Neuron,

Vol. 4, 313-321,

February,

1990, Copyright

0 1990 by Cell Press

Mutational and Gene Dosage Analysis of CalciumActivated Potassium Channels in Drosophila: Correlation of Micro- and Macroscopic- Currents Akira Komatsu: Satpal Singh,+ Perry Rathe, and Chun-Fang Wu Department of Biology University of Iowa Iowa City, Iowa 52242

Summary In Drosophila, two Caz+-activated K+ currents, IcF and lcs, have previously been distinguished in conventional voltage clamp experiments. The slowpoke (s/o) mutation eliminates IcF specifically. We report that in patch clamp recordings a single-channel Caz+-activated K+ current is readily distinguished from other channel activities in normal larval muscle membrane, whereas no such current is observed in s/o muscles. This singlechannel current thus correlates with the macroscopic IcF. No obvious differences in amplitude or properties were detected between normal (+/+I and heterozygous (s/o/+) IcF channels in whole-cell voltage clamp recordings or single-channel patch clamp recordings. These results are consistent with the hypothesis that s/o is a structural gene for the IcF channels only under certain conditions. The selective effect of the s/o mutation may reflect a defect in a regulatory mechanism that is specific for the functioning of the Icr channel protein. Introduction Ca*+-activated K+ channels play an important role in the regulation of neuronal function and a variety of cellular processes (Meech, 1978; Petersen and Maruyama, 1984; Rudy, 1988; Latorre et al., 1989). Yet unlike other types of ion channels, molecular genetic analysis of their mechanisms is still not available. Drosophila mutations affecting specific ion currents provide a powerful approach to the identification and localization of the relevant genes and subsequently allow structure-function analysis of ion channels and channel-related proteins by gene cloning and DNA sequencing. This approach is especially useful for the study of K+ channels, for which protein chemistry data are still lacking. As demonstrated by the cloning and sequencing of the Shaker (Sh) gene, characterization of a collection of Drosophila mutants with defects in a voltage-dependent K’ current (IA) ultimately led to crucial information about K+channel structure (Baumann et al., 1987; Kamb et al., 1987, 1988; Papazian et al., 1987; Pongs et al., 1988; Schwarz et al., 1988). Mutations of a separate gene, slowpoke (do), have *Present address: Department of Physiology, Tokyo Women’s Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan. +Present address: Department of Biochemical Pharmacology, State University of New York at Buffalo, Buffalo, New York 14260.

been shown to eliminate a macroscopic Cal+-activated K+ current, IcF (Elkins et al., 1986; Singh and Wu, 1989), and could be useful for analyzing the molecular mechanisms of the Ca2+-activated K+ channels. !n this paper we examine the properties and the effect of the s/o mutation on Icy at both the microscopic and the macroscopic levels. Patch clamp studies generally reveal a larger variety of single channels than the macroscopic currents identified with voltage clamp techniques. (See Byerly and Leung [I9881 and Sole and Aldrich [I9881 for K+ currents in cultured Drosophila neurons.) This makes it difficult to correlate a particular type of single channel with a macroscopic current. Such a correlation requires a unique way of identifying a particular macroscopic current (e.g., by means of pharmacological specificity, physiological properties, cellular distribution, etc.) so that the same criteria of identification can be applied at the single-channel level. This analysis is easier in well-studied preparations like acetylcholine receptor channels in vertebrate skeletal neuromuscular junctions, which have already been characterized in considerable detail at the macroscopic level, but is relatively difficult in more complex systems, like vertebrate central neurons, or more recently established systems that have not been characterized in detail. Drosophila mutations that selectively eliminate particular macroscopic currents can be used as important criteria to identify single channels corresponding to these currents. In Drosophila, membrane currents have been characterized mostly in muscle fibers because of their suitable size for conventional voltage clamp experiments. In muscle cells, five distinct types of currents have been distinguished by differences in physiological properties and sensitivity to pharmacological agents and mutations (Salkoff and Wyman, 1981; Salkoff, 1983; Wu et al., 1983; Wu and Haugland, 1985; Gho and Mallart, 1986; Wei and Salkoff, 1986; Elkins et al., 1986; Singh and Wu, 1989). In addition to an inward Ca2+ current (I&, different types of voltageactivated and Ca2+-activated outward K+ currents are present. The voltage-activated K+ currents include a fast transient current called the A current (IA) and a slow delayed rectifier current (IK)* The Ca2+-activated K+ currents, generally called the C currents (Ic), consist of two separate components, a fast transient current (ICE) and a slowly augmenting current (1~) (Singh and Wu, 1989). These currents are sensitive to specific pharmacological agents: IA is blocked by 4-aminopyridine (Salkoff, 1983), IcF by charybdotoxin (Elkins et al., 1986) and tetraethylammonium ion (Gho and Mallart, 1986), and IK by quinidine (Singh and Wu, 1987, Sot. Neurosci., abstract; 1989). To record single-channel currents that can be compared with the macroscopic currents within the same cell type, we worked out a membrane vesicle prepara-

NWKNl 314

fer how the sio gene product contributes ture and function of the IcF channels.

20mV -60mV i

to the struc-

L

Results

a

The muscle vesicles were prepared according to the procedure of Standen et al. (1984, 1985), which has been shown to form vesicles in the outside-out configuration. As in muscle vesicles of other species (Berger et al., 1984; Standen et al., 1984), several different types of single-channel currents could be recorded in the larval muscle vesicles. Voltage-activated K+ channels are among the distinct and readily identifiable channel types. Since channels with similar properties have been described in other preparations from Drosophila (Sole and Aldrich, 1988; Zagotta et al., 1988; Yamamoto and Suzuki, 1989), only a brief description of these channels is given below.

