549

Journal of Physiology (1992), 445, pp. 549-568 With 10 figures Printed in Great Britain

THE EFFECT OF INTRACELLULAR pH ON ATP-DEPENDENT POTASSIUM CHANNELS OF FROG SKELETAL MUSCLE

BY N. W. DAVIES, N. B. STANDEN AND P. R. STANFIELD From the Ion Channel Group, Department of Physiology, University of Leicester, PO Box 138, Leicester LE1 9HN

(Received 25 April 1991) SUMMARY

1. We have used patch-clamp methods to study the effects of pH at the cytoplasmic surface of the membrane on ATP-dependent K+ channels (KATP channels) in patches excised from frog (Rana temporaria) skeletal muscle, and to study the kinetics of ATP binding. 2. In the absence of ATP, a reduction in pH led to a slight decrease in singlechannel current amplitude, an increase in the number of very brief closings, an increase in the apparent mean open time, and an increase in burst duration. After correction for missed closings, the change in mean open time was slight. Despite these changes in detailed kinetics, the channel open-state probability, Popen' changed little with changes in pH in the absence of ATP. 3. In the presence of ATP, a decrease in internal pH (pHi) reduced the degree of channel inhibition by ATP, shifting the curve relating Popen and [ATP] to higher concentrations of ATP without altering its steepness. The ATP concentration for half-inhibition of channel activity (Ki) was 17 /SM at pH 7-2 and 260 /M at pH 6-3. 4. The effect of pH could be modelled by assuming that one or two protons bind to the channel and prevent ATP binding to exert its effect of causing channel closure. The predicted dissociation constants for ATP and H+ respectively were 5-4 and 0-11

JM.

5. The rate constants for binding and unbinding of ATP were estimated from the dependence of the mean open time on [ATP] and from the Ki. The apparent rate constants for ATP binding were 0-6 and 0-04 mm-' ms-1 at pH 7-2 and 6-3 respectively, while the rate constant for unbinding was 0 01 ms-'. In terms of our model the calculated true rate constant for ATP binding was 1X85 mM-' ms-'. ATP binding also led to a reduction in burst duration. 6. The effect of pH described here differs from findings in cardiac muscle and pancreatic B-cells. The results are discussed in relation to the possible function of KATP channels in skeletal muscle during exercise. INTRODUCTION

K+ channels closed by internal adenosine-5'-triphosphate (ATP) have been found in cardiac, skeletal and smooth muscle cells (Noma, 1983; Spruce, Standen & Stanfield, 1985; Standen, Quayle, Davies, Brayden, Huang & Nelson, 1989) and also MS 9340

N. W. DAVIES, N. B. STANDEN AND P. R. STANFIELD in pancreatic B-cells and neurones (Cook & Hales, 1984; Ashford, Sturgess, Trout, Gardner & Hales, 1988). The concentration of ATP required to half-close ATPdependent K+ channels (KATP channels) is much lower than the intracellular concentration of ATP under physiological conditions. Furthermore, in both cardiac and skeletal muscle the concentration of ATP is maintained at 5-10 mM, even during vigorous activity, by the buffering action of creatine phosphate and creatine kinase (Carlson & Siger, 1960). At rest the open state probability (P'pen) of these channels is very low in cardiac and skeletal muscle. Recently it was shown that a decrease in internal pH (pHi) reduced the inhibitory effect of ATP on KATP channels in isolated inside-out patches of frog skeletal muscle (Davies, 1990). Since pHi has been shown to decrease by up to 1 unit in exercising skeletal muscle (Pan, Hamm, Rothman & Shulman, 1988; Renaud, 1989) it is likely that internal protons are an important regulatory mechanism linking P1pen of KATP channels with metabolic activity of skeletal muscle. In this paper we present a detailed investigation into the changes of the kinetics of KATP channels produced by altering pHi. We also show that the main effect of pH1 is on the inhibitory action of ATP, so that a decrease in pHi increases the ATP concentration for half-inhibition (Ki) of channel activity without any change in the stoichiometry of the block. This effect can be modelled in terms of two protons competing with ATP for the site at which it inhibits activity. We have used the same model to estimate the kinetics of ATP binding to the channel. Preliminary reports of some of these findings have been communicated to the Physiological Society (Davies, Standen & Stanfield, 1989b, 1991 a). 550

METHODS

Preparation Sarcolemmal vesicles of the cutaneous pectoris muscle of the frog Rana temporaria were prepared

by KCl loading and enzymatic treatment (collagenase followed by protease) as described in detail by Standen, Stanfield, Ward & Wilson (1984). The frogs were killed by decapitation followed by destruction of the brain and spinal cord.

