels to external Ba* reflects the sensitivity of whole-cell M-current to these substances, as reported in this paper, and is inconsistent with the high sensitivity shown by the delayed rectifier to rl-aminopyridine (Marsh and Brown, 1991) and its relative insensitivity to Ba*. Furthermore, multichannel patches exhibited delayed rectifier-like activity from channels of over 10 pS when the mem-

Neuron

650

A

B

Acetylcholine I

GTWS AIF4BeF3 G

J ,

I I Receptorcoupled G-protein

GTP-binding moiety

r-l I

I

1

I

\ \ \ slow garing

closure

J

lost in isolated patches Figure 10. Schematic trol of the M-Channel

can be preserved in isolated patches Representation

of the Proposed

Dual Con-

Pathway (A) is the muscarinic receptor/G protein-linked pathway, which may use a diffusible messenger in closure of the M-channel. This sourceof control seems to be lost in outside-out patches. Pathway (B) consists of a GTP-binding moiety closely associated with the channel protein, which is either activated or stabilized by the presence of GTPTS or AIFI-. It governs the slow gating of the M-channel. This pathway can be preserved in an outside-out patch.

brane voltage was stepped to positive potentials, and some patches exhibited only this type of behavior. For these reasons, we do not consider the unitary activity described to be delayed rectifier channels. The possibility that the channels might be A-channels seems unlikely, in view of the fact that A-current-like ensemble activity is readily seen and can clearly be separated from M-channel ensembles, in terms of inactivation and deactivation characteristics. The occurrence of channel activity in low to negligible Caz+ levels and relative insensitivity to tetraethylammonium, apamin, and d-tubocurarine exclude the involvement of Ca2+-activated channels. Channel Behavior The single M-channels showed a number of interesting features, as follows: -Channels exhibited multiple conductance states, .the basis of which is unlikely to originate in independent gating of multiple pores. -M-channels exhibited more than one mode of gating, so that activation was termed, for convenience, fast or slow, according to whether the activation time constant at wound -30 mV was -10,or -100 ms, respectively. The fast gating mode readily occurred in patches with 1 or few active channels. The slow

mode was more readily seen in patches where several active channels were observed. -The fast or slow behavior of the channel could be influenced by activators of G proteins; their presence made the slow mode more likely, as long as multiple channels were present. -The kinetic behavior of the channel was correlated with the channel conductances; the slow gating mode was associated with the lower conductance states, mostly below 6 pS. -The measured single-channel gating was similar for fast and slow patches, indicating that at -20 to -30 mV, we have not measured the rate-limiting steps involved in activation and deactivation. -The channels exhibited slow inactivation, which was not readily modified by cysteine oxidation/reduction. Fast/Slow Gafing We could consider the fast gating mode to be artifactual in these patches, since it was inconsistentwith the whole-cell currents seen at the same voltages: -50 to -20 mV. Yet, its origin is unclear. It readily appeared in cell-attached as well as outside-out patches, irrespective of the direction of K+ current flow. However, in the cell-attached mode, slow kinetics were much more predictable and stable when very large patch pipettes were used (<2MSZ), together with a very gentle sealing technique, implying that the channels were readily disturbed by patch formation per se, not merely by changes in solution bathing the inner membrane surface. There is the potentially enigmatic observation that we were able to sustain appropriate M-channel-like gating in outside-out patches only in the presence of G protein activators: circumstances that in thewholecell mode would close the M-channels (Pfaffinger, 1988; Lopez and Adams, 1989; Brown et al., 1989). This leads us to propose that there may be two G proteins involved here (Figure IO). One would be closely associated with the channel (but presumably also readily dislodged on patch formation) and controls the gating pattern, though apparently without governing its open-closed state. The second G protein would be associated with the muscarinic receptor, at a distance from the M-channel itself. This G protein would require a second messenger to govern channel availability. The loss of slow gating in small cell-attached patches perhaps then reflects the fragilityof a submembrane structural association, resulting inthelossofeitheraGproteinorachannel-associated GTP-binding structure. It is well to recall here that in the outside-out patches which lost their slow gating, it was not later recovered despite the continued presence of the G protein activators. Thus, the AIF4-, and GTPyS seemed to be capable of stabilizing a molecular conformation, rather than recovering it once lost. In this context, we note that one of the identifying criteria for the M-current has been its slow kinetic behavior. But the above observations suggest that, in fact, these kinetics may be under a regulatory control that