Y

Voltage-Activated

b

- 0.6PA

1OOmsec

- 0.4 - 0.2 A0

2.0 -

C I (PA) pipette/bath .: zSK1130K . : 85K1130K /*/ b=:

-60

c-

*/*

:,: 0’ -40

.

/’

:

I 0

:

: 40

:

1 60

V LmV) Figure 1. A Rapidly Channel in Muscle

Inactivating Membrane

Single Vesicles

Voltage-Dependent K+ from Normal Larvae

Patch clamp recordings were made from unexcised patches. The bath contained 130 mM KC. (a) Representative current traces in response to voltage pulses from -80 mV to +20 mV. The pipette solution contained 65 mM K+. (b) Ensemble current average from 87 such traces. (c) Current-voltage relation of the single channel current. In addition to the patch presented in (a) and (b), a different patch with 2.5 mM KC in the pipette is presented.

tion derived from larval muscles based on procedures developed in other species (Berger et al., 1984; Standen et al., 1984,1985). In spite of the various currents that coexist in the cells, the use of the s/o mutation, which selectively eliminates lcF, enabled us to correlate a single-channel current with the whole-cell IcF. This also permits an integration of information gathered at both microscopic and macroscopic levels. Analysis was performed on both homozygous (+/+ and s/o/s/o) and heterozygous (s/o/+) individuals to in-

K+ Channels

Both inactivating and non-inactivating voltagedependent K+ channels were observed when voltage steps were applied to patches with seals of over 50 CCL The properties of these channels in the s/o preparations were not obviously different from those in the normal preparations. Figure la shows responses of a rapidly inactivating channel to voltage steps in a cellattached (unexcised) patch from a normal larva. The ensemble average (Figure lb) indicated that the inactivation was nearly complete during a 400 ms pulse to +20 mV. The channel had a unit conductance of about IO pS in 130 mM K+ solution with 2.5 mM K+ in the pipette (Figure Ic). In the other patch shown in the figure, the pipette solution contained a high K+ concentration (65 mM). This decrease in K+ gradient shifted the extrapolated reversal potential by about 55 mV and reduced the slope conductance (Figure lc). Among the macroscopic currents identified in previous voltage clamp studies of these muscle fibers, IA is the only voltage-activated K+ current that shows rapid inactivation (Wu and Haugland, 1985; Haugland, 1987; Singh and Wu, 1989). However, the single channels reported here inactivated with a time constant in the range of 100 ms at +20 mV, which is much longer than that of the macroscopic IA in larval muscle (about IO ms) (Wu and Haugland, 1985) and those of the inactivating single K+ channels characterized in cultured embryonic myotubes (Zagotta et al., 1988) and larval neurons (Sole and Aldrich, 1988) in Drosophila. At present, it is not possible to conclude whether these differences arise as a result of the procedures adopted for vesicle preparation or whether the single channels described here represent an inactivating current not previously identified at the macroscopic level. Figure 2 shows a channel with a larger conductance recorded in an unexcised patch from normal muscle. The ensemble averages (Figure 2b) indicate that the channel did not inactivate during a pulse of 500 ms to 0 mV, and showed only a slight inactivation during a

Calcium-Activated 315

Potassium

0. 20. -60

Channels

in Drosophila

40 mV

mV

a

I2 PA -3

b

PA

-2

-1

4

100

msec -0

4

C

I (PA) 0

3

-60t

-40

-20

0

0

20

1 60

40 V (mV)

Figure 2. Recordings from a Non-Inactivating Dependent K+ Channel in an Unexcised larvae

Patch

Single from

VoltageNormal

The bath contained 130 mM K+ and the pipette solution 2.5 mM K+. (a) Current traces in response to voltage steps from -80 mV to +20 mV. (b) Ensemble averages obtained for voltage steps to 0 mV (129 traces), +20 mV (107 traces), and +40 mV (95 traces). (C) Current-voltage relation for the single-channel current from the same patch.

stronger pulse to +40 mV. The single-channel slope conductance was about 30 pS near 0 mV in 130 mM K+ solution with 2.5 mM K+ in the pipette (Figure 2~). The properties of this channel correspond to those of the delayed rectifier IK. Similar channels have also been described in cultured myotubes and intact larval muscles (Zagotta et al., 1988) and in cultured larval neurons (Sole and Aldrich, 1988; Yamamoto and Suzuki, 1989).