Recording methods Currents through single KATP channels were recorded using the inside-out configuration of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Patch pipettes were made from 1-5 mm o.d. borosilicate capillaries (GC150F, Clark Electromedical Ltd) pulled on a

Kopf puller, coated with Sylgard resin to reduce background noise, and fire-polished. Their resistances were 5-20 MCI when filled with electrolyte solution. After seal formation the patch was excised and placed in the outlet stream of a multibarrelled perfusion pipette consisting of a common outlet connected to four or five different reservoirs. Currents were measured using a List-electronic EPC-7 amplifier with a 50 GQ feedback resistor. The membrane potential was set by application of a steady voltage to the pipette. Membrane potentials are expressed conventionally, as inside (of the membrane) relative to outside, and outward currents are defined positive and plotted upwards. Solutions The solution in the patch pipette, which bathed the extracellular side of the membrane, contained KCl at 2-5, 10 or 60 mm, NaCl so that [Na++K+] = 120 mm, 1-8 mM-CaCl2, and 10 mmHEPES buffer; pH was adjusted to 7-2 with HCl or NaOH. Intracellular solutions contained 5 mMEGTA neutralized with KOH, KCl to bring [K+] to 120 mM, 10 mM-HEPES for pHi 8.0 and 7-2. For pHi 6-8 and 6-3 10 mM-PIPES (piperazine-N,N'-bis[2-ethanesulphonic acid] was used instead of

EFFECTS OF pH ON MUSCLE KATP CHANANELS

551

HEPES, while 10 mM-MES (2-[N-morpholino]ethanesulphonic acid) was used for pH. 5-7. The bath solution in which vesicles were formed and seals made was the same as the intracellular solution at pH 7-2. ATP (dipotassium salt), which was weighed and dissolved just before an experiment, was added to the intracellular solutions and the pH readjusted. Data collection KATP channel currents were filtered at 10 kHz by the EPC-7 amplifier and recorded on videotape using a modified Sony PCM-701 digital audio processor (Lamb, 1985). For single-channel analysis the videotape was later replayed through an 8-pole Bessel filter at a cut-off frequency (-3 dB) of between 2 and 4 kHz, and digitized at 20 kHz using a CED 502 A-to-D converter interfaced to a PDP 11/73 computer. For analysis of P1pen or fractional block in multichannel patches the replayed currents were filtered at 1 kHz and digitized at 3-33 kHz. Analysis Binning and open and closed time distributions. Measurements of the distribution of open and closed times and burst durations were confined to patches containing a single active channel. Events were detected using the 50% threshold method (Colquhoun & Sigworth, 1983), and the resulting idealized trace could then be compared to the original data. If necessary, records were refiltered digitally with a Gaussian filter to ensure that the detection threshold was at least three times the standard deviation of the noise. Event durations were stored in a file as real positive numbers for openings and real negative numbers for closings. Minimum resolution could then be imposed on this file by ignoring all events shorter than a time, tmin, of 150 /as (Colquhoun, 1987; see Fig. 2). This enabled direct comparison between dwell-time histograms of data filtered at different cut-off frequencies. We have used logarithmic binning of data to allow a wide range of times to be included (McManus, Blatz & Magleby, 1987). Open and closed time distributions were obtained by binning events at a resolution of 25 bins per log unit according to the formula: bin = 1 + integer (25 x logl0(event duration in sampling points)). (1) Since bin width has a complex relation to bin number it was necessary to form a decoding file by binning all integers between 1 and 10000 (the maximum duration in sampling points binned). The width of a bin in sampling points is then the number of integers in the bin and their mean gives the mid-point of that bin. Owing to the integer operation in eqn (1), an event of duration one sampling point falls in bin 1 as expected, but an event of two sample points falls in bin 8, an event of three sample points in bin 12 and so on until no further null bins are encountered. Null bins are excluded from the fitting routines, and for display purposes the number of events in a bin is divided by 1 + the number of following consecutive null bins, with the width adjusted accordingly. The distributions are plotted with a logarithmic abscissa and the ordinate is the square root of the number of events in a bin (see Sigworth & Sine, 1987). Such plots give peaks close to the time constants of the exponential components, and make it easier to see components with relatively small areas. After binning, the open and closed time distributions were fitted, using the method of maximum likelihood (Colquhoun & Sigworth, 1983; Sigworth & Sine, 1987), to the probability density function: m

f(t) =

E

j=1

(aj/lr)exp(-t/Tj),

(2)

where ai is the area of component j, rT is the time constant of component j and m is the number of components. The log likelihood of the binned data is given by: I

log likelihood =

ni log i=k

ti+1

f(t) dt/B,

(3)

J ti

where k is the first and I is the last bin to be fitted, ti is the time at the beginning of bin i, ni is the number of events in bin i and B is the integral of f(t) between the limits of the fit. The set of parameters which maximized the likelihood was found iteratively using the downhill simplex method (Press, Flannery, Teukolsky & Vetterling, 1986). Correcting for missed closings. Since many brief closings occur during a burst of openings the measured mean open time (1.o') is an overestimate of true to. An indication of the true mean open

552

N. W. DAVIES, N. B. STANDEN AND P. R. STANFIELD

time was obtained as the product of the measured mean open time and the proportion of closed events detected, the latter being given by integrating the fitted closed time distribution between the minimum resolution and infinity. Thus:

to = to E aj exp ( tmin/i)),

(4)

where tmin is the minimum resolution and a, is the area of component j, r1 is the time constant of component j and m the number of components in the closed time distribution. Brief openings were much rarer and consequently the measured mean closed time was not corrected. Analysis of bursts. Bursts were defined in the recordings as an opening or group of openings separated by closings shorter than a specified critical time, t,. Thus closings longer than t, were classified as interburst gaps. The closed time distribution has four components (see Results) and we assigned the longest two time constants of the distribution as interburst gaps. We chose the method of Colquhoun & Sakmann (1985) to calculate t,, namely that of equalizing the proportion of long intervals misclassified as short ones with the proportion of misclassified short intervals. This was achieved by solving the equation, 4

