Changes in densities and kinetics of delayed rectifier potassium channels during neuronal differentiation

Neuron, Vol. 1, 739-750,

October, 1988, Copyright 0 1988 by Cell Press

Changes in Densities and Kinetics of Delayed Rectifier Potassium Channels during Neuronal Differentiation Greg 1. Harris, Leslie l? Henderson: and Nicholas C. Spitzer

al., 1988). Of the three, IKv changes the most, increasing

Department

the first day in culture-the

time during which the major

change in impulseduration

occurs. Thesechanges

and the Center for Molecular University

3-fold in density

of Biology of California,

La Jolla, California

Genetics

and 2-fold in rate of activation during

reflect modifications

San Diego

92093

of the underlying

likely

population

of KC

channels. Patch clamp analysis of single channels from a variety of cell types has revealed that multiple channels

Summary

can underlie

dent currents (Dubois,

Single-channel K+ currents were recorded from young and mature spinal neurons cultured from Xenopus embryos to examine the bases of the developmental increases in density and in rate of activation of the macroscopic voltage-dependent delayed rectifier K+ current (I,& K+ channels of three conductance classes (40, 30, and 15 pS) are present at both ages, but only the intermediate and small conductance classes are voltagedependent and thus underlie Ix”. The increase in the density of Ix” is due to increases in the numbers of intermediate and small channels per cell, but not to changes in their open probabilities. The increase in rate of activation of Ix, results from a change in the activation kinetics of the intermediate channel class alone.

Gardner,

1986;

1988).

Thus,

at early stages of development frequently

pos-

and Aldrich,

1988a).

of a given channel the differentiation

Moreover,

class may change 1985; Yool et al.,

of K+ currents

at the

whole cell level may involve changes in the properties and/or the numbers of one or more channel types. In the present study, we report changes in both the properties and the numbers

of voltage-dependent,

Ca*+-insensi-

tive K+ channels in developing Xenopus spinal neurons.

Results Three Classes of K+-Selective Channels in Young and Mature Neurons conductance classes of K+ channels

at depolarized Neurons

Hoshi

1983; Marty, 1983b

1984; Marty and Neher, 1985;

during development (Blair and Dionne,

Three

Introduction

1981; Hamill,

Peterson and Maruyama, the properties

species of K+

both voltage- and Ca*+-depen-

potentials

in excised,

from young or mature neurons

were active

inside out patches

(6-9

hr or 1 day in cul-

sess long duration action potentials for which the inward

ture; Figures 1A and 1B). The same three channel classes

current

were observed in cell-attached patches of mature neu-

is carried

primarily

by Ca*+ ions

(see Spitzer,

1985, for review). K+ currents can play a role in terminat-

rons (data not shown).

Slope conductances

ing these impulses

stant when determined

over a range of membrane po-

(Baccaglini and Spitzer,

1977) and,

were con-

indeed, have been shown to appear before inward cur-

tentials from -10 to 40 mV. Mean conductances for the

rents in neurons

three channel classes were 77 (large), 31 (intermediate),

excitability

in which the developmental

has been examined

man and Spitzer,

(Warner,

1973;

Good-

1979, 1981; Bader et al., 1983; Ahmed

et al., 1986). During

development,

the action potential

in cultured amphibian spinal neurons duration

and largely Na+-dependent

borghini,

1976; Blair, 1983; O’Dowd,

Na+ current

onset of

density

doubles

inward Ca*+ current density

1983; Barish,

1986).

during this time, and the

et al., 1988) or shows

a small however,

crease in both density

and Lam-

remains constant (O’Dowd

Outward

K+ currents,

becomes brief in (Spitzer

increase (Barish,

1986).

undergo a marked in-

and rate of activation

(Barish,

and 16 pS (small) in young cells and 84, 29, and 14 pS in mature cells (Table 1). Small amplituide channels had conductances as low as 6 pS; 20 pS was taken as the upper limit of this class, based on the frequency distribution of all conductances in the range of 6-50 ences in other channel characteristics further

justify

this division.

The large channel class ex-

hibited conductances greater than 50 pS and displayed kinetic properties

markedly

different from those of the

two smaller channel classes. Any combination

1986; O’Dowd et al., 1988). Much of the decrease in the

taneous openings of small and intermediate nels were frequently

dependence is consequent

depolarized

to changes in K+ currents.

spinal

neurons

from Xenopus

are prominent embryos

day in vitro: a voltage-dependent

in cultured

during the first

delayed current

(I,),

of chan-

nel types could be found in a patch, and multiple simul-

duration of the action potential and the change in its ionic Three outward K+ currents

pS. Differ-

described below

potentials.

Multiple

simultaneous

of large channels were seen less frequently. both large and intermediate

in young

and mature

a rapidly inactivating Ca*+-dependent current (IKcr), and

neurons.

a sustained

apparent substate of 13 pS; the large channel appeared

Ca*+-dependent

current

(I&

(O’Dowd

et

intermediate

openings

Openings of

class channels to subcon-

ductance states were observed The

class chan-

observed at the onset of steps to

to have several substates.

channel class exhibited However,

mediate and small channels * Present address: Department of Physiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111.

openings

of inter-

to their principal

conduc-

tance state were seen in isolation quencies

than the substates

an

at much higher fre-

of large and intermediate

Neuron 740

A YOUNG

Figure 1. Single-Channel K+ Currents and Conductances from Excised, Inside Out Patches from Young and Mature Neurons

’ LARGE

Dashes at left margin of records in this and subsequent figures indicate unitary current levels. (A) Records from three young neurons. Currents of three amplitudes are apparent. Step potential, 30 mV. Open durations of the large class are briefer than those ofthe intermediate and small classes. I-V relations and slope conductances are illustrated for the channels giving rise to the three currents. The extrapolated reversal potentials W,,) for currents of small (m) and intermediate (A) channels are close to the K+ equilibrium potential (EK = -47 mV); V,,, for the current of the large channel (o} extrapolates to a more positive voltage. (6) Single-channel currents and conductances in three mature neurons are similar to those seen in young neurons. Step potential, 30 mV. I-V relations yield slope conductance of the three classes of channel and the V,,, values of their currents; large CO), intermediate (A), and small (0). (C and D) K+ selectivity and TEA sensitivity of large, intermediate, and small conductance channels in mature neurons. Each panel illustrates data from a single inside out patch. (C)The conductance of a large (61 pS) channel is reduced (to 41 pS), and the V,,, value of the current is shifted to the right by a 25% reduction in [K], (replaced with Na+). The V, values of the currents of intermediate and small channels (25 and 17 pS) are shifted to the right by 50% reductions in [K],. (D) Large and intermediate conductance (75 and 32 pS) channels are reversibly reduced in apparent amplitude (to 55 and 17 pS) by bath application of 10 mM TEA. A small (11 pS) channel is completely but reversibly blocked by 30 mM TEA. Step potential, 50 mV.