CaZ+-Activated K+ Channels A major type of single-channel current in this preparation showed characteristic kinetic behavior and could be easily differentiated from the others. In the present paper, we describe the salient properties of this channel that are relevant to the s/o mutation. Other details of this channel will appear elsewhere (Komatsu and Wu, unpublished data.) In unexcised patches of the vesicle membrane bathed in 130 mM K+ solution, this channel was very active and showed frequent brief closures during a burst (Figure 3a). The active states were occasionally interrupted by relatively long closures (over several hundred milliseconds). The current increased upon depolarization of the patch membrane and decreased upon hyperpolarization. The single-channel currentvoltage relations shown in Figure 3c (open circles) reveal outward-going rectification with a low K+ concentration (2.5 mM) in pipettes. The open-time histogram was fit by a single exponential with a time constant of about 5 ms at 10 mV (Figure 3d and open circles in Figure 3f). The closed-time histogram had three time constants: the shortest one was about O.l0.2 ms, the middle one about 0.5-2.0 ms, and the longest one over hundreds of milliseconds (data not shown). The open-time constant showed voltage dependency. It became longer when the patch membrane was depolarized (Figure 3f, open circles). With 130 mM K+ in the bath, the conductance of this channel depended on K+ concentration in the pipette, the slope conductance at 0 mV being 54 + 10 pS (n = 9) at 2.5 mM (Figure 3c) and 88 + 13 pS (n = 12) at 130 mM (data not shown). In inside-out excised patches the activity pattern was the same as in cell-attached patches at appropriate free Ca*+ concentrations. The current-voltage relations for different K+ concentrations could be described by the Goldman-Hodgkin-Katz constant-field current equation (Goldman, 1943; Hodgkin and Katz, 1949). A permeability to K+ of 1.7 x IO-l3 cm/s was calculated from the single-channel conductance of 84 + 6 pS (n = 6) under symmetrical 130 mM K+ solutions (Benham et al., 1986). The selectivity ratio for Nat over K+, as estimated from the reversal potential shift (Katz, 1966) of 15 mV for the reduction in internal K+ from 130 mM to 75 mM by Na+ replacement, was less than 0.05, suggesting high selectivity for K+. The channels are Ca*+ activated, as shown by varying the free Ca*+ concentration on the cytoplasmic side in the bath. An experiment demonstrating channel activation by Ca2+ in an excised inside-out patch is shown in Figure 4a. This membrane patch contained at least four Ca2+-activated K+ channels. At a free Ca2+ concentration of IO-7 M, the channels were in active burst states. At 2 x IO-* M they stayed in the closed state(s). Figure 4c (open symbols) shows the probability of channels being open (P,) derived from patches containing this type of channel at different free Ca*+ concentrations. At lOmE M free Ca*+, P, was almost zero. When free Ca2+ increased from IOva to IO-’ M, P, in-

a V ImVl

b +/+

V CmV)

d

I

dOi+

0

e

+/+ at 1OmV

at 1OmV

Tau=4.9msec

Tau=5.7msec

10

20

SlOl+

30

40

Open

5 PA

0

time

IO

20

30

40

lmsec)

1 set 3

p*

1 -60

( -40

-20

I 0 T .

I

I 0

20

4 60

40

20

mV

Figure

3. Patch

Clamp

Recording

of Single-Channel

40 Voltage

Ca2+-Activated

(a) Currents in an unexcised normal (+/+) patch. (b) Currents of the single-channel current in normal (open circles, n = 6) and e) Open-time histograms at a membrane potential of +I0 exponential of similar time constants. (f) Voltage dependence (open circles) and heterozygous (filled circles) patches.

K+ Currents

in Normal

and

60

(mV)

Heterozygous

Drosophila

Larvae

in an unexcised heterozygous (s/o/+) patch. (c) Current-voltage relation and heterozygous (closed circles, n = 3) membranes (mean + SD). (d mV in normal (d) and heterozygous (e) patches could be fit by a single of the time constant determined from open-time histograms in normal

creased steeply. At levels above IO-’ M Ca*+, P, reached a plateau level of about 0.6 to 0.9. These results indicated that this type of singlechannel current was mediated by Ca2+-activated K+ channels. However, as mentioned above, voltage clamp studies of the larval muscle have identified two Ca2+-activated K+ currents, L-r and Its. How would this type of single-channel current be related to the two macroscopic Ca*+-activated K+ currents, lcr and Its? The fact that the mutation s/o blocks only IcF but not Its (Singh and Wu, 1989) provided a simple, clearcut solution to this problem. If the single-channel Ca2+-activated K+ current recorded in normal muscle vesicle preparations corresponds to macroscopic 1,-r, this microscopic current would be missing in s/o preparations. If it corresponds to macroscopic lcs, on the other hand, it would be detected in the mutant preparation, where the density of the channel would be expected to be the same as in the normal preparation. We compiled the statistics of all patches, including inactive ones, recorded in a series of experiments designed specifically for the purpose of comparing the channel activity in normal and mutant preparations (Table 1). In muscle vesicles from the s/o mutant,

no such type of single-channel K+ current corded, while several other types of currents countered as in the normal preparation, that the microscopic current missing in sponds to the macroscopic Icr.

was rewere enindicating slo corre-

Gene Dosage Analysis The use of slo and Sh mutants that specifically eliminate 1,-r and IA also enables extraction of these currents from the voltage clamp data (Haugland and Wu, 1986; Singh and Wu, 1989). Subtraction of the membrane current in the mutant muscle lacking L-r from the one having it (e.g., Sh;slo vs. Sh) gives us the whole-cell current trace of pure IcF, which can be effectively used to estimate the density and kinetics of these channels. The procedures of current subtraction and density computations (see Experimental Procedures) were used previously in the cases of iA and IK in larval muscle (Haugland and Wu, 1986; Singh and Wu, 1989; Wu et al., 1989) and give a reliable quantitative estimate of these currents. Moreover, certain features of the structure and/or function of a channel can be inferred from analysis of mutants and of heterozygotes that express a mixture of normal and

Calcium-Activated 317

Free-Ca

Potassium

Channels

in Drosophila

a

(M) 2x10-8

10-7

2x10-8

-

Table 1. Frequencies of Channel Activity

10-7

2x10-

of Patches

Exhibiting

Different

Tvoes II

s Types

Ca2+-Activated K+ Channel

Others

No Activity

Total

Normal slow

24 0

68 54

36 34

128

88

I 3 PA Free-Ca

30sec,300msec

b

(M)

3x10-e

5x10-8

C

A a

.

.

‘3 6 8

0.6

-

Z

0.6

-

n .

$v

x g

v O

.