M-Channels

in Outside-Out

Patches

651

is separate from, and additional to, that governing channel availability (Figure IO). Conductance and Gating In principle, slow kinetics could result from slow voltage control. However, there are three points to consider here. First, we did not observe unusually high filtering of unit current rise times nor slow current changes in blank or “leak” records. Second, the conversion of channel kinetic behavior from slow to fast certainly has nothing to do with signal filtering and was readily seen with large pipettes as well as with small pipettes. Third, apparent open times were the same for slow or fast patches. A high access resistance would be likely to result in a false estimation of patch voltage, and since the channel kinetics are highly voltage sensitive (Figure SA), we should have seen a difference if our predicted voltagewas inaccurate. It seems more likely that our observation of smaller conductances for the longer time constants may be real for our recording conditions, but we have yet to determine whether this holds true for cell-attached patches. A very low unitary conductance, l-3 pS, has been predicted from spectral analysis (Neher et al., 1988; Owen et al., 1990; Marrion et al., 1992). Estimation of single-channel conductance from fluctuation analysis assumesasingleunitaryconductance,which isclearly not true of these channels. The channel gating is also clearly more complex than predicted from the noise analysis. Although transitions between substates might contribute to the low estimations of channel amplitude from noise analysis, in this context our own observation that lower channel conductance correlates with slower time constants is a particularly interesting one. Spectral analysis of muscarine-sensitive currents in these same cells (Owen et al., 1990; Marrion et al., 1992) detected a low frequency component with a time constant of 162 ms at -30 mV, which may relate to the burst duration of 158 ms seen here. We can also see apparent burst activity from slowly deactivating channels at -50 mV (Figure 2). However, an interesting outcome of the single-channel measurements was that we did not find a difference in open or shut times between fast and slow activating channels. The similarity in the shut times for fast and slow patches suggests that the rate-limiting step in the slow M-current kinetics is unlikely to be the channel opening rate constant and is more likely to lie between two closed states. Multiple closed states are also suggested by the evident contribution of burst behavior to the slow deactivation tail currents (Figure 2) (see dual deactivation time constants seen forwhole-cell M-current; see also Marrion et al., 1992). The measured closed times imply the presence of at least three closed states. These, plusthe inactivated stateand its accompanying altered open state, give us a starting model consisting of four closed and two open states. However, since the same open and closed times were measured for

fast and slow activating patches, this implies that there must bean additional closed state-one that isvoltage sensitive and rate limiting in the activation process. In other words, measurement of first event latencies is more appropriate than open/closed state measurement in estimation of the limiting-rate constants for current activation. Muscarine The principal discrepancy between the properties of these isolated M-channels and the macroscopic current is clearly the resistance of the isolated channels to muscarine. The reason for this is probably that M-channel closure by muscarine requires the production of a cytoplasmic messenger and that this production mechanism is ineffective in isolated patches. Thus, in parallel experiments using cell-attached patch electrodes, we have detected channels with two main conductance levels (7 and 12 pS); the former corresponds to those described here from isolated patches (Selyanko et al., 1992). As in outside-out patches, channels in cell-attached patches showed persistent activity with muscarine present directly on the patch surface. However, in the cell-attached patches, they could be closed by applying muscarine to the cell membrane outside the patch. In the present experiments, the higher conductance channels, 12-18 pS in cell-attached patches, have not been observed togive rise to the longertime constants associated with the whole-cell recorded M-current relaxations, whereas they have been associated with activation time constants of IO-50 ms. These are nevertheless slower than those observed for similar conductance channels in the outside-out patches. Thus, in the present studies the 12-18 pS channels have not been included in the analysis because they were never observed to show slow activation in outside-out patches, although they certainly display the same sensitivity to Ba* as the smaller channels. On the other hand, in cell-attached patches, channels of 7 pS have been observed to generate an average current with an activation time constant of 200 ms close to -30 mV, a value much closer to that measured in the whole-cell currents reported here. Until a detailed analysis of the relationship of conductance to activation time constants in cell-attached patches is made, we cannot make more precise comment. Thus, to summarize our main findings, we have presented the following evidence: -On the bases of their kinetics and pharmacology, the channels described here can be identified as M-channels. -Under a patch pipette, the M-channels readily lost their slow gating behavior and became fast activating and deactivating (a slow inactivation may also be an induced artifact). -The smaller conductances exhibited by the channels were more prominent in those patches giving the slowest activation kinetics.