L 5pA

lOms

B MATURE

C

LARGE

INTERMEDIATE

IOmM TEA

Table 1. Conductances

SMALL

IOmM TEA

and Reversal Potentialsa

30mM

of Potassium

TEA

Channels

Small Conductance

(pS)

Vre, (mWb

a Mean b Mean

* SEM (number * SEM (number

Young Mature Young Mature of observations) of observations)

16 f 14 f

Intermediate 1 (20) 1 (18)

31 f 29 f

- 55 f 2 (19) - 52 f 2 (14)

- 34 * 2 (22) - 39 f 2 (26)

from inside out patches. determined by linear extrapolation;

channels, suggesting that me pnnclpal conductance states are not simply subconductance states of a single channel class. A fourth conductance class was also evident at depolarized potentials, but the amplitude of the outward current was too small to measure accurately except at strongly depolarized voltages, and these channels were not studied further. Reversal potentials were extrapolated for the three channel classes (Table 1). Based on these values, it appeared that the small and intermediate classes were primarily selective for K+, while the large class exhibited a lower K+ selectivity (EK = -47 mV). This proposition

2 (23) 1 (31)

EK = -47

Large 77 f 8 (3) 84 f 6 (18) -29 -23

f 11 (3) + 2 (18)

mV.

was tested more rigorously by performing ion substitution experiments on excised patches from mature cells in which [K]i (bath solution) was reduced by one-half through equimolar substitution of Na+ (Figure 1C). For this substitution, the predicted shift in the reversal potential from the Nernst relation for a purely K+-selective channel is 17.5 mV. Reducing [K]i resulted in a shift of 16 f 2 mV for the intermediate conductance channel (Mean f SEM; n - 4) and 18 f 3 mV for the small conductance channel (n = 3); slope conductances were not markedly reduced. Substitution with Na+ or Tris for 75% of [K]i caused a reversible block of these channels. The

Developmental 741

Changes in Neuronal

A

K+ Channels

Table 2. Closed Times of Potassium

LARGE 300

Channel Smallb Young Mature lntermediateb Young Mature LargeC Mature

0 0 IO 20 OPEN DURATION lms)

0

0-

CLOSED DURATION Figure 2. Kinetics Neurons

~2(ms)

~3(ms)

0.2 * 0.01 (3) 0.2 f 0.01 (3)

1.3 f 0.04 (3) 1.3 f 0.2 (3)

9.0 (1) 10 f 0.3 (2)

0.2 f 0.03 (6) 0.2 f 0.01 (3)

1.1 it 0.3 (6) 0.9 * 0.1 13)

19 f 6.7 (3) 8.1 (1)

0.2 * 0.01 (7)

0.9 f 0.1 (7)

29 f

71

(ms)

a Mean f SEM (number b At 40 mV. c At O-70 mV.

tms:

0

of Large Conductance

0

I

2

3

TIME IICC I

Channels

from

4

Mature

(A) Histogram of open times for a 67 pS channel from an excised patch at 30 mV; 1291 events were analyzed. The distribution was fit by a single exponential with a time constant of 2.4 ms. (B) Mean open time as a function of step potential is independent of voltage over the range examined. Data from 6 cells; values are means f SEM in most cases for n > 3 patches. (C) Histogram of closed times for a 70 pS channel from an excised patch at 20 mV; 1296 events were analyzed. The distribution was fit by two exponentials with time constants of 0.2 and 1.1 ms and relative amplitudes of 0.65 and 0.35. The minimum duration for computation of the second exponential was 1000 us; durations >5 ms were not considered. ( D) Time dependence of relative open probabilities of large channels; records from excised patches from two mature neurons. Top, unitary activity of a large (82 pS) channel continues throughout the duration of a 3s sample at 40 mV. Bottom, NP,,, of large channels from mature neurons appears to be independent of voltage and time over the ranges examined. Current from two patches was averaged over 150 ms time periods during several depolarizations of 3-4 s, and NPopenwas normalized to the greatest values for each patch. Interruptions in plots were time required to store the data.

large conductance class was more susceptible to block induced by lowering [K],; reduction by one-half usually abolished all activity (n = 4 of 5 cases). Reducing [K]i to 75% of normal led to a 5.0 f 1 mV (n = 2) shift in the reversal potential (versus prediction of 7.2 mV) and a 30% reduction in slope conductance, which may reflect channel blockade by Na+ (Marty, 1983a; Yellen, 1984). The currents through the three channel classes were reduced to different extents by lo-30 mM TEA applied to the internal face of the membrane of patches excised from mature neurons (n > 3 for each class; Figure 1D). The small channels were the most sensitive since they were fully blocked in 2 of 3 cases. The intermediate and large channels were less sensitive (apparent conductances were reduced by 50% and 35%, respectively).

Channel@

3.3 (3)

of observations).