9

0.4-

g2

0.2

-

0

8

;

l

0

8

0

Open symbols: Closed symbols:

+I+ slol*

“0

0 L-&(3)-

1-F

10-B

10-7 Free-Ca

concentration

Figure 4. Responses of the Ca2+-Activated ent Free Ca*+ Concentrations Recorded Patches

10-e

2x10-3

(M) K+ Channel to Differin Excised Inside-Out

(a) Single-channel current from a normal patch obtained with 65 mM K+ in the pipette solution, and bath perfusion of high-K+ (130 mM) solution containing 2 x 1Om8 or IO-’ M free Ca2+ as shown. This patch contained at least four active channels that were activated by IO-’ but not 2 x 1Om8 M free Ca2+. (b) Single-channel currents from a heterozygous patch obtained with 2.5 mM K+ in the pipette and high-K+ solutions in the bath containing 2, 3, and 5 x 1O-8 M free Ca2+. (c) Sensitivity of the channel to free Ca*+ in normal (+I+, open symbols) and heterozygous (s/o/+, filled symbols) patches. Different symbols represent different patches. When a patch contained more than one channel (some of the open symbols), the averages of probability of open are presented. The free Ca*+ concentration was buffered to the desired level with EGTA.

mutant gene products. The strength of this approach has been seen in studies on different Sh mutations and their heterozygous combinations (Timpe and Jan, 1987; Haugland, 1987) and by subsequent analysis by gene cloning, sequencing, and recording IA currents expressed in mRNA-injected Xenopus oocytes (Timpe et al., 1988; lverson et al., 1988). Drosophila is a diploid organism, and for structural genes of many enzymes (Stewart and Merriam, 1980) and IA channels (Haug-

land and Wu, 1986; Haugland, 1987; Timpe and Jan, 1987), the two copies of the gene contribute equal amounts of the product. Although IcF is totally removed in the homozygous s/o mutant (s/o/s/o), the remaining current in the heterozygote (s/o/+) cannot be predicted from this fact and is determined by the nature of the s/o gene product. For example, if the IcF channel consists of a single s/o polypeptide, without the contribution of additional subunits encoded by other genes, the heterozygote may be expected to reveal Icr current, but with only one-half the amplitude seen in normal larvae. If the channel is constructed from homomultimeric subunits encoded by the s/o gene, the heterozygote may contain channels made of different combinations of normal and mutant subunits and these channels may express characteristics different from one anolher. Voltage clamp analysis of the macroscopic Icr was undertaken in slo homozygous (s/o/s/o), heterozygous (s/o/+), and normal (+/+) larval muscles. The Ca2+activated currents recorded in normal saline with 1.8 mM Ca2+ showed large variations, which were of a similar range in the heterozygous and the normal fibers, and made it difficult to make quantitative comparisons between different genotypes. However, recordings made with 20 mM Caz+ in saline uroduced larger currents with smaller variations, alloiing more reliable quantitative conclusions (Singh and Wu, 1989). The measurements were facilitated by removing IA when the above genotypes are placed in a Sh background (by recombinations with Shfi’33 in double mutants). Thus the initial inward Ica was revealed in the double mutant (Sh;s/o/s/o, Figure 5a), and the difference between the double mutant and the Sh single mutant (Sh;+J+, Figure 5c) yielded the normal lc~ (Figure 5, c - a). Under these conditions, the fast Ca2+activated K+ current, lcF, recorded from heterozygous (s/o/+) fibers was similar to that from normal (+I+) fibers (Figure 5, c - a and b - a). It showed nearly normal amplitude, kinetics (Figure 5, b - a compared with c - a), and current-voltage relationship (Figure 5, inset) and did not indicate a 50% reduction in amplitude (the two current-voltage relations do not show statistically significant differences). These current traces obtained from the voltage clamp measurements, however, do not show the exact kinetic properties of the IcF channel, because the intracellular free Ca2+ concentration changes with time owing to the activation and inactivation of Ca2+ channels and the Ca2+ sequestering mechanism. To over-

NellrOil 318

Figure 5. Macroscopic Membrane Currents Measured with a Two-Electrode Voltage Clamp in Larval Muscle Fibers at 4OC in Saline Containing 20 mM CaZ+

mV +30 -40

(a) Average current densities in a Sh;s/o/s/o double mutant. Number of fibers (n) = 7; number of larvae (I) = 3. (b) s/o heterozygote in a 53 background (Sh;s/o/+). n = 5; i = 4. (c) Sh single mutant (Sh;+/+). n = 10; I = 4. (b - a) Extracted average current traces of fast Ca2+-activated K+ current (Icr) produced by a single copy of the normal s/o gene in the heterozygote obtained by subtraction of Sh;sloisio (a) from Sh;s/o/+ (b). (c - a) Extracted average current traces of Icr produced by two copies of the normal gene by subtraction of Sh;s/o/s/o (a) from Sh;+l+ (c). The inset shows current-voltage relations for the peak values of the average currents in Sh;slo/s/o (filled circles) and of the extracted average currents in S/I;+/+ (filled squares) and Sh;slo/+ mutants (open squares). Means rt SEM are from the same data as shown in (a-c). The membrane was stepped from a holding potential of -80 mV to various voltages. Calculations of active current density are based on the membrane capacity of each fiber and subtraction of the leakage current from the total current (Singh and Wu, 1989).

-80 E nA/nF

a

0

Sh;slo/slo

40

80

0

.