Neuron 652

-The slow-gating mode may be under the control of a closely associated G protein, presumably separate from and additional to that governing channel availability through the muscarinic receptor. -Finally, the measured open and closed times from channels in either slow or fast gating mode apparently did not differ, implyingthatthe limiting-rateconstants are difficult to detect at these membrane potentials, without the use of nonstationary analysis of first event latencies or averaging. Experimental

Procedures

The Preparation Rats (Sprague-Dawley) 17 days old were used. Superior cervical ganglia were dissociated using enzyme and light mechanical treatment, and the cells were plated onto laminincoated plastic 35 mm culture dishes (Marrion et al., 1987). Recordings were made within 24 hr of culture. Solutions External solutions consisted of 144 mM NaCI, 2.5 mM KCI, 5 mM MgCh, 10 mM glucose, 5 mM HEPES, buffered with Tris base to pH 7.4. Tetrodotoxin (1 PM) was usually present. In a number of experiments, the MgCl2 was replaced by 0.5 mM MgCb, 2.5 mM CaCb. For symmetrical K+ conditions, the external Na+ and K+ was modified to 130 mM KCI. Occasional additions to the external medium were Qaminopyridine, tetraethylammonium chloride, d-tubocurarine, Bach, and apamin (Sigma), all at concentrations defined in the text. The basic internal solution (solution 1) consisted of 100 mM potassium gluconate, 30 mM KCI, 2 mM MgClz, ECTA (Sigma) or BAPTA (Sigma) at various concentrations (0.2-2 mM) (Ca* was added to give a calculated free Ca* concentration of IO-80 nM; see below), 2 mM ATP-MgZ’ (Sigma), 0.5 mM CTP-Na+ (Sigma), 10 mM HEPES (Sigma), buffered with Tris base (Sigma) to pH 6.8. In some experiments, the ATP and GTP were replaced by 0.5 mM GTPyS (Sigma) (solution 2). Many experiments used a modification of the basic solution, replacing the KCI with 20 mM KF and 0.01 mM AICId- or BeCI, for solutions 3 and 4, respectively (see Chabre, 1990; Yatani and Brown, 1991). In these solutions, the CaCh, ATP, and CTP were omitted, and EGTA (1 mM) was added. Chloramine-Tand reduced glutathione were from Sigma. All chemicals, unless specified, were from BDH. Internal solutions were designed to maintain Caz+ within the range IO-80 nM. Our estimations of free Ca*+ concentration was obtained using the program React, version 2.01, designed by G. L. Smith (Department Physiology, University of Glasgow, Scotland). We used it to calculate the total amount of Ca2+ required for solutions of specified free Ca2+ concentrations, using specified OH, ionic strenath, total ATP, and ECTA or BAPTA. The various affinity constaits for H+, Ca2+, and MgZ’ were as given by Smith and Miller (1985) for EGTA and by Tsien (1980) for BAPTA. Drugs were applied by continuous close perfusion from a pipette of approximately 200 pm diameter placed within 200 urn of the patch, with a flow rateof around 1 mllmin. No corrections for liquid junction potentials were made. Pipettes Borosilicate glass pipettes (Clark Electromedical) were pulled to resistances of around 10 MO and then polished to 30 MD for outside-out patches, but for cell-attached patches, much larger pipettes of around 1 MD were used. Good resolution of the channels was aided by tight seal resistances of above 50 CD, Sylgard coating (Dow Corning), and shallow penetration of electrodes in the perfusion fluid. Whole-Cell Recording and Analysis The whole-cell records were made using an Axoclamp2A switched clamp amplifier (Axon Instruments) operating at 5 kHz.

Current analyzed

records using

were acquired on line on an IBM-type pCLAMP software (V5.5).