Open and Closed Durations of large Channels t_arge conductance channels in mature neurons had a mean channel open time of 2.3 f 0.3 ms (n = 6) that was independent of voltage between -10 and 40 mV (Figures 2A and 28). Analysis of closed times within clustered periods of activity yielded histograms that were fit by the sum of two exponentials (Figure 2C) corresponding to brief closures or flickers and longer closed times; neither component changed as a function of voltage. In patches in which clustered activity was interrupted by long closures, a third, slow voltage-independent time constant was evaluated (Table 2). Further evidence for the absence of voltage dependence of this channel is provided by the observation that it was active at potentials as negative as -80 mV. Histograms ofi normalized, averaged activity were complied during 3-4 s depolarizations (as described in Experimental Procedures) in order to evaluate the relative open probability (NP,,,) as a function of time for this channel class in mature cells. NP,,, was constant over the voltage range -10 to 40 mV and showed no decline over a period of several seconds (Figure 2D; n = 2), indicating the absence of a prolonged inactive state. A similar analysis of this channel class in young cells was not performed due to the low level of channel activity (see below). Open and Closed Durations of Intevmediate and Small Channels For both the intermediate and the sirnaIl conductance channels, the mean channel open time was dependent on voltage in both young and mature neurons (Figure 3), being 2-3 times longer at 40 mV than at 0 mV. The mean channel open time of the intermediate class channel in young cells was significantly longer than that for the same channel class in mature cells, when measured at 10 and 30 mV (Figure 3C; p < 0.001). At 30 mV, ‘5 was 12.4 f 0.6 ms in young cells (n - 4)mand 7.3 f 0.5 ms in mature cells (n - 6). There were no significant differences in values of r between young and mature neurons for the small conductance class (Figures 3D-3F); at 30 mV, these values were 8.9 f 2.1 ms and 6.2 f 1.1 ms for young (n = 3) and mature (n- 4) cells, respectively. Intermediate and small channels both appeared to exhibit at least three types of closed durations, correspond-

Neuron 742

A

the course of several seconds (Figures 4C and 4F). How-

INTERMEDIATE

ever, the extent and time course of inactivation of intermediate and small channels were similar in patches from young and mature neurons Long sets of stimuli

(n = 2 for each case).

elicited records in which channel

activity appeared to be clustered.

Presentations

of 20 ms

voltage steps at 1 Hz yielded sequences of traces without any channel openings OPEN DURATION (msl

D

W&L

n

. Ywng 0 Mature

E

1984; Standen et al., 1985; Hoshi

T

Three

12

and mature neurons

Tou B Ims) P

f

y

20

40

0

IO

20

-IO

IO

30

occurrence

ImVl

did not vary with

class. These

but that its

development

for either

data indicate the existence

For both channel classes, data are derived from 7 young and 6 mature neurons; in most cases each point in (C) and (F) is the mean f SEM of values from 3 or more patches. (A and B) Histograms of open times for intermediate channels in excised patches from a young ([A];21 pS) cell and a mature ([B];28 pS) cell. Step potential, 10 mV for both; 412 and 1283 events were analyzed. Distributions were fit by single exponentials; values for T are 7.9 and 4.2 ms, respectively. (0 Mean open durations over a range of voltages for intermediate conductance channels; open times are voltage-dependent for both young (A) and mature (A) cells. (0 and E) Histograms of open times for small channels in patches excised from a young ([D];17 pS) and a mature ([El;18 pS) neuron. Step potential, 20 mV for both; 513 and 340 events were analyzed. Single exponential fits yield values for T of 5.1 and 4.1 ms, respectively. (F) Mean open duration of small conductance channels increases with depolarization for both young (W and mature (El) cells.

activation may underlie the time-dependent NPopen observed tion (Figure 4).

during

depolarizations

decrease in

of long dura-

Developmental Changes in the Densities of K+-Selective Channels The developmental

increases in current density of inter-

mediate and small

channels

were estimated from en-

semble-averaged records of currents

of a single channel

class. The increase in current density of the large channels was not evaluated by this method since they often opened too infrequently The

during the sampling procedure.

ensemble-averaged

currents

small channels were multiplied exhibiting

of intermediate

channel activity (62% for young and 89% for

mature cell patches) to correct for the numbers of patches lacking activity. Pipettes used in these experiments tances of 19.5

and closures occurring within bursts of acbetween bursts

(>5

ms). The

cessation of channel activity during depolarizations

last-

and 17.7 MQ

averaged currents

respectively.

patch membrane areas determined sistances (Sakmann

and Neher,

tude of the ensemble-averaged

the existence of a fourth class of closures,

intermediate

an

to ex-

had average resis-

were accordingly

ing longer than a few seconds (see below) may indicate comprising

and

by the fraction of patches

amine young and mature neurons

tivity and longer closures

of a

slow inactivation process present for each channel class in both young and mature cells. This slow process of in-

Figure 3. Open Durations of Intermediate and Small Conductance Channels from Young and Mature Neurons

ing to flickers

of

degree of clustered

,”

. Ybvnq 0 MotYre

OPEN DURATION Cmsl

Calculations

activity at 20 or 40 mV (Z 2 2.3; a = 0.01) channel

0

were considered.

p

I

1988b).

of the number of runs, Z, indicated that

most patches showed a significant 1

1982; Horn et al.,

and Aldrich,

to 5 patches for each channel class from young

the distribution

,‘y

that the clustering

occurred by chance (Patlak and Horn,

F n

(Figure 5). Runs analysis was

performed to evaluate the probability

Ensemble-

normalized

to the

from the pipette re-

1983). The peak amplicurrents

in patches with

channels from young cells at 40 mV is 1.0

pA (n = 7), compared with 2.5 f 0.7 pA (n = 8)

inactivated state. For both the intermediate and the small

f

channels,

in patches from mature cells. The peak amplitude of the

a satisfactory

double

exponential

fit for the

0.1

two shortest closed time distributions

was obtained from

ensemble-averaged

currents

at the same potential

in

durations

of the two shorter

patches with small channels

from young cells is 0.4

+

of less than 5 ms. Neither

closed times differed mature

cells

significantly

for either

between young and

the intermediate

or the small

0.1

pA (n = lo), compared with 0.8

+ 0.1 pA (n = 9)

in patches from mature cells. Thus there is a 2- to 3-fold increase in peak current

magnitudes for both classes of

4A, 46, 4D, and 4E; Table 2). The third class of closures,

channel (Figure 6B). This

change could be attributable

comprising

to increases in the numbers

channel class when examined at 40 mV (n = 3-6; the interburst

closed durations

curred with such low relative frequency titative developmental

comparison

NPopen

relative

open

>5 ms, oc-

that no quan-

could be made for

either channel class (Table 2, mean values). dependent

Figures

probabilities

The time-

as assessed

by

of the intermediate and small conductance chan-

of channels

changes in their open probabilities Calculations of channels ways.