4-o

come this difficulty, single-channel analysis is necessary for a more precise comparison of the properties of these channels. Thus patch clamp recordings were made in both normal and heterozygous larval muscles. Although no specific experiments were undertaken to assess the exact percentage of patches containing Ca2+-activated K+ channels in s/o/+ preparations, about one-quarter of the active patches in the preparations throughout this study showed a singlechannel current similar to the Ca*+-activated K+ channel described above. This frequency of occurrence is also similar to that observed in normal preparations (Table 1). Figure 3b shows a record of the single-channel currents obtained with the cell-attached configuration in a heterozygous (s/o/+) patch. Its activity and amplitude appear to be similar to those in normal patches (Figure 3a). At a depolarization of 40 mV this patch showed long closed states over 1 s. Such long closed states were also observed in normal patches at similar depolarization (Komatsu and Wu, unpublished data). The current-voltage characteristics (Figure 3c) of normal and heterozygous patches were similar, at least in the voltage range between -40 mV and +40 mV. Furthermore, the open and closed times of the normal and heterozygous channels showed similar distributions. The open-time his-

8-O ms

togram in the heterozygote was fit by a single exponential whose time constant was about 5 ms at 10 mV (Figure 3e), and its voltage dependence was similar to normal (Figure 3f). The closed-time histogram reveals at least three time constants, 0.1-0.2 ms, 1-2 ms, and several hundred milliseconds (data not shown). These values are similar to those in normal preparations. The heterozygous channel was also activated by free Ca*+ from the cytoplasmic side (Figure 4b). It had a similar dependence on free Ca*+ concentration, being activated at about 2 x 10m8 M and saturated above IO-’ M (Figure 4~). In all these respects, the normal and the heterozygous channels were indistinguishable. Discussion identification of Ca*+-Activated Single-Channei K+ Currents The effect of the s/o mutation on a Ca*+-activated K+ current (L-r) in muscle fibers of Drosophila has been well characterized at the macroscopic level using voltage clamp recordings (Elkins et al., 1986; Singh and Wu, 1989). One purpose of the present study was to identify single channels corresponding to icr and to analyze the properties of these channels. Patch clamp recordings from a vesicle preparation

Calcium-Activated 319

Potassium

Channels

in Drosophila

of Drosophila larval muscle show different types of single-channel currents. One Ca*+-activated and two voltage-activated K+ channels have been presented here. A stretch-activated channel has also been observed (Gorczyca and Wu, unpublished data). The Ca*+-activated channels studied here could correspond to either the fast (I,,) or the slow (I& macroscopic current. Therefore, the single-channel studies in normal preparations alone cannot positively identify these channels with either IcF or Its. Selective elimination of the Ca*+-activated singlechannel currents in s/o mutants, which are known to lack Icr at the macroscopic level, provides a useful tool for this purpose. A clear correlation between the absence of Icr in s/o larval muscles and the absence of the single channels in vesicle preparations from the same cell types in s/o larvae provides a correspondence between these channels and IcF. A major class of channel activity (nearly one-fourth in active patches) observed in the vesicle membranes of normal preparations was fortunately derived from the Ca*+-activated Icr channels (Table 1). In s/o preparations, these channels were not observed, nor was a new major type of channel seen in their place. So far we have not observed any other type of microscopic Ca*+-activated K+ current in vesicles, although a current corresponding to Its would have been expected. The reason for this is not clear at present. One possibility is a differential distribution of channels on the membrane, with the vesicles being derived from the regions of the membrane rich in the Icr channels but not the Its channels. For example, the vesicles may be derived from the T-tubular system of the muscle cell or they may not have a cytoskeletal structure after the enzyme treatment (Tank et al., 1982). Another possibility is that the enzymatic treatment or the high K+ solution may affect the membrane and/or the Its channel properties such that the channels either lose Ca*+ sensitivity or become nonfunctional. Alternatively, it can be argued that the single channels recorded actually correspond to Its and it is the 1,-r channels that are missing even in the normal vesicle preparation. In that case, the s/o mutation would have to alter the subcellular distribution of Its channels or increase their sensitivity to the enzyme treatment during vesicle preparation, but eliminate only Icr at the macroscopic level. Gene

Dosage

Analysis

Another purpose of our study was to examine the hypothesis that s/o is the structural gene for the Icr channels. A complete lack of Icr in homozygous s/o mutants suggests that the mutant protein is completely nonfunctional. However, IcF recorded from s/01+ heterozygotes did not show a gene dosage effect. Furthermore, a new allele of s/o has been reported recently. This allele, s/04, arose by an inversion that has one breakpoint in the s/o locus (Atkinson et al., 1989, Sot. Neurosci., abstract). Voltage clamp recordings from homozygous and heterozy-

gous s/o4 fibers give results that are essentially similar to those of s/o reported here (G. A. Robertson, N. S. Atkinson, and B. Ganetzky, personal communication). These observations indicate that there is no strict relationship between s/o gene dosage and the amplitude of Icr in the larval muscle. If a single s/o polypeptide constitutes the entire channel itself and the mutant protein is indeed totally nonfunctional, heterozygous larvae bearing one slo allele and one normal allele are likely to show a gene dosage effect, expressing half the macroscopic IcF recorded in normal larvae, as has been demonstrated in Sh-coded IA channels (Haugland and Wu, 1986; Haugland, 1897; Timpe and Jan, 1987) and for many enzymes in Drosophila (Stewart and Merriam, 1980). A similar logic would apply for heteromultimeric channels in which s/o contributes only one subunit. If the Icr channel is homomultimeric, mutation in a structural gene would lead to the presence of normal, mutant, and mosaic channels in heterozygotes, and affect the amplitude and/or the properties of both the macroscopic and the single-channel currents. A linear relationship between the gene dosage and the current amplitude and an effect of mutation on the channel properties would strongly argue for s/o being a structural gene for 1,-r. On the other hand, the absence of any marked differences in amplitude, kinetics, and voltage dependence of the macroscopic currents and in the current-voltage characteristics, kinetics, and Ca*+ dependence of the single-channel currents between heterozygous and normal preparations would not conclusively rule out this hypothesis. However, these observations make the hypothesis less likely because it would hold true only under certain conditions. One possibility is that the s/o gene produces the Icr channel protein in excess for limited membrane sites or other resources. Under such conditions, if the mutant copy of the s/o gene in the heterozygote simply yields no product or grossly defective products excluded from membrane incorporation, the, . one-half normal channel proteins in heterozygotes could be sufficient to generate a nearly normal Icr. A similar argument can be made for a multimeric Icr channel. For example, excess production of an acetylcholine receptor subunit and a Na+ channel subunit has been demonstrated (Schmidt and Catterall, 1986; Witzemann et al., 1989). In a similar way, the s/o gene could encode a subunit of Icr that is produced in excess. Another possibility is that a mosaic multimeric channel may be functional even if only one of the subunits is normal. As an alternative possibility, the s/o gene may encode a channel-associated protein, such as a calmodulin-like sensor for Ca2+ (Hinrichsen et al., 1986; Schaefer et al., 1987) or a cytoskeletal component for channel anchoring within the membrane (Changeux and Revah, 1987). A calmodulin gene (Smith et al., 1987; Yamanaka et al., 1987) and genes for a number of cytoskeletal proteins (Kemphues et al., 1980; Beall et