X exp (-tjr) = 2,

(5)

iteratively for t,. Measurement of Popen' Measurement of Popen was performed on patches containing up to eight channels by measuring the times, tj, spent at current levels corresponding to j = 0,1,2 ... N channels open. The overall Popen was then obtained using:

Popen = ( tij) /TN,

(6)

where the duration of the recording, T, was usually 30-60 s. The maximum number of channels in a patch, N, was taken as the maximum number of simultaneously open events seen under control (no ATP) conditions. Some patches had twenty to thirty active channels. Such patches were often used to obtain the concentration dependence of inhibition by ATP by averaging the current remaining under different ATP concentrations and normalizing this to the value obtained in the absence of ATP. Unless otherwise stated, results are expressed as means + S.E.M. Experiments were done at room temperature, 17-23 'C. RESULTS

The effect of pHi on KATP channels in the absence of ATP KATP channels were studied in excised inside-out patches at a holding potential of 0 mV (to avoid any currents caused by the opening of Cl- channels) and with 10 mMK+ in the pipette solution. Under these conditions, channels were spontaneously active with an open-state probability, Popen, of about 0X6. Figure 1A shows examples of the activity of a single KATP channel when the cytoplasmic face of the patch was exposed successively to solutions of different pH. Decreasing pHi had two noticeable effects: it caused a slight decrease in the amplitude of the single-channel current and it altered the kinetic behaviour of the channels. Figure lB shows amplitude histograms in pHi 8-0 and 5-7. Mean unitary current was reduced from its value of 1-79+0-04 pA (n = 5) at the normal pHi of 7-2 to 1-48+0-03 pA (n = 5) at pHi 5.7 (Fig. 5B). The reduction in unitary amplitude caused by increasing proton concentration was not associated with an increase in open-channel noise (Fig. 1B). These results suggest that protons either block the channel with very fast kinetics or that they alter the surface charge at the internal side of the channel, reducing its conductance by repulsion of permeant ions.

EFFECTS OF pH ON MUSCLE KATP CHANNELS

553

A

op. C , 1 i U¶1 ig A'!1 f# II 1

0

ImArfilmill f1JI

pHi 8 0

I I

1

1

0.

o~~~

C M1tX O

iw"l f

pHi 72

I'*444.'

pHi 6 3

_

, 1*-T

pHi57

2

tI1

t

150 ms

30

ms

B

pH1 8 0

pHi 5.7 800

V,

4-

0 CL 0

250

0

E z

0 0

1 Current (pA)

2

3

0

1

2

3

Current (pA)

Fig. 1. A, examples of KATP channel activity recorded from an inside-out patch at the four different pH, values indicated. The traces have been filtered at 2 kHz for display and those on the right show the portion of the left-hand traces between the arrows at an expanded time scale. The holding potential was 0 mV, the patch pipette contained 10 mmK+ and the bathing solution contained 120 mM-K+. 0, open; C, closed. B, amplitude histograms obtained at pH, 8&0 and 5&7 under the same conditions as in A. Fitting the data with Gaussian distributions gave values for the baseline and open channel current of 0 00 + 0 19 and 1P77 + 0X22 pA respectively at pHi 8&0 and 0 00 + 0-23 and 1P37 + 0-24 pA respectively at pH, 5f7.

pA

N. W. DAVIES, N. B. STANDEN AND P. R. STANFIELD

554

Open and closed time distributions Distributions of open and closed times were obtained from patches containing a single active channel. After appropriate filtering, the current records were used to obtain an idealized trace showing the transitions between the open and closed levels

C *.

2 pA

5 40 ms

No imposed resolution

Minimum resolution = 150 ,s

Fig. 2. Detection of events and the effects of setting a minimum time resolution. The activity of a KATP channel in pH, 7-2 at the original filter cut-off frequency of 4 kHz is shown at the top. The dashed line is the detection threshold for both opening (0) and closing (C) and was set at half the single-channel amplitude. The two traces below show the idealized record reconstructed from crossings of this threshold and the idealized record after ignoring all events shorter than 150 #us.

as described in the Methods. Data, together with the idealized trace, were displayed in 51-2 ms segments, and a decision made to accept or reject the idealized trace or to reset the threshold. In this way any baseline drift could be compensated for and occasional unsuitable segments of recording, caused for example by extraneous noise, could be rejected. Open and closed times were computed from the idealized trace and stored for analysis. Figure 2 shows an example of a current record at pHi 7-2 together with the idealized traces before and after imposing a minimum resolution of 150 Fs. Open and closed times were binned according to eqn (1) after imposition of a consistent resolution of 150 ,us and were fitted with probability density functions (PDFs) given by eqn (2) using maximum likelihood as described in the Methods. Figure 3 shows fitted histograms of the open and closed time distributions obtained from a patch exposed to pHis of 7-2 and 6-3. The distribution of open times consisted of two exponential components while that of the closed times had four components, indicating the presence of at least two open and four closed states. Spruce, Standen & Stanfield (1987) identified two open and at least three closed states; here the use of log binning has allowed us to fit a fourth component of the closed time distribution. We made similar fits to open and closed time distributions in fourteen other patches. Because the number of events recorded from different patches varied considerably (from 380 to 9604), we have calculated weighted mean values for the parameters of the fitted PDFs (ajs and rs in eqn (2)) by weighting the values from