PC and

Recording from Membrane Patches An Axopatch-ID or Axopatch-200 amplifier was used for singlechannel recording, records were low pass filtered at 10 kHz (-3 dB), and the signal was stored on videotape using a Sony Betamax recorder and a modified Sony PCM. The Axopatch-200 amplifier has an integrating headstage, so some of our records contain the resetting transient artifacts from the headstage. In actual practice, the steady holding current for the patches was usually less than 0.4 pA, so the resetting of the integratorcircuitoccurred at less than 30 s intervals. The transients themselves did not resemble single-channel events. The average of 100 transients (sampled at 10 kHz and low pass filtered at 2 kHz) measured 0.25 pA with a decay time constant of 1.8 ms. Thus, with the bandwidth used for the M-channel analysis, the transients are insignificant. Analysis of Patch Currents Signals from the tape were filtered at between 250 Hz and 1 kHz (-3 dB) before further amplification and digitization via a CED-502 interface (Cambridge Electronic Design), usually at 4 times the filter frequency. Filter cut-off frequencies given are the -3 dB frequency, which is half the value shown on the front panel of our filter, an 8 pole Barr and Stroud bessel-type filter. Averaged records of the current response to voltage steps wereobtained using a modificationof DA5 (1. Wise, Sandoz Institute for Medical Research) software, DA8 (Dr. David Owen, Wyeth Research, England), running on a PDP-IV23 computer. This program permits triggered acquisition of episodes (512 sample points per record), leak subtraction, and averaging. It also enables visualization of every leak-subtracted trace before its entry to the average. The averages themselves were normally composed of around 100 records, using about 90% of those available. Curve fitting to the leak-subtracted averaged activation and tail currents was completed by pCLAMP programs (V5.5, by minimization of least squares error) after exporting the files from the PDP computer to an IBM-type PC. Single-Channel Analysis Single-channel analysis was carried out on a PDP-II/23 computer running PAT4 software (John Dempster, Strathclyde University) and on a PDP-II/73 computer running a suite of programs created by David Colquhoun(UniversityCollege, London, England). Both steady-state conditions and events evoked during depolarizing steps were analyzed. For the latter, the latency of events from step onset has not been taken into account. Analysis for amplitudes, open point histograms, and open times each required slightly different selection of data and hence separate analysis. Event amplitudes were measured manually using potentiometer-controlled cursors to select unambiguous open states, avoiding possible doubleopenings and obvious fast flicker between open states (usually a total of 512 openings). The selected open levels were then subjected to mean low variance analysis (Patlak, 1988), the criterion here being that the standard deviation of 10 or moreconsecutive sample points of open channel noise should not be greater than 1.5 times the baseline rootmean-square noise. With 10 data points used as the running mean, this represents a minimum duration of 5 ms for acceptable open periods. The sum of 2-4 Gaussian components could normally be fitted to the resultant histogram. This method permits detection of clean conductance levels and was employed for those patches in which slope conductance measurement was required. In 14 patches, the root-mean-square baseline noise was 0.055 * 0.019 PA, and the ratio of the smallest event amplitude (fitted Gaussian component) to root-mean-square noise was 5.8 f 1.99. The method described above would obviously bias any estimate of occupancy of any particular conductance level, so a separate analysis was done for those patches in which an estimate of dominant sublevel was required, accepting all events

M-Channels 653

in Outside-Out

Patches

that showed closures to baseline and including long events up to, but excluding, superimpositions. These data were subjected initally to mean. low variance analysis in order to establish the principal peaks, by fitting the sum of 2-4 Gaussian components. Having established the number and values of the main amplitudes, those values were fixed when fitting the original open point datawith summed Gaussian components (leaving the magnitude and SD of each one free during the fit) to obtain the relative proportion of each component. A weighted mean value for conductance (y) (Figure 7) was calculated from this, taking the total area under the curves = 1. Fork Gaussian components, if u, = mean value for the j ‘h Gaussian component and ai = fractional area for the th Gaussian component, then the mean amplitude value i =

;: p,a,. -1 Measurement of open times t&d the method described by Colquhoun and Sigworth (1983), in which the event time course was fitted using the measured step response functions of the patch amplifier, recorder, and filters. In 10 patches, the mean rise time was 332 us. Brief closures within bursts have, as far as possible, been taken into account and treated as closures. The minimum resolvable duration was taken to be 2 ms; however in the data measurement process, periods of noisy, flickering open events briefer than 3 ms have been largely omitted to enable adequate measurement of the longer events. This bias has been taken into account by using only durations longer than 8 ms when fitting exponential components to thedata bythe method of maximum likelihood (Colquhoun and Sigworth, 1983). No attempt has been made to separate the various conductance levels in the open time analysis of any individual patch, all events with clear closures to baseline being treated equally. Events that could be interpreted as simultaneous openings of 2 channels were omitted. Bursts were identified by using a critical gap length (T,,,,) calculated from components 2 and 3 of the closed time distribution, as described by Colquhoun and Sakmann (1985). The effect of Ba2+ on channel activity at steady state was measured in two ways, each using a thresholdcrossing criterion: in patches with superimposed activity, the suppression of total charge transfer in patches was measured; for nonsuperimposed channel activity, the total open time was measured. For both methods, a 2 min period was used each for control, test, and recovery conditions. The computer programs used for this were written by one of us (S. 1. M.) and by J. Dempster (Strathclyde).

hereby marked “advertisement”in tion 1734 solely to indicate this Received

August

11, 1992; revised

accordance fact. January

with

18 USC Sec-

26, 1993.