Neuron 320

al., 1989) have been identified in Drosophila at chromosomal loci different from that of s/o. If s/o codes for any such component, it would have to be different from these products or a variant of one of these. Alternatively, s/o may affect an enzyme for channel modification or for producing molecules important to channel regulation or the membrane environment (Levitan, 1988). Whatever mechanism is postulated, it must account for the observation that s/o affects Icr but no other currents (Elkins et al., 1986; Singh and Wu, 1989). Thus the s/o product must be specific to the 1,-r channel and may be crucial to the understanding of the Caz+-activated K+ channels. Further studies using DNA cloning and sequence analysis of the normal and mutant s/o genes (Atkinson et al., 1989, Sot. Neurosci., abstract) in conjunction with functional assay by physiological methods should ultimately elucidate the function and regulation of this type of channel. Experimental

Procedures

Animals Mature third-instar larvae of the fruit fly Drosophila melanogaster were used in both voltage clamp and patch clamp experiments. The Canton-S strain was used as normal control and the s/o and Sh mutants were in a Canton-S background (over 95% of the genome was replaced by recombination during genetic mapping). The macroscopic currents in larval muscles have been described in several Sh alleles (Wu and Haugland, 1985) and a single s/o allele (Singh and Wu, 1989). The mutant strain used for eliminating iA was ShW133. Thus voltage clamp recordings for L-r were made from Sh w133;+/+ larvae and compared to recordings from Sh”133;s/o/slo and ShKS733;slo/+ larvae (see Figure 5). Voltage Clamp Recording Two-microelectrode voltage clamp recordings were made from the body wall muscles of third-instar larvae at a holding potential of -80 mV as previously described (Wu and Haugland, 1985; Singh and Wu, 1989). The preparation was cooled to 4°C with a Peltier junction. Physiological saline contained 128 mM NaCI, 2 mM KCI, 2il mM CaCI,, 4 mM MgCb, 35.5 mM sucrose, and 5 mM HEPES and was buffered at pH %I. The voltage electrode was filled with 2.5 M KCI and the current electrode with a mixture of 2.5 M KCI and 2.0 M potassium citrate. The electrode resistance was 5 to 15 MD in saline. Current densities were obtained by normalizing current to the fiber capacitance, and the leakage current was subtracted digitally using a personal computer as described previously (Haugland and Wu, 1986; Singh and Wu, 1989; Wu et al., 1989). The clamp took 2-3 ms to stabilize after the initiation of the voltage step. Capacitive transients during this period have been omitted from voltage clamp traces for the sake of clarity. Single-Channel Recording Single-channel currents were recorded from muscle cell membrane vesicles or evaginations by the standard patch clamp technique as described by Hamill et al. (1981). Membrane vesicles were prepared by enzymatic treatment and KCI loading of muscle cells of third-instar larvae, a procedure similar to the method developed for frog smooth and striated muscle cells (Berger et al., 1984; Standen et al., 1984). After removal of the internal organs (central nervous system, digestive system, fat bodies, etc.) and washingwith normal saline (128 mM NaCI, 2 mM KCI, 4 mM MgClz, 1.8 mM CaClz, 35.5 mM sucrose, 5 mM HEPES [pH 7.1, titrated with NaOH]), the body wall muscle cells were incubated in high-K+ solution (130 mM KCI, 1.8 mM CaCb, 4 mM MgCb,

5 mM HEPES [pti 7.2, titrated with KOH]) containing 1 mgiml coilagenase (Sigma, type IA; about 400 U/ml) for 60 min at room temperature. After two washes in high-K+ solution, muscle cells and vesicles were transferred to an experimental chamber. Pipettes were double-pulled from 1.5 mm Pyrex fiber-filled capillaries (WPI, lB150F). The shank portions of the pipettes were coated with resin (Dow Corning, Sylgard 184) and the tip was then fire-polished using a Narishige microforge. The pipette was filled with 2.5 mM KCI, 127.5 mM NaCI, 1.8 mM CaClz, 4 mM MgClz, 5 mM HEPES (pH 7.2, titrated with NaOH) except where otherwise mentioned. When the K+ concentration was changed, osmolarityand ionic strength were maintained by substituting K+ with Na+ to keep the sum of K+ and Na+ at 130 mM. The high-K+ solution was used for the bath in the cell-attached configuration to maintain the resting potential near zero. The pipette resistance was IO-20 MS2 in high-K+ solution. Seals of about 5 to20 GQ were usually formed byapplyinggentle suction after the pipette tip was brought in contact with the vesicle membrane. When seals over 50 CD were formed, voltage steps were applied to reveal voltage-activated channels. inside-out patches were made by quick pulling of the electrode from the vesicle and were then moved to a second chamber (volume 0.07 ml) for perfusion experiments. To determine the effects of varying free Ca2+ concentration on the inner surface of the membrane, perfusion solutions were prepared with assumed apparent dissociation constants of 0.131 mM for Ca-ECTA and of 18.7 mM for Mg-ECTA at pH 7.2, and free Ca2+ concentrations were calculated according to the method of Caldwell (1970). Experiments were done at room temperature (22OC * 2OC). Single-channel currents were recorded with a patch clamp amplifier (List Electronics, EPC-5) and stored on an FM tape recorder (Store-d, Lockheed Electronics Co.) with a frequency response of 2.5 kHz. Played-back signals were filtered at 1 kHz using a fourpole Bessel filter, digitized at 100 ps, and then analyzed using pCLAMP software (Axon Instruments, inc.) with an IBM XT or AT personal computer.