EFFECTS OF pH ON MUSCLE KATP CHANNELS Open times

555

Closed times 256-

144

pHi 7.2 U,

64-

w- 360

0-1

10

1000

0.1

10

1000

c,

L)

wl

10 10 1000 0.1 1000 Time (ms) Time (ms) Fig. 3. Histograms of open and closed time distributions of a KATP channel recorded from an inside-out patch held at 0 mV and exposed to pH. 7-2 and 6-3. A minimum resolution of 150 ,ts was imposed on the data and durations were binned according to eqn (1). Two exponential components were required to fit the open times and four were needed for the closed times. Mean values for the parameters of probability density functions fitted to the distributions from fifteen patches are given in Table 1.

0*1

TABLE 1. Open and closed time distributions of KATP channels at 0 mV in the absence of ATP pHi 7-2 pHi 6-3 pHi 5-7 pH, 8-0 Open times 14493 6881 6330 2718 no 0-12 +0-02 0-024 + 0-01 0-16+0-01 0-10+0-01 al 1-45+0-13 0-84+0-04 0-58+0-10 0050+0003 Iri 0-98 + 0-01 0-84+0-01 0-90+0-01 0-89+0-02 a2 12-24+ 1-20 15-51+0050 6-66+0-14 21-73 +0 73 T2 Closed times 2717 6886 5483 14492 nc 0-78+0-01 0-78+0-03 095+000 0-92+0-01 al 009+000 0-08+0 00 0-08+0 00 o011+0o00 I1 007 +0-01 003+000 0-20+0 00 0-19+0-03 a2 0 50+0-08 0-92 + 0-02 0-45+0-06 0-55+0003 T2 001 +0-00 003+000 0-02 + 000 0-02 + 0*00 a3 739+0*84 15-18+1-13 10-07 + 0-42 6-30+0-42 T3 0-002 + 0000 0 009 + 0-001 0 004 + 0000 0-002 + 0000 a4 9990 + 330 5 301-4+25-6 343-9 + 50-6 317-5+34-7 74 The distributions were fitted by eqn (2) with the a- and Tr values below; nO and n, are the number of openings and closings respectively.

N. W. DAVIES, N. B. STANDEN AND P. R. STANFIELD

556 A

pHi 7 2

oB lht

CF

Ilb

10.

II-1L_

pHi 63

300

B

pHi 7-2

36

C', :3

0

01

10

1000

10

1000

pHi 63

U1)

co 0 0.1

Time (ms) Fig. 4. For legend see facing

page.

is

EFFECTS OF pH ON MUSCLE KATP CHANNELS 557 each patch in proportion to the total number of events recorded from that patch. We calculated the weighted variance as

a2 = (xi

wi

(7)

where wi is the weight for the ith patch, and so the standard deviation and standard error of the mean. These weighted values are given in Table 1. TABLE 2. Distribution of burst durations of K ATP channels at 0 mV in the absence of ATP pHi 7T2 pHi 5-7 pHi 80 pHi 6-3 1974 1272 879 142 nb 008+0-03 005+0-01 0-13+002 0-15+002 al 0-31+003 0-51+006 082+008 0-50+0-14 Ir1 025+0-02 0-25+0004 0-21+0005 024+0003 a2 35-98+6620 22-53+9-26 11-36+ 163 21-71+3-97 32 0-61+0-02 0-71+0415 0-70+0-04 0-64+0-04 a3 198-4+20-6 4209+ 131-7 58-6+ 1-8 88-5+9-1 Ir. The distributions were fitted by eqn (2) with the a, and T values below; nb is the number of bursts at each pH.

Decreasing pHi leads to an increase in the number of very short closings corresponding to an increase in a, and a small decrease in r1, as shown in Table 1, and an increase in the measured mean open time (to'). However, most of the increase in to' seems to result from the increased number of missed closings, since the mean open time obtained after correction for these using eqn (4), f,o changed much less with pHi, being 2-26, 2-65, 3-83 and 3-63 ms respectively at pHi 8-0, 7-2, 6-3 and 5-7 (see also Table 3). One possibility is that the increased number of short closings results from a blocking action of protons on open channels different from that which reduces the unitary current. Such a mechanism would lead to an additional short duration component in the closed time distribution. We have not seen such a component, although we would expect it to be hard to detect because of difficulties in measuring very short closed times. It seems more likely that protons exert their effects by changing some of the rate constants for channel gating. Certainly the increase in burst duration seen with decreased pHi (described below) is too large to be explained by open channel block. Burst kinetics Openings of muscle KATP channels, whether in the absence or presence of ATP, occur in bursts (Spruce et al. 1987; Fig. 4A). The critical time, tc, used to detect bursts was calculated for each patch from the closed time distribution by solving eqn (5), giving values in the range from 0O58 to 2-54 ms, and histograms of burst length Fig. 4. Burst distributions in pH, 7-2 and 6-3. A, examples of records with (below) the corresponding idealized burst traces, calculated using critical times of 1-04 and 1-23 ms at pHi 7 2 and 6-3 respectively to distinguish bursts (eqn (5)). 0, open; C, closed; B, burst. B, histograms of burst length distribution. Mean values for the burst parameters in fifteen patches are given in Table 2.