The

of P require determination

per patch (n), which maximum

number

per cell (N), to

(P), or to both. of the number

was assessed

of channels

in two

simultane-

ously open was noted from inspection of currents elicited

nels at both ages were quite different from those of the

by each of 50-200

large channel class in that they decreased markedly over

gently, binomial

depolarizing

analysis

commands.

More co-

of the same records was used

Developmental 743

Changes in Neuronal

K+ Channels

INTERMEDIATE

6

INTERMEDIATE YOUNG lr, o.zm* To+ O.Tmr

100

6 CLOSED D”RATION(m5: D SMALL

150

200 MATURE Tm, 0.2ms

nut

I.lrn,

loo

b CLOSED WRATIONkn:) Figure 4. Closed Durations

of Intermediate

and Small Conductance

Channels

from Young and Mature

Neurons

(A, 6, D, and E) Data from four patches, each of which appeared to contain activity of only one channel. Step potential, 40 mV. In each case, distributions of the two shortest closed durations were fit by the sum of twu exponentials, durations >5 ms were omitted, and the minimum duration for computation of the second exponential was 800 ps. (A) Histogramof closed times for a 21 pS channel from a young neuron; 279 events were analyzed. T, = 0.2; r2 - 0.7 ms. Relative amplitudes were 0.89 and 0.11. (B) Histogram of closed times for a 28 pS channel from a mature neuron; 1058 events were analyzed. 5, - 0.2; r2 - 1.0 ms. Relative amplitudes were 0.87 and 0.13. (D) Histogram of closed times for a 12 pS channel from a young neuron; 402 events were analzyed. rl - 0.2; f2 = 1.2 ms. Relative amplitudes were 0~67 and 0.33. (E) Histogram of closed times for a 12 pS channel from a mature neuron; 375 events were analyzed. T, - 0.2; 72 = 1.1 ms. Relative amplitudes were 0.94 and 0.06. (C and F) Time dependence of relative open probabilities of intermediate and small channels; records from excised patches from 10 mature neurons. Multiple levels of activity of intermediate and small (31 and 18 pS) channels are seen at the onset of voltage steps to 40 mV; activity declines during the sample interval. NPop,, at 30 mV for intermediate and small channels in two patches each from young (filled symbols) and from mature (open symbols) neurons declines toward zero in a time-dependent manner that is similar at both stages of development.

to derive the most frequent mate of the number ensemble-averaged

current.

timates

number

of channel

cases examined

maximum

of channels

likelihood

contributing

These two independent agreed

in the majority

channels;

6C). P was then evaluated number

by binomial

open probability

Figure

from these instances in which

of channels analysis.

The

per patch was conmaximum

channel

was found to increase slightly with volt-

age but did not change during deveilopment channel

of

n = 3 of 3 and 4 of

6 patches for young and mature small channels; the observed

es-

(n = 2 of 3 and 2 of 5 patches for young

and mature intermediate

firmed

estito the

for either

class (Figure 6D).

These results indicate that an increase in the number of channels

rather than a change in their open probabil-

ity accounts for the increase in ensemble-averaged rents. The open probability L

I

I

I

0

I

I loo

SWEEP NUMBER Figure 5. Clustering

of Channel

Activity

Records of intermediate (27 pS) channels in an excised patch from a mature neuron. (A) Twenty-four consecutive records elicited by 20 ms voltage steps to 20 mV presented at 1 Hz reveal a group of traces lacking channel activity. (8) Distribution of responses to the full sequence of 100 voltage pulses. Bracket refers to interval illus-

uate the absolute number

cur-

estimates were used to evalof channels

per neuron from

the relationship N = /l(i.P) where N equals the number of channlels per cell,

(1)

I is the

trated in (A). Z = 4.3; values ofZ B 2.3 were obtained for 50% and 75% of young and mature intermediate channels and for 66% and 60% of young and mature small channels.

NeUMXl

744

0

2

0.5

INTERMEDIATE

I& s 0”

INTERMEDIATE

I

OL

05r

@ f ::

E c

x 0

0

Figure 6. Ensemble-Averaged and Mature Neurons

Currents

and Open Channel

Probabilities

of Intermediate

SMALL

40 (mV)

_

b? 0

and Small Channels

v

1

20

0

20

40 imV)

in Excised Patches from Young

(A) Upper traces are currents from a single class of channels in a patch from a mature neuron. Two channels (~27 pS) are active in the patch following steps to 20 mV. Bottom trace is the ensemble average of currents recorded from the same patch following 89 steps. (B) Ensembleaveraged currents of intermediate (A and A) and small (Hand 0) channels increase with depolarization at both ages and are larger in mature (open symbols) than in young (filled symbols) neurons (note difference in ordinates). Data were obtained from 9-11 neurons for each channel class at each age, and values are mean f SEM usually for 6 or more patches. (C) Probabilities that 0, 1, or 2 channels are open were evaluated for a patch from a young neuron exhibiting activity of two small channels at 40 mV (11 pS; dotted lines). These may be compared with values predicted by binomial analysis when n = 2 (solid lines). See Experimental Procedures for details of analysis. (D) Voltage dependence of open probabilities of intermediate and small channel classes from young (filled symbols) and mature (open symbols) neurons. The maximum ensemble-averaged current at each voltage, including traces lacking channel activity, was divided by the product of the measured unitary current amplitude and the estimated number of channels in the patch. Each point represents the mean f SEM for n = 2-4 patches. For both channel classes at each age, P increases over the range of 0 to 40 mV. The open probability does not change during this developmental period.

whole cell current,

i is the single-channel

current,

and

2.1-fold during the first day in culture. The number of in-

f is the probability that a single channel is open (channel

termediate conductance channels

open probability).

2.5-fold

This

procedure

assumes

that all K+

channels active in the whole cell configuration

are also

active in the excised patch. Given the relative densities

of intermediate

and small channels (see below) and the

(Table 3). Records

per patch increased

of the large conductance

channel class elicited by steady depolarization

revealed

an increase in the number of channels by a factor of 7.8 during the first day in culture.