Acknowledgments We thank 6. Jacobson and P. Taft for technical assistance and M. Gorczyca for discussion. The support services provided by University House, the University of Iowa, are gratefully acknowledged. This work was supported by U.S. Public Health Service grants NS15350, NS18500, and NS26528. Received

August

29, 1989;

revised

November

29, 1989.

References Baumann, A., Krah-Jentgens, I., Mueller, R., Mueller-Holtkamp, F., Seidel, R., Kecskemethy, N., Casal, J., Ferrus, A., and Pongs, 0. (1987). Molecular organization of the maternal effect region of the Shaker complex of Drosophila: characterization of an IA channel transcript with homology to vertebrate Na+ channei. EMBO J. 6, 3419-3429. Beall, C. J., Sepanski, M. A., and Fyrberg, E. A. (1989). Genetic dissection of Drosophila myofibril formation: effects of actin and myosin heavy chain null alleles. Genes Dev. 3, 131-140. Benham, C. D., Bolton, T. B., Lang, R. I., and Takewaki, T. (1986). Calcium-activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery. J. Physiol. 377, 45-67. Berger, W., Grygorcyk, R., and Schwarz, W. (1984). Single K+ channels in membrane evaginations of smooth muscle cells. Pfluegers Arch. 402, 18-23. Byerly, neurons

L., and Leung, in embryonic

H.-T (1988). Ionic currents of Drosophila cultures. 1. Neurosci. 8, 4379-4393.

Caldwell, P. C. (1970). Calcium chelation and Cellular Function, A. W. Cuthbert, tin’s Press), pp. 10-16. Changeux,

J.-P, and Revah,

F. (1987).

and buffers. In Calcium ed. (New York: St. MarThe acetylcholine

receptor

Calcium-Activated h

Potassium

molecule: allosteric IO, 245-250.

Channels

in Drosophila

sites and the ion channel.

Trends

Neurosci.

Elkins, T., Ganetzky, B., and Wu, C.-F. (1986). A Drosophila mutation that eliminates a calcium-dependent potassium current. Proc. Natl. Acad. Sci. USA 83, 8415-8419. Gho, M., and Mallart, A. (1986). Two distinct calcium-activated potassium currents in larval muscle fibers of Drosophila melanogaster. Pfluegers Arch. 407, 526-533. Goldman, D. E. (1943). Potential, impedance, membranes. J. Gen. Physiol. 27, 37-60.

and

rectification

in

Hamill, 0. P., Marty, A., Neher, E., Sackmann, B., and Sigworth, F. J. (1981). Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch. 397, 85-100.

Schmidt, J. W., and Catterall, W. A. (1986). Biosynthesis cessing of the a subunit of the voltage-sensitive sodium in rat brain neurons. Cell 46, 437-445.

and prochannel

Schwarz, T L., Tempel, B. L., Papazian, D. M., Jan, Y. N., and Jan, L. Y. (1988). Multiple potassium-channel components are produced by alternative splicing at the Shaker locus in Drosophila. Nature 337, 137-142. Schaefer, W. H., Hinrichsen, R. D., Burgess-Cassler, A., Kung, C., Blair, I. A., and Watterson, D. M. (1987’). A mutant Paramecium with a defectivecalcium-dependent potassium conductance has an altered calmodulin: a nonlethal selective alteration in calmodulin regulation. Proc. Natl. Acad. Sci. USA 84, 3931-3935. Singh, S., and Wu, C.-F. (1989). potassium currents in Drosophila.

Complete Neuron

separation of four 2, 1325-1329.

Haugland, F. N. (1987). A voltage clamp analysis of membrane potassium currents in larval muscle fibers of the Shaker mutants of Drosophila. Ph.D. thesis, University of Iowa, Iowa City, Iowa.

Smith, V. L., Doyle, K. E., Maune, J. F., Munjaal, R. P., and Beckingham, K. (1987). Structure and sequence of the Drosophila melanogaster calmodulin gene. J. Mol. Biol. 796, 471-485.

Haugland, K+ current

Sole, C. K., and Aldrich, channels in larval CNS 2556-2570.

F. N., and Wu, in Drosophila.

C.-F. (1986). Gene-dosage Biophys. J. 49, 168a.

effects

on a

R. W. (1988). Voltage-gated potassium neurons of Drosophila. J. Neurosci. 8,

Hinrichsen, R. D., Burgess-Cassler, A., Soltvedt, B. C., Hennessey, T, and Kung, C. (1986). Restoration by calmodulin of a Gas+dependent K’ current missing in a mutant of Paramecium. Science 232, 503-506.

Standen, N. B., Stanfield, P. R., Ward, T. A., and Wilson, S. W. (1984). A new preparation for recording single-channel currents from skeletal muscle. Proc. R. Sot. (Lond.) B 227, 455-464.