558

N. W. DAVIES, N. B. STANDEN AND P. R. STANFIELD

were formed. The distribution of burst lengths was best fitted by the sum of three exponential components. Figure 4B shows examples of the fits to burst durations at pHi 7-2 and 6-3 for the same patch as that shown in Fig. 3. Mean values for the distribution of burst lengths, weighted as described above, are given in Table 2. Mean A

1.0

I

T

0.5

a 0~

I T

of

5.0

6.0

7.0

8.0

7-0

8.0

B

2.0 0.

G1)6

1.5 1.0 _

C.)

0.51oo

5.0

6.0 pHj

Fig. 5. The effect of pHi on Popen and unitary current amplitude in the absence of ATP. A, * show mean+ S.E.M. values of Popen pooled from fifteen patches. B, mean unitary current amplitudes (@) measured from five patches. The standard errors of the mean were smaller than the symbol in each case. 0, unitary current multiplied by popen from the results shown in A and B.

burst lengths were 43 9, 61-5, 134-3 and 299 6 ms in pHi 8-0, 7-2, 6 3 and 5-7. As will be shown later, burst lengths were markedly reduced by ATP (see also Spruce et al. 1987).

Popen changes little with pHi in the absence of ATP Despite the changes in kinetics described above, Popen in the absence of ATP remained fairly constant over the range of pHi values studied (Fig. 5A). This can also be seen in Table 1, where Popen calculated from the fitted open and closed time + distributions as /(to+tc) changes little with pH. Figure 5B shows that the mean current flowing through KATP channels, calculated as iP0pen, where i is the unitary current, also changes relatively little with pHi. Thus the observed change in burst

EFFECTS OF pH O'N MUSCLE KATP CHANNVELS 559 length with pH does not affect Popen to any great extent because of other changes in channel kinetics. As we would expect, changing pHi seems to affect several of the transitions between states of the channel. The effect of pHi on channel inhibition by ATP A much more striking effect of pH on Popen was seen in the presence of ATP; under these conditions a decrease in pHi reduced the inhibition by a given concentration TABLE 3. Corrected mean open time (fo), mean closed time (t), mean burst duration (tn), and Popen at different pHi in the absence of ATP pHi 80 pHi 7-2 pHi 6-3 pHi 5-7 tO 2-26+006 265+0-21 383+0-12 3-63+011 1-29+003 230+003 162+0-01 1-59+0-01 flC 45-7 + 1-9 61-5 + 9-2 134-3 + 157 299-6+ 89-2 tb 064 054 070 070 Popen

of ATP. Figure 6A shows recordings from a patch exposed to ATP at 1 and 3 mm at pHi 7 2 and from a patch exposed to the same concentrations of ATP at pHi 6 3. At pHi 7-2, 1 mM-ATP reduced activity to very occasional openings, while even fewer openings were seen in 3 mM-ATP. When pHi was lowered to 6 3 substantial channel activity remained at both ATP concentrations. We measured the dependence of channel inhibition on intracellular [ATP] at different values of pHi either by measuring the ratio Popen, ATP/Popen control or, in patches with more than eight channels, the ratio of the mean currents, IATP/Icontrol. In either case the control value is that in the absence of ATP. Patches were exposed to a maximum of four different ATP concentrations, and during the course of the experiment the control solution was frequently applied to correct for 'run-down' which is, however, much less marked in patches from skeletal muscle than has been reported in other tissues. The control F or Popen was taken as the mean of those bracketing a set of ATP applications. The dependence of channel activity on [ATP]i at different pHi values is shown in Fig. 6B. The concentration dependence of the action of ATP at each pHi could be fitted by the expression IATP = 1-

[ATP]n

(8) 8

'control [AP tK~ with a Hill coefficient, n, of 1. At pH 7 2, application of ATP to the cytoplasmic side of inside-out patches inhibited the activity of KATP channels with a Ki of 17 JCM, a value that is little affected by membrane potential (N. W. Davies, unpublished observations). Decreasing pHi shifted the curve to higher ATP concentrations, an effect that also occurs at other voltages (Davies, 1990). The blocking stoichiometry remained 1:1 at all pHi values tested (in each case n = 1 gave the best fit), while Ki increased as pHi was reduced, being 260 ,aM at pHi 6-3 (see Fig. 8). We have considered whether these effects of pH could be explained by the alteration in the relative concentrations of ATP complexes which are expected to occur as pH is changed. This might be so, for example, if the most important complex