However, the number of

ratio of their unitary conductances (Table 11, three-quar-

large channels per patch from young cells was less than

ters of the whole cell current

one-tenth that of either small or intermediate

(O’Dowd et al., 1988) can

be attributed to the activity of intermediate channels. At a holding potential of 20 mV, the relationship

yields esti-

(Table 3). Given

neurons

channels

selected to have a develop-

mentally constant surface area (A) (O’Dowd et al., 1988),

mates of 1200 and 4400 intermediate channels for young

the patch areas (a), determined

and mature neurons,

tances, and the observed number of channels per patch

respectively

lar estimates for small channels

(3.7x

increase). Simi-

indicate 1100 and 3400

(n), if a homogeneous

channels for young and mature neurons (3.1x increase).

sumed,

These estimates of channel number may be low if P was

channels:

overestimated

by restricting

the probability

analysis

For example, records in which channel activity

is not well

described

by a binomial

arise as a result of either clustering

distribution

may

of openings due to

of channels

have 5200

is as-

intermediate

N = A.n/a

patches in which channel activity behaved in a binomial fashion.

distribution

mature neurons would

to

from the pipette resis-

(2)

This value is similar to that obtained by the first method. It may be unrealistically

high if channels

trated in the perinuclear

region of these neurons,

are concenlike

a slow process of inactivation (see above) or cessation of channel function during the recording interval. An alternative approach for determination of channel

Table 3. Densities

number per cell involves estimates derived from the observed maximum

number

of channels

simultaneously

open in all patches from which ensemble-averages compiled.

After correcting for differences

were

in pipette re-

sistance and for the percentage of patches lacking channel activity as described

above, the number

of small

conductance channels per patch was found to increase

Small Intermediate Large

of Potassium

Channels”

Young

Mature

Increase

0.9 * 0.1 (11) 1.1 it 0.1 (6) 0.06 + 0.03 (45)

1.9 * 0.1 (11) 2.7 f 0.3 (10) 0.47 f 0.10 (53)

2.1 X 2.5x 7.8x

d Number of channels of patches).

per patch; values are mean f SEM (number

Developmental Changes in Neuronal K+ Channels 745

C

B

A

INTERMEDIATE

SMALL

INTERMEDIATE 40 -60

I .4 ms

(31)

YOUNG

MATURE 0.6ms

(53)

MATURE

YOUNG

-20

0

20

40 (mV1

1.6ms ”

-20

I.4 ms

0

20

40 ImV)

Figure 7. Activation Kinetics of Ensemble-Averaged Currents of Intermediate

0

20

40 IrnV)

and Small Channels from Young and Mature Neurons

(A and B) Currents were elicited by 20 ms steps to 40 mV (only the first 10 ms after the voltage steps are shown). Number of steps averaged for ensemble-averaged cutrents of intermediate is indicated in parentheses below each trace; sweeps lacking activity were omitted. (A) t v2max conductance channels from young (21 pS) and mature (27 pS) neurons are 1.4 and 0.6 ms, respectively. (B) ta,zmax for thie ensemble-averaged currents of small conductance channels from young (12 pS) and mature (17 pS) neurons are 1.6 and 1.4 ms, respectively. (Below) Voltage dependence of tI,zmax.Data for each class of channel were obtained from at least 9 young and 9 mature neurons. Each point is the mean value from 3-11 patches; SEM is shown when greater than symbol size. Young intermediate (A); mature intermediate [Ah);young small (W; mature small (0). The intermediate class of channels activates 2 to 3 times more rapidly in mature cells than in young cel~ls.There is no change in the rate of activation of the small channel class. (C) Cumulative distributions of first latencies of intermediate channels from young and mature neurons. The probability that a channel has opened by any particular time after the onset of depolarization is’plotted as a function of time (dotted limes). Data at each voltage (O-40 mV) were combined from 2 patches, both of which displayed activity of 2 channels. First latencies were evaluated from single-level records only, however, and those lacking activity were not considered. The probability of a channel opening after any given time incrrases with voltage over the range of voltages examined and is greater for channels from mature neurons. of time Curves (continuous lines) are fits to P(t) = 1 - (Ae- Et - BemA’)/(A - 6); A and B are rate constants. (Below) Voltage’dependence constants; T, - l/A; 52 = l/B. r, IS brief and relatively insensitive to voltage or age (dashed lines). ~2 is voltage-dependent and decreases during development (solid lines). Young neurons (A); mature neurons (A).

acetylcholine receptors in striated muscle (Fischbach and Cohen, 1973; Englander and Rubin, 1987). There is extensive evidence for nonuniform localization of ion channels in neurons (Almers and Stirling, 1984), and K+ channels may exist in aggregates in molluscan neurons (Kazachenko and Geletyuk, 1984; Taylor, 1987). Developmental Changes in the Kinetics of K+-Selectiw Channels Ensemble averages of single-channel currents elicited by 20 ms duration depolarizing voltage commands were compiled to compare activation rates for the intermediate and small conductance channels in young and mature neurons. The activation of the large channel class in mature neurons appeared to be independent of voltage, since the channels were frequently maximally activated by the end of the capacitative artifact, irrespective of the holding potential (data not shown). A developmental comparison was not attempted due to the low frequency of appearance of large channel activity in young cells. Over the voltage range of 0 to 40 mV, the time to half-maximal current (tI/2max)due to intermedi-

ate channel activity in patches from young cells was 2-to 3-fold greater than tI,2maxmeasured in patches from mature cells. For example, at 20 mV, tj,Zmaxfor the intermediate channel ensemble-averaged currents in young cells was 3.1 * 0.6 ms (n = 8) compared with 1.1 f 0.1 ms (n = 6) for the intermediate channel class in mature cells (Figure 7). The activation rates of the small channel ensemble-averaged currents did not change during development; values of tI,Zmaxfor the small channel class in young and mature cells were 2.5 f 0.4 and 2.0 f 0.2 ms, respectively (n = 7,6; 20 mV; Figure 7). Thus, in mature cells the two classes of voltage-dependent channels are further distinguished by the faster activation kinetics of the intermediate class. The ensemble-averaged currents of both the intermediate and rhe small conductance channels of young neurons showed no inactivation during the voltage step to 20 mV (n = 17). For mature neurons, only 1 of 15 ensemble-averaged currents analyzed at 20 mV displayed inactivation (SO%, over 20 ms) and was excluded from further analysis. This inactivation is more rapid than the decline in NPopen (Figure 4) but is consistent with the time constant of de-

NelJKN746

cay of A-current neurons

at the time of its first expression

(Ribera

The

and Spitzer,

probability

occurred

that

by any time

command

is given

First latencies from

of the distribution

the minimum channels

number

opening

of closed

side at rest. This analysis change

during

are achieved dures,

channels

reveals which

the identichannels

also reveals the transitions as well

7) derived

as those

that

from

start in one closed

ure 7C). Although

the

the

existence

the open

and mature

from

for the first latency

this equation.

ms for young channels

intermediate

ized

potentials

over

these distributions voltages However,

the larger voltage

an-

closed appear

neurons.