Hodgkin, A. L., and Katz, B. (1949). The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol. 108, 37-77.

Standen, of single colemma

Iverson, L. E., Tanouye, M. A., Lester, H. A., Davidson, N., and Rudy, B. (1988). A-type potassium channels expressed from Shaker locus cDNA. Proc. Natl. Acad. Sci. USA 85, 5723-5727.

Stewart, B., and Merriam, J. (1980). Dosage compensation. In The Genetics and Biology of Drosophila, Vol. 2d, M. Ashburner and T. R. F. Wright, eds. (London: Academic Press), pp. 107-140.

Kamb, A., Iverson, L. E., and Tanouye,hh characterization of Shaker, a Drosophila potassium channel. Cell 50, 405-413.

Tank, D. W., Wu, E.-S., and Webb, W. W. (1982). ular diffusibility in muscle membrane blebs: constraints. J. Cell Biol. 92, 207-212.

M.A. (1987). Molecular gene that encodes a

N. B., Stanfield, P. R., and Ward, T A. (1985). Properties potassium channels in vesicles formed from the sarof frog skeletal muscle. J. Physiol. 364, 339-358.

Enhanced molecrelease of lateral

Kamb, A., Tseng-Crank, J., and Tanouye, M. A. (1988). Multiple products of the Drosophila Shaker gene may contribute to potassium channel diversity. Neuron 7, 421-430.

Timpe, L. C., and Jan, L. Y. (1987). Gene dosage and complementation analysis of the Shaker locus in Drosophila. J. Neurosci. 1307-1317.

Katz, B. (1966). Hill).

Timpe, L. C., Schwarz, T L., Tempel, B. L., Papazian, Y. N., and Jan, L. Y. (1988). Expression of functional channels from Shaker cDNA in Xenopos oocytes. 143-145.

Nerve,

Muscle

and Synapse

(New

York:

McGraw-

Kemphues, K. J., Raff, E. C., Raff, R. A., and Kaufman, T. C. (1980). Mutation in a testis-specific B-tubulin in Drosophila: analysis of its effects on meiosis and map location of the gene. Cell 27, 445-451. Latorre, Varieties Physiol.

R., Oberhauser, A., Labarca, P., and Alvarez, 0. (1989). of calcium-activated potassium channels. Annu. Rev. 51, 385-399.

Levitan, I. B. (1988). Modulation other cells. Annu. Rev. Neurosci.

of ion channels 77, 119-136.

Meech, R. W. (1978). Calcium-dependent in nervous tissues. Annu. Rev. Biophys.

in neuron

and

potassium activation Bioeng. 7, 1-18.

Papazian, D. M., Schwarz, T L., Tempel, B. L., Jan, Y. N., and Jan, L. Y. (1987). Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila. Science 237, 749-753. Petersen, potassium 693-696.

0. H., and Maruyama, channels and their

role

Y. (1984). Calcium-activated in secretion. Nature 307,

Pongs, O., Kecskemethy, N., Mueller, R., Krah-Jentgens, mann, A., Kiltz, H. H., Canal, I., Llamazares, S., and (1988) Shaker encodes a family of putative potassium proteins in the nervous system of Drosophila. EMBO 1096.

I., BauFerrus, A. channel J. Z 1087-

Rudy, B. (1988). Diversity ence 25, 729-749.

Neurosdi-

Salkoff, L. (1983). sophila potassium Biol. 48, 221-231.

and

ubiquity

of K channels.

Genetic and voltage-clamp analysis of a Drochannel. Cold Spring Harbor Symp. Quant.

Salkoff, L., and Wyman, R. (1981). Genetic sium channels in Drosophila Shaker 228-230.

modification of potasmutants. Nature 293,

Wei, A., and Salkoff, L. (1986). Occult Drosophila nels and twinning of calcium and voltage-activated channels. Science 233, 780-782. Witzemann, V, Barg, (1989). Developmental encoding acetylcholine Lett. 242, 419-424.

B., Criado, regulation receptor

Z

D. M., Jan, potassium Nature 337,

calcium chanpotassium

M., Stein, E., and Sakmann, B. of five subunit specific mRNAs subtypes in rat muscle. FEBS

Wu, C.-F., and Haugland, F. N. (1985). Voltage-clamp analysis of membrane currents in larval muscle fibers of Drosophila: alteration of potassium currents in Shaker mutants. J. Neurosci. 5, 2626-2640. Wu, C-F., Ganetzky, Potassium currents fected by mutations

B., Haugland, in Drosophila: of two genes.

F. N., and Liu, A.-X. (1983). different components afScience 220, 1076-1078.

Wu, C.-F., Tsai, M.-C., Chen, M.-L., Zhong, Y., Singh, S., and Lee, C. Y. (1989). Actions of dendrotoxin on K+ channels and neuromuscular transmission in Drosophila melanogaster, and its effects in synergy with K+ channel-specific drugs and mutations. J. Exp. Biol., in press. Yamamoto, D., and Suzuki, are responsible for single ethylammonium ions. Am.

N. (1989). K channel J. Physiol.

Two distinct mechanisms block by internal tetra256, C683-C687.

Yamanaka, M. K., Saugstad, J. A., Hanson-Painton, O., McCarthy, B. J., and Tobin, S. L. (1987). Structure and expression of the Drosophila calmodulin gene. Nucl. Acids Res. 75, 3335-3348. Zagotta, W. N., Brainard, M. S., and Aldrich, R. W. (1988). Singlechannel analysis of four distinct classes of potassium channels in Drosophila muscle. J. Neurosci. 8, 4765-4779.