560

N. W. DAVIES, N. B. STANDEN ANVD P. R. STANFIELD

for regulation of channel activity were ATP4-, as has been suggested in insulinsecreting cells (Dunne, West-Jordan, Abraham, Edwards & Petersen, 1988). In the bathing solutions we used, the main complexes of ATP present are: ATP4-, HATP3-, H2ATP2-, and KATP3-. The concentrations of these complexes were calculated using pHi 6.3

pHi 7.2

A

1 mM-ATP

1 mM-ATP

]10 pA

_

3i,X mM

T

3 mM-ATP

0 3 mM-ATP

I0lO pA

5s

B 1 C

0

or

0.8[

._

._

n 0.6p C

0 C.)

r-

0.4 [ 0.2p

o.0 L

0.1 10 1 [ATP] (mM) Fig. 6. Effect of pH1 on channel inhibition by ATP. A, left-hand panel: records from a multichannel patch at pHi 7 2 throughout, exposed first to control solution and then to 1 and 3 mM-ATP respectively. Right-hand panel: records from a different patch exposed to the same [ATP]s, but maintained at pHi 6 3 throughout. B, concentration dependence of the block of KATP channel activity by ATP at pHi 7 2 (i), 6 8 (0) and 6 3 (V). The data were obtained from a total of twenty-five patches and the curves are least-squares fits to eqn (8) giving K1 values of 17, 42 and 260 /tM for pHi 7 2, 6 8 and 6 3 respectively. A Hill coefficient of 1 was used in each case. the iterative procedure described by Storer & Cornish-Bowden (1976) using the stability constants given by Martell & Smith (1974). A plot of the concentration of these various complexes as a function of pH is shown in Fig. 7. None of the individual

EFFECTS OF pH ON MUSCLE KATP CHANNELS

561

complexes change as much as the measured Ki values for ATP; for example, a change in pHi from 7-2 to 6-3 would reduce [ATP4-] by less than half, while we found that Ki for ATP changed 15-fold. This is consistent with the previous observation in skeletal muscle that comparable effects of pHi were seen even in the presence of