The

were ob-

values for z are 1.0 and

range

with

more depolar-

of 0 to 40 mV, at both to steeper

rates of rise of

7C). The rate is greater

in patches

from

mature

and changes

at all

neurons.

of the two time constants

dependence

the

sparse

frequency,

during

state transition

A state diagram

that is consistent

and four

exhibits

more during

Discussion

This is like the model

suggested

for a delayed

from frog skeletal

similar

rectifier

to that advanced

for a delayed channel

upon

opening.

from

in a closed

In young

inactivated voltage

of this study

classes of voltage-dependent

rent

to the larger amplitude neurons,

does not change.

membrane, metabolic potential

inhibition

scopic

current

The increase it could

of which

(O’Dowd,

cur-

open

and concur-

probability

of one of the two

consistent

with

cell current

proteins

K+ channels

is in accord

inhibitors

thesis

channel

of channel

some

rectifier

the

in mature

of these results is that transcrip-

functional

This account

their

of the whole

tion and translation

the

of the macroscopic

while

also increases,

more rapid activation

in two

underlie

increases

The rate of activation

cells. One explanation of more

delayed

of these channels

classes of channels

tion

are that changes

K+ channels

of the macroscopic

in mature

have

with

block

and Aldrich

prevents

to transit

cells the

to interburst

any one of the closed Three Their

delay

be generated

which

another

it

before

is longer

intervals

between

toward

the open

classes

of

properties

marized

and may lead to an

At the termination

may be in the open

K+ channels

in mature during

were

characterized.

development

in both young

classes of channels neurons,

prooerties.

the maturation

that

of the action

differentiation

result,

of the macro-

1987).

channels

neurons

inactivation

slowly

have a different 1985; Ribera and

nor the intermediate

(Connor

than

since their

A-currents

and Stevens,

of A-currents

neurons

is largely

al., unpublished

cur-

recorded

1971; Cooper

nels studied

in a variety

such as squid axon

lymphocytes tionary node

from

Xenopus

by 100 ms (Ribera et

of the intermediate

here are similar

nels described

recorded

complete

data).

The properties

chromaffin

of first laten-

single-channel

neurons

the small

in the

of 10 mM

and Shrier, 1985; Kasai et al., 1986; Taylor, 1987). In addi-

of the intermediate the time constant

extremely

recorded

to be K+ A-channels

more

rons (Kazachenko

of one or more of the closed states. Analysis

were

(Blair and Dionne,

are likely

from other

these

Neither

rents inactivate

they

are un-

have

the Ca2+-dependent from

slope conductance Spitzer,

since

channels

or must

Ca2+ and in the presence

recorded

submitted).

since one way in which

is by decreasing

of added

EGTA. Furthermore,

spinal

neuronal

Ca2+ sensitivity

currents

tion,

different

and Spitzer,

high

absence

as indi-

and reversal po-

of all three

to be Ca2+-dependent,

param-

have been sum-

and mature

currents

poten-

state at the far left.

cells as well as their

(Table 4). The same three

Unitary

of the

state or in

states. At hyperpolarized

it resets to the closed

eters that change

in the

the demonstrations

in rate of activation

is an intriguing

(1988b)

state at the left,

state at the right.

tials, however,

lead to the inser-

1983; Blair, 1983) and that RNA syn-

(Ribera

et al. (1985)

muscle and very

and this initial transition

step, the channel

likely

tributes

a se-

states:

rat PC12 cells. At rest, the

depolarization

depolarization

by Standen

by Hoshi

rectifier

resides

leaves

tentials.

rent. The density

specifies

closed

cated by analysis of slope conductances

differentiation

contributing

for the intermediate

with the findings

of one open

at

steady

Closed*Closed*Open=Closed=Closed.

are present

findings

method,

state. The closed state on the other side of the open state

at 0 mV; for mature

development.

The major

was not

of 40 mV by the latter

of the initial

model

of T, and ~~

of closed times. A de-

corresponds

(Figure

the longer

analysis

due in part to a small difference

and

to the first latencies. channel

age. The two

to values

in the longer time constant

probably

depolarization,

con-

Values for T are 2.6 and 0.5

leading

for channels

stationary

change

voltage

two time

on developmental

at a potential

however, this

quential

states from

distributions

decrease

the

stages of development,

detected

and

are comparable

from

velopmental

indicated

channels

at the same potential,

0.5 ms. The first latencies

obtained

are

to account

channels

of two closed

tained

on voltage constants

that

1982; Fig-

of additional

Thus intermediate

state in both young

channels

that one of these is more dependent

in which

state and pass through

to reside at rest at a distance time constants

re-

both time

Proce-

model

they are not necessary

for the observations.

of intermediate

fits of the distributions (see Experimental

other to reach the open state (Patlak and Horn, states is not excluded,

were

rates of activa-

and indicates

an equation

equation

channels

dis-

states through

Satisfactory

with

first latency

states in which

development

voltage-dependent.

has

of first latencies

of closed

pass before

ties and durations

opening

used to examine

ties

stants and revealed

of a depolarizing

of intermediate

records

in these

data).

first

by the cumulative

evaluated

Analysis

a channel’s after the onset

tribution. tion.