References Adams, P. R., and Brown, D. A. (1982). Synaptic inhibition of the M-current: slow excitatory post-synaptic potential mechanism in bullfrog sympathetic neurones. I. Physiol. 332,263-272. Adams, P. R., Brown, D. A., and Constanti, A. (1982). and other potassium currents in bullfrog sympathetic J. Physiol. 330, 537-562.

Belluzzi, O., and Sacchi, 0. (1991). A five-conductance model of the action potential in the rat sympathetic neurone. In Progress in Biophysics and Molecular Biology, Volume 55, D. Noble and T. L. Blundell, eds. (Oxford: Pergamon Press, Inc.), pp. I-30. Belluzzi, O., Sacchi, O., and Wanke, outward current in the rat sympathetic voltage-clamp conditions. J. Physiol.

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Brown, D. A. (1988a). M-current. In Ion Channels, Volume 1, T. Narahashi, ed. (New York: Plenum Publishing Corp.), pp. 55-99. Brown, D. A. (1988b). 11, 294-299.

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Brown, D. A., Marrion, N. V., and Smart, T. C. (1989). On the transduction mechanism for muscarinic-induced inhibition of M-current in cultured rat sympathetic neurones. J. Physiol. 413, 469488. Chabre, M. (1990). Aluminofluoride and beryllofluoride complexes: new phosphateanalogs in enzymology. Trends Biochem. Sci. 75, 6-10. Colquhoun, D., and Sakmann, B. (1985). Fast events in single channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J. Physiol. 369, 501-557. Colquhoun, D., and Sigworth, F. J. (1983). Fitting and statistical analysis of single-channel records. In Single Channel Recording, B. Sakmann and E. Neher, eds. (New York: Plenum Publishing Corp.), pp. 191-263. Constanti, clamped 289-294.

A., and Brown, D. A. (1981). M-currents in voltagemammalian sympathetic neurones. Neurosci. Lett. 24,

A Non-K+ Membrane Conductance Also Gives Slow Current Relaxations These membranes were observed to possess a presumed Clconductance, possibly Ca2+ dependent, since it was seen almost exclusively in leaky patches.Awarenessof itwas important, since the unitary conductance was extremely low, activating on depolarization as a smooth, time-dependent relaxation, even in a small patch, with a slow time constant in excess of 100 ms. It reversed close to -20 mV so that at -30 mV the relaxation was inward, but positive to -20 mV it relaxed outward (i.e., in the same direction as the M-current). Normally this feature was absent, but even when barely present, it could be very readily detected as an inward, slowly deactivating tail current on repolarizing to -SO mV. Patches exhibiting this phenomenon were discarded.

Calvan, clamped

Acknowledgments

Marsh, S. J., and Brown, D.A. (1991). Potassium currents uting to action potential repolarization in dissociated rat superior cervical sympathetic neurones. Neurosci. 298-302.

We thank Yvonne Vallis for the excellent superior cervical ganglion cell cultures and Professor David Colquhoun, John Dempster, and David Booth for the use of their computer programs. The support of the Wellcome Trust and Medical Research Council is gratefully acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be

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Lopez, H., and Adams, P. R. (1989). A G-protein mediates the inhibition of the voltage-dependent potassium M-current by muscarine, LHRH, substance P and UTP in bullfrog sympathetic neurons. Eur. J. Neurosci. 7, 529-542. Marrion, currents culture.

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Matsuda, H. (1988). Open-state substructure of inwardly rectifying potassium channels revealed by magnesium block in guineapig heart cells. J. Physiol. 397, 237-258. McManus, 0. B., Blatz, A. L., and Magleby, log binning, fitting and plotting durations

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Neher, E., Marty, A., Fukuda, K., Kubo, T., and Numa, S. (1988). Intracellular calcium release mediated by two muscarinic recep tor subtypes. FEBS Lett. 240,88-94. Owen, D. C., Marsh, S., and Brown, D.A. (1990). M-current noise and putative M-channels in cultured rat sympathetic ganglion cells. J. Physiol. 437, 269-290. Patlak, J. (1988). Sodium channel sured with a new variance-mean 413-430. Pfaffinger, P. J. (1988). Muscarine by activating an IAP-insensitive 3353.

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