1 0

0.8-

E

0.6

-

KATPS

E0 0

~~~~~~~~~ATp4-

0

-

0.4-

0.2-

/H2ATp2 °L

HTP

HATp35.6

6.4

7.2

8.0

pHi Fig. 7. The pH dependence of the concentrations of the various complexes of ATP calculated for a total ATP concentration of 1 mm using the methods of Storer & CornishBowden (1976).

excess Mg2+, when the change in the relative concentrations of different forms of ATP will be much smaller (Davies, 1990). Similarly, Spruce et al. (1987) showed that relatively large changes in the nucleotide structure were needed to diminish significantly the inhibitory effect on these channels.

A model for the effect of pHi on inhibition by ATP Changes in pH shift the concentration dependence of channel inhibition by ATP without changing the steepness of the relation. Thus the most economical model for the effect of pHi is one in which protons compete with ATP for a site at which it binds to close the channel. We found that a competition between one H+ and one molecule of ATP did not give a sufficiently steep dependence of Ki, the inhibition constant for ATP, on pHi. We therefore propose a model in which two protons compete with one ATP molecule for the site. This model may be represented by the scheme: 2h-1 h.1 a[ATP]

h[H+]

2h[H+]

a-1

N. W. DAVIES, N B. STANDEN AND P. R. STANFIELD where S is the site at which ATP binds, SATP, S1H, and S2H represent the states in which it has ATP or one or two protons bound respectively, and a, a-,, and h, h-1 are the binding and unbinding rate constants for ATP and H+ respectively. We assume that the fractional inhibition by ATP depends on the occupancy of state SATP. From 562

1000

100

10

1

,

.

.

5.6

6*0

6.4

6.8

7-2 7.6 8.0 pH; Fig. 8. Dependence of the apparent inhibition constant for ATP, K,, on pH,. The symbols show values at pH, 7-2, 6-8 and 6-3 obtained as described in the legend to Fig. 6, plus a value at pH, 8-0 obtained from the fit to results from four patches. The curve is drawn to eqn (10) with Kd = 5-4 #M and KH = 0 1 #M, and was fitted by eye.

this scheme Ki, the measured inhibition constant for ATP (the [ATP] for halfinhibition), will be given by K1

=

Kd( ± [H]±2Hf K2 KH)

(10)

where Kd = a-,/a and KH = h-1/h. Figure 8 shows that our experimental values for Ki are well fitted by eqn (10) with Kd = 5X4 /tM and KH = 0-11 /M, assuming an activity coefficient for H+ of 0-78 as defined by the US National Bureau of Standards at 04 M ionic strength. The rate of ATP binding and dissociation For a scheme like that of eqn (9) in which ATP binds to cause channel closure, the channel mean open time, i0, will fall as [ATP] is increased and the apparent rate constant for ATP binding can be obtained from the relation between to and [ATP]. In the presence of ATP the reciprocal of to is given by: + hto = { #xi} Po ([ATP] -0) {Y PO at '} [ATP],

(11)

where Pi is the equilibrium occupancy of the ith open state, PO is the sum of the equilibrium occupancies of all open states, xi is the sum of the rate constants by

EFFECTS OF pH ON MUSCLE KATP CHANNELS

563

which the ith state can shut and a'? is the apparent rate constant for ATP binding to the ith open state. This expression, which predicts a linear relation between 1/Io and [ATP], is general provided that ATP does not affect the relative occupancy within the set of open states (Colquhoun & Hawkes, 1977; Davies, Spruce, Standen 3

2-

E

0 0

2

6

4

8

10

[ATP] (mM) Fig. 9. Plot of I/tI, the reciprocal of the corrected mean open time, against ATP concentration at pHi 7 2 (i) and 6-3 (0). Values in the presence of ATP are weighted results from nine patches at pH, 7-2 and seven patches at pH, 6-3, with a further six and seven patches respectively in the absence of ATP. The lines are drawn to eqn (12) taking a rate constant for unbinding of 0 01 ms-' and apparent rate constants for binding of 0-6 and 0-04 mM-' ms-' respectively to give K, values of 17 and 260 ,UM.

& Stanfield, 1989 a). Assuming that ATP binding rates are independent of the kinetic state of the channel, eqn (11) gives t0 to

itoI([ATP]=O + a'[ATP], °

(12)

([ATP]=o

so that the slope of the relation between 1/to and [ATP] will be a', the apparent rate constant for ATP binding. For the scheme of eqn (9) this will be the product of a, the true rate constant for ATP binding, and the probability that the channel is in state S rather than states 1H or S2H, so that

{P[S]+P[SH]+P[S2H

(13)

This leads to an expression relating a and a' exactly equivalent to that relating Ki to Kd given in eqn (10). Figure 9 shows plots of 1/of as a function of [ATP] at pHi 7 2 and 6-3. As expected, the relation has a steeper slope at pHi 7-2, corresponding to a higher apparent rate constant for ATP binding at this pH. The lines in Fig. 9 are drawn as calculated from eqn (12). Our model for the effect of pHi predicts that the unbinding rate for ATP should be unaffected by pH, and we have taken the value

N. W. DAVIES, N. B. STANDEN AND P. R. STANFIELD

564

of the rate constant a-1 as 0 01 ms-1 to give a reasonable fit to the experimental values in Fig. 9 with our experimental Kis of 17 and 260,UM at pHi 7-2 and 6-3 respectively. The apparent rate constant for ATP binding, a', corresponding to the slope of the line at each pH is then 0-6 mm-'ms-1 at pHi 7-2 and 0 04 mm-1 ms-1 at 160

120

E 80

E

40

-0

0 0

2

10 6 8 [ATP] (mM) Fig. 10. Effect of ATP on mean burst length (tb) at pHi 7-2 (-) and 6-3 (0). Points show weighted means for results pooled from ten patches exposed to ATP at pHi 7-2 plus a further six patches in the absence of ATP, while those at pHi 6-3 were obtained from five patches in ATP and seven in its absence.

4

pHi 6 3. Assuming the model of eqn (9), the estimate for the true rate constant for ATP binding a of eqn (9), which is independent of pHi, is 185 mm-' ms-'. The effect of A TP on burst length Figure 10 shows that burst durations were markedly reduced by the application of ATP (see also Spruce et al. 1987). As we would expect, the reduction was more pronounced at pHi 7-2, where 1 mM-ATP decreased mean burst duration, ib, to 41 % of control, compared to a reduction to 29-5 % with the same [ATP] at pHi 6-3. Low [ATP]s cause a greater reduction in tb than they do in mean open time to. For example, fo in 1 mm-ATP at pHi 7 2 was 35-5 % of its value in the absence of ATP. This may be explained in terms of four states contributing to the bursts we measure, two open and the briefer two closed states. If ATP can bind to any of these states (subject to protons not being bound to the channel) then ATP binding provides four additional routes for termination of a burst. DISCUSSION

In this paper we have examined the effect of pHi on both the kinetics of KATP channels and on their inhibition by ATP. While changing pHi causes many changes in kinetic behaviour in the absence of ATP, our results show that its major effect on Popen occurs by changing the degree of channel inhibition by ATP.

EFFECTS OF pH ON MUSCLE KATP CHANNELS

565