unpublished

(Conti

(Cahalan

fluctuation of Ranvier

of other

rectifier

mature

and Neher,

and Celetyuk,

cells (Marty

and small K+ chan-

to delayed

1980), snail neu-

19841, bovine

and Neher,

analysis

of K+ conductance

myelinated

adrenal

19851, and human

et al., 1985). In particular,

of frog

chan-

preparations,

axons

T

nonstaat the

revealed

13

Developmental Changes in Neuronal K+ Channels 747

Table 4. Potassium Channels Properties in Mature Cells Channel Class

Conductance

Small

Lowest, 14pS Intermediate, 29ps Highest, 84pS

Intermediate Large

Developmentally

Potassium Selectivity

Sensitivity to TEA

Voltage Dependence

Greatest

Most

Activation ft,,,,,,,,),

1 ~ownr%ored

~,pnr%redra

Inactivates over seconds

Less

Intermediate Least

Less

Kinetic Behavior

Popen

Activation,&,J,

1 ~opnr%ored

%pen,%redra

Inactivates over seconds

Po,x-n

None

1 ~openr~~clored

No inactivation

Regulated Properties

Channel Class

Density

Topen

QorPd

t‘,>ltUX

Small Intermediate

Increases 2-3x Increases 2-3x

No change Decreasesh

No change 1 of 4 decreases’

No change Decreases

Large

Increases 8x

NDd

NDd

NDd

First Latency Distribution Not examined Two time constants; longer one decreases NW

a The voltage dependence is inferred from the voltage dependence of tKmax. h This shortening of the open time of intermediate channels would appear to be developmentally anomalous since the result could be to reduce the contribution of this current in mature cells. However, an increase in the frequency of flickers, which constitute ‘most of the closures, would reduce the mean open time with only a small reduction in the total time spent open. Open probabilities of intermediate and small channels exhibited a similar voltgage dependence and increased slightly over the range of 0 to 40 mV in both young and mature neurons. This observation suggests that the decrease in channel open time of the intermediate class that occurs during development does not significantly alter the contribution of these channels to the macroscopic IKv. c The decrease is inferred from the first latency distribution, although it is not detected by stationary analysis. d Due to the low frequency of observation of channel activity, these parameters were not studied in young neurons.

and 30 pS channels

differing

vation (Conti et al., 1984).

in the kinetics of their actiDetailed analysis

of a 15 pS

may, however, contribute to the developmental decrease in action potential duration by promotiing repolarization

channel from frog skeletal muscle in lipid vesicles dem-

of the cell. It may also account in part for a decrease in

onstrated

input resistance

a single voltage-dependent

least three closed times.

The

open time and at

open probability

channel increased with depolarization, slow

inactivation

Close scrutiny

was evident

of this

and a process of

(Standen

et al.,

1985).

of a 12 pS channel from rat PC12 cells re-

vealed two open times,

one of which

was voltage-de-

(Baccaglini and Spitz&,

The developmental

increases

intermediate

channels

creases in amplitude

can largely account for the inand activation rate of the macro-

scope voltage-dependent 1988). Thus

to decrease with depolarization.

to other voltage-dependent

The open probability

voltage-dependent,

and clustering

of

to the longest closed state,

erties are not known.

(Hoshi

roles of transcriptional

1988b).

Delayed rectifier channels

scribed in embryonic ing murine

1988a,

have also been de-

or immature cells such as develop-

macrophages

(Ypey and Clapham,

1984),

et al.,

K+ channels. The molecular

O’Dowd,

It will be important to ascertain the mechanisms

(Grampp et al., 1972;

1983; Mishina et al., 1986; Ito et al., 1986; Bris-

mar and Gilly, posttranslational

1987; Ribera and Spitzar, modification

submitted) and

(Seelig aind Kendig, 1982;

guinea pig dorsal root ganglion cells (Kasai et al., 1986),

Strong et al., 1987; Armstrong

and chick ciliary ganglion cells (Gardner,

et al., 1987) in the differentiation

1986). Investi-

(O’Dowd

can be assigned

bases of the developmental changes in K+ channel prop-

responsible

and Aldrich,

K+ current

only a small contribution

activity seemed attributable for inactivation

of the inter-

mediate and small channels and in rate of activation the

pendent, and four closed times, two of which appeared was accordingly

1977).

in density

and Eckert, 1987; Choquet of these channels. Mo-

gation of K+ channels in differentiating

rat Purkinje

neu-

lecular probes specific for K+ channel Igenes CTempel et

rons indicated the existence of 27,44,

and 70 pS chan-

al., 1987, 1988) may provide useful tools with which to

nels; two of these exhibited

developmental

their frequency of occurrence,

changes in

and one exhibited an in-

crease in its mean channel open time (Gruel,

Experimental

produced by the large channels

not voltage-dependent,

it should

not contribute

macroscopic delayed rectifier current. would

most likely

This

to the

component

have been subtracted as part of the

et al., 1988).

Current

through

of these channels

Procedures

is

leakage current recorded in the whole cell configuration (O’Dowd

of expression

1984; Yool

et al., 1988). Since the current

investigate the control

during neuronal development.

large channels

The methods used to prepare dissociated cell cultures of stage 15 Xenopus embryos (Nieuwkoop and Faber, 1956) were similar to those previously described (Spitzer and Lamborghini, 1976; Blair, 1983). Data were obtained from the heterogeneous population of sensory, motor, and interneurons (Spitzer and Lamborghini, 1976; Lamborghini, 1980; Bixby and Spitzer, 1984; Lamborghini and Iles,