The effect of pHi on KATP channels in the absence of ATP In the absence of ATP the changes in kinetics produced by decreasing pHi are subtle and result in relatively little change in Popen- Measured changes in mean open time were significant, but the true mean open time, given by the product of the proportion of closed times detected and the measured mean open time (eqn (4)), was only slightly affected. This was also manifest as an increase in short (and undetected) closings observed at low pHi values and illustrates the substantial effect that missed events can have on measured distributions. At all pHi values tested two components were detected in the open time distribution and four in the closed time distribution. Two of the closed time components were fast (see Table 1) and we considered these as originating from gaps within bursts of openings. The predicted distribution of burst length in this case should consist of four exponential components (Colquhoun & Hawkes, 1982). We could easily observe three components but the fourth component was possibly too small for detection. Mean burst length increased with decreasing pHi.

Effects of pH in the presence of ATP Intracellular pH has a much greater effect on Popen in the presence of ATP. Lowered pHi causes a reduction in channel inhibition by ATP, seen as a shift of the ATP inhibition curve to higher [ATP]s. Although it is likely that protons could affect protein structure in many ways, we find that a quite simple model, where H' binding prevents ATP binding from closing the channel, predicts the observed change in Ki well if we assume that two protons are able to bind. Of course, H+ would not necessarily have to bind to the same site as ATP to produce such an effect. It is interesting that the dependence of Ki on pH is steep, and that the calculated KH (the dissociation constant for H+) of 011 /tM is close to the normal [H+]i of about 0O085 ftM (based on a pHi of 7418: Abercrombie, Putnam & Roos, 1983), giving maximal sensitivity to changes in pHi in the physiological range.

Comparison with effects of pH on KATP in other tissues The effect of intracellular pH on KATP channels of skeletal muscle differs either in degree or in nature from findings on this type of channel in cardiac and pancreatic B-cells. In ventricular myocytes Lederer & Nicholls (1989) found that lowered pH1 increased channel activity by shifting its ATP dependence essentially as we report here. Their effect was very much weaker, however, with a decrease in pH, from 7-25 to 6-25 doubling the Ki for ATP compared to the 15-fold increase that we measured in skeletal muscle for about the same change in pHi. In addition, the effect of low pHi in cardiac muscle appeared to be accompanied by a change in the Hill coefficient for inhibition by ATP (Lederer & Nicolls, 1989). In B-cells, Misler, Gillis & Tabcharani (1989) found little effect of changing pH in the range above pH 6-5 on channel activity in the absence of ATP. In the presence of ATP, however, lowering pH1 decreased channel activity, apparently by increasing ATP inhibition.

566

N. W. DAVIES, N. B. STANDEN AND P. R. STANFIELD

Kinetics of ATP binding We estimate an apparent rate constant for ATP binding at pH 7-2 of 0-6 mM-1 msfrom the change in mean open time with [ATP]. Qin, Takano & Noma (1989) estimated this rate constant in cardiac muscle using an oil-gate method to produce a jump in [ATP]. They found that the decrease in Popen followed one of two rates, corresponding to rate constants of 0-052 and 0-006 mM-1 ms-'. Thus our rate constant is about 11*5-fold higher than their faster constant. The discrepancy could in part reflect the rate at which the concentration can be changed using the oil gate. Diffusion delay at the pipette tip leads to a half-time for a change in concentration of about 6-5 ms (Qin et al. 1989). An alternative approach is to use flash photolysis of caged ATP. Using 2 mM-caged ATP, Nicholls, Niggli & Lederer (1990) measured a time constant of 300 ms in ventricular cells, corresponding to a considerably slower rate than either our value or that of Qin et al. (1989). The results from flash photolysis are somewhat complicated by the inhibitory effect on the channel of the caged ATP itself, by uncertainty about the free [ATP] produced by photolysis of 2 mM-caged ATP, and by simultaneous release of protons. In view of other differences in the properties of KATP channels of cardiac and skeletal muscle, for example in the effects of pH and stoichiometry of ATP binding, it is also quite possible that rate constants for ATP binding do differ between the tissues.

Possible physiological significance The notion that KATP channels of skeletal muscle are regulated mainly by changes in intracellular [ATP] has always suffered from the difficulty that [ATP]i is well buffered, as we described in the Introduction, so that bulk [ATP]i, at least, changes significantly only in extreme exercise. Intracellular pH is a more attractive candidate for a regulator of KATP activity under physiological conditions as it changes progressively and quite rapidly during exercise. As we have pointed out above, KATP channels are maximally pH sensitive at physiological pHi. The actions of pH occur by way of changing the sensitivity of the channel to inhibition by ATP. Thus we can think of ATP as setting a background level of channel inhibition, corresponding to a very low Popen in muscle, against which pH, and possibly other intracellular factors, acts to regulate channel activity. We show in this paper that pH opens channels by lowering their affinity for ATP, and we consider it possible that other channel openers act in the same way. Indeed the channels may be thought of as ATP-binding proteins, regulated by changing their affinity for the nucleotide. In support of this idea, pharmacological openers such as RP 49356 are also known to open the channels by lowering their affinity for ATP (Thuringer & Escande, 1989). In fatigued muscle, channel opening in response to a fall in pHi could lead to electrical inexcitability in some fibres, causing them to rest. Other possible functions for KATP channels in exercise are related to the K+ efflux that may occur through them. The rise in muscle [K+]O that occurs in exercise is thought to act both locally to contribute to vasodilatation in muscle, and remotely to increase respiratory effort (for further discussion see Davies, Standen & Stanfield, 1991 b). It is not yet clear how much KATP channels contribute to this K+ efflux. We have recently found, however, that lowering pHi in intact frog muscle fibres gives rise to a component of whole cell K+

EFFECTS OF pH ON MUSCLE KATP CHANVNVELS

567

current that can be blocked by the sulphonylurea glibenclamide, and which therefore is likely to flow through KATP channels (N. B. Standen, A. Pettit and P. R. Stanfield, unpublished observations). We thank Mr WV. King for expert technical assistance, and Professor A. G. Hawkes for advice on weighted variance. We also thank the Medical Research Council and the Wellcome Trust for their support. REFERENCES

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