Neuron 748

1985) since it was not possible to distinguish cell types on the basis of their morphology. However, the population appears homogenous with respect to changes in the macroscopic voltage-dependent K+ current, irrespective of the length of neurites (O’Dowd et al., 1988). Young neurons were studied at 6-9 hr and mature neurons at 18-30 hr in culture. For recordings made from inside out excised patches, the culture medium was replaced with a high K+ solution (100 mM KCI, 3 mM NaCI, 10 mM HEPES, 10 mM EGTA, pH adjusted to 7.4 with 29 mM KOH) immediately prior to the onset of recording. For recording from cell-attached patches, culture medium was replaced with a high Na+ solution (3 mM KCI, 100 mM NaCI, 5 mM MgC12, 5 mM HEPES, 10 mM ECTA, pH adjusted to 7.4 with NaOH). Solution exchange entailed mu35 serial dilutions of bath volume to ensure complete replacement and thus removal of extracellular Cal+. Culture dishes were superfused via gravity flow with external solutions during the course of an experiment in order to change the ionic environment or to apply and remove drugs that were simply added to the external solution. Fire-polished, Sylgard-coated (Dow Corning) pipettes (VWR blue band micro-hematocrit) were filled with a low K+ solution without added Caz+ (20 mM KCI, 60 mM NaCI, 5 mM MgClz, 5 mM HEPES, pH adjusted to 7.4 with NaOH) and had resistances of lo-30 MD. Cigohm seals were made on the perinuclear region of the cell soma. Experiments were performed at room temperature (20”C-22°C). Excised patches were generally held at -80 mV and stepped to more positive potentials for several seconds. Potentials indicated for cell-attached experiments give the membrane voltage assuming a resting membrane potential of -80 mV (Spitzer and Lamborghini, 1976; O’Dowd, 1983; Blair, 1983). Depolarizing voltage steps of 20 ms duration were also applied to excised patches to determine the number of channels active in a patch and to examine the kinetics of activation of channels. Currents were recorded with a Dagan 8900 amplifier (Dagan Instruments) using a 10 CD headstage, filtered at 2 or 2.5 KHz, and sampled at intervals of 100 ps. A PDP 11/23 computer and Cheshire Data interface board (Indec Systems) were used to generate voltage steps and to digitize and store the records. Data were analyzed with interactive computer programs. Single-channel conductances and extrapolated reversal potentials were determined from least squares best fit to I-V relationships (23 voltages; usually mean of 100 or more single-channel currents at each voltage). Mean channel open and closed times were estimated (>lOO openings at each voltage) using maximum likelihood analysis (Colquhoun and Sakmann, 1983) in which the time constant IS equal to the difference between the mean open or closed time and the minimum resolvable dwell time (200 t.rs). This method can yield time constants less than the minimum dwell time. Open times were analyzed from patches containing only one level of channel activity or with records of multiple channels from which all but unitary openings were excluded. In the latter case, exclusion of overlapping events could give nonexponential distributions or underestimates of open times (Horn and Standen, 1983). Closed times were usually analyzed in patches displaying only one active channel; the two shortest closed times of intermediate and small channels were sometimes evaluated from patches containing more than one channel when analysis was confined to bursts displaying unitary activity presumed to arise from a single channel. Significance of differences between means was assessed by Student’s t test. Relative amplitudes of the contributions of closed states to the distributions were calculated as A.T,/(A,~, + 8r,) and t?.sJ (A.r, + 8.~~) from values in the equations describing the fits. The (NP,,,,)was evalutime dependence of relative open probability ated from patches that exhibited channel activity of a single class during 3-4 s depolarizing voltage commands. Current was averaged over intervals of 150 ms, and values are the means of 3-5 such intervals. NP,,, was calculated as the quotient of the average current at a given voltage and the unitary current determined from the I-V relationship of channels in the patch. Ensemble averages of single-channel records (usually the mean of 50 or more traces) elicited by 20 ms steps were generally compiled from patches exhibiting activity of only a single channel class. For those cases in which multiple channel classes were present in a patch, averages were compiled only from traces exhibiting activity

of a single class. Leakage and capacitative currents were removed by averaging the responses to 40 mV steps that did not elicit channel activity and scaling and subtracting these currents from the original records. The number of intermediate or small channels active in a patch was evaluated from the greatest multiple of unitary current amplitude observed shortly after steps (n 3 50) to depolarized voltages; the number of active large channels was evaluated during periods of sustained depolarization. The membrane area in a patch was calculated from an empirical relation of pipette resistances to capacitance measurements (Sakmann and Neher, 1983), assuming 1 pFlcm2, given as a = 12.6 (l/R

+ 0.018)

(3)

where a is the area and R is the resistance. Runs analysis of responses to sets of 50-200 stimuli was performed (Swed and Eisenhart, 1943; Gibbons, 1971) to determine the likelihood that clustered activity arose by chance. Z, the distribution of the number of runs, is given by Z = -(R

-

2npfl

- p)/2n”pfl

- p))

(4)

Where R is the observed number of runs, n is the number of trials, and p is the probability of observing at least one channel opening. Z is small if events are randomly ordered. Binomial analysis of idealized records was also performed to determine the number of channels active in a patch. The probability of channels’ being open at a given time, P(t), was first determined from the data. The probability P, of observing x channels open out of a total number N was derived from P, The maximum

(0 =

likelihood

($Pft)X.[l

-

Pft)]‘N

was then evaluated

L(N) =

x

x1 as the maximum

(5) of

go

cx.ln IP,(t)l

where E,Xis the total number of observations at level x (Patlak and Horn, 1982). Open probabilities (P) were computed from ensembleaveraged records of responses including traces lacking channel activity. P was calculated as the quotient of the maximum value of the averaged current at a given voltage with the product of the unitary current and the number of channels determined by binomial analysis. Values for tKmax of ensemble-averaged currents excluding traces lacking channel activity were measured directly from hard copies. First latencies (times to first opening) were used to generate cumulative histogram distributions. Measurements were made from singlelevel records only. If more than one channel were active in a single-level record, the mean values of first latencies would have been underestimated. Curves of the form P(t) = 1 -

(Ae - sr - Be -At)/(A -

6)

(7)

were fit to the distributions by nonlinear regression, where A and Bare rate constants. This equation is derived from a model in which channels start in one closed state and pass through another to reach the open state (Patlak and Horn, 1982). Satisfactory fits indicate that the data are consistent with the model and allow evaluations of A and 8.

Acknowledgments We thank V. Dionne and R. Maue for advice during the initial phases of the work and V. Dionne, M. Liebowitz, and R. Maue for supplying some of the software. We are grateful to R. de Baca and J. Schimke for technical assistance and to R. W. Aldrich, P Brehm, A. B. Ribera, 5. A. Siegelbaum, and J. H. Steinbach for critical reviews of the manuscript. This work was supported by a USPHS fellowship to C. L. H., by an MDA fellowship to L. F? H. and by NS15918 to N. C. S. Received July 12, 1988; revised August

17, 1988

Developmental Changes in Neuronal K+ Channels 749

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