Auxiliary Hyperkinetic beta  Subunit of K+ Channels: Regulation of Firing Properties and K+ Currents in Drosophila Neurons

Wei-Dong Yao and Chun-Fang Wu

Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242


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Yao, Wei-Dong and Chun-Fang Wu. Auxiliary Hyperkinetic beta  subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons. Molecular analysis and heterologous expression have shown that K+ channel beta  subunits regulate the properties of the pore-forming alpha  subunits, although how they influence neuronal K+ currents and excitability remains to be explored. We studied cultured Drosophila "giant" neurons derived from mutants of the Hyperkinetic (Hk) gene, which codes for a K+ channel beta  subunit. Whole cell patch-clamp recording revealed broadened action potentials and, more strikingly, persistent rhythmic spontaneous activities in a portion of mutant neurons. Voltage-clamp analysis demonstrated extensive alterations in the kinetics and voltage dependence of K+ current activation and inactivation, especially at subthreshold membrane potentials, suggesting a role in regulating the quiescent state of neurons that are capable of tonic firing. Altered sensitivity of Hk currents to classical K+ channel blockers (4-aminopyridine, alpha -dendrotoxin, and TEA) indicated that Hk mutations modify interactions between voltage-activated K+ channels and these pharmacological probes, apparently by changing both the intra- and extracellular regions of the channel pore. Correlation of voltage- and current-clamp data from the same cells indicated that Hk mutations affect not only the persistently active neurons, but also other neuronal categories. Shaker (Sh) mutations, which alter K+ channel alpha  subunits, increased neuronal excitability but did not cause the robust spontaneous activity characteristic of some Hk neurons. Significantly, Hk Sh double mutants were indistinguishable from Sh single mutants, implying that the rhythmic Hk firing pattern is conferred by intact Shalpha subunits in a distinct neuronal subpopulation. Our results suggest that alterations in beta  subunit regulation, rather than elimination or addition of alpha  subunits, may cause striking modifications in the excitability state of neurons, which may be important for complex neuronal function and plasticity.


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INTRODUCTION
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The proper functioning of the nervous system depends on a large variety of neurons displaying distinct excitability patterns. The firing properties of neurons are regulated by potassium channels (Hille 1992; Rudy 1988), which represent one of the most diverse groups of ion channels. K+ channels have been implicated in activity-dependent neural plasticity, including learning in Aplysia (Byrne and Kandel 1996) and habituation in a defined neural circuit in Drosophila (Engel and Wu 1998). Screening for leg-shaking mutants in Drosophila has led to the identification of a number of behavioral genes (Kaplan and Trout 1969), such as Shaker (Sh) and ether a go-go (eag), which encode different membrane-spanning, pore-forming alpha  subunits of K+ channels (Kamb et al. 1988; Pongs et al. 1988; Schwarz et al. 1988; Warmke et al. 1991), and Hyperkinetic (Hk), which recently was identified as a cytoplasmic auxiliary beta  subunit (Chouinard et al. 1995). Homo- and hetero-multimeric assembly among Sh splicing variants provides one mechanism for generating diverse K+ channels (Christie et al. 1990; Haugland and Wu 1990; Isacoff et al. 1990; McComack et al. 1990; Ruppersberg et al. 1990; Wu and Ganetzky 1986, 1988). Heteromultimeric interactions of K+ channel alpha  subunits across different families, e.g., Sh, Slo, and Eag, could contribute further to structural and functional diversity (Chen et al. 1996; Schopperle et al. 1998; Wu and Chen 1995; Zhong and Wu 1991, 1993).

Conceivably, association of beta  subunits with different alpha  subunits can contribute further to channel functional diversity. The structural complexity of voltage-gated Na+, Ca2+, and K+ channels is enhanced by the auxiliary or beta  subunits, which are cytoplasmic proteins in tight association with alpha  subunits (Catterall 1991, 1992; Isom et al. 1994). For K+ channels, at least four subtypes of beta  subunit polypeptides, Kvbeta 1, Kvbeta 2, Kvbeta 3, and Kvbeta 4, have been identified biochemically and molecularly in mammalian species, which are associated with specific alpha  subunits (England et al. 1995; Fink et al. 1996; Majumder et al. 1995; McCormack et al. 1995; Morales et al. 1995; Nakahira et al. 1996; Parcej and Dolly 1989; Scott et al. 1994; Sewing et al. 1996; Yu et al. 1996). Extensive in vitro studies using heterologous coexpression with alpha  subunits have demonstrated that beta  subunits regulate surface expression of alpha  subunits (Accili et al. 1997b; Shi et al. 1996) and their inactivation kinetics and voltage sensitivity (Chouinard et al. 1995; Rettig et al. 1994). Furthermore, interaction among different types of beta  subunits potentially generates additional complexity in K+ channel properties (Accili et al. 1997a; Xu and Li 1997).

Although the regulatory role of K+ channel beta  subunits has been well established in heterologous expression systems, the physiological consequences of beta -subunit regulation have not been determined in neurons. How are K+ currents in native neurons modulated by the beta  subunit? What are the functional consequences of such modulation as reflected in excitability and firing patterns? Are neurons of different functional categories differentially regulated by the beta  subunits? Such questions are largely unanswered, and exploring these issues can provide insights into the underlying molecular and cellular basis.

Defects in non-pore-forming auxiliary subunits of Na+ and Ca2+ channels are responsible for certain types of mammalian epileptic disorders (Burgess et al. 1997; Letts et al. 1998; Wallace et al. 1998). Mutations of the K+ channel beta  subunit may further our understanding of certain neurological conditions and behavioral defects. Drosophila Hk mutants provide a unique opportunity for defining the in vivo function of a K+ channel beta  subunit in neurons to bridge the gap between the molecular interactions (Chouinard et al. 1995) and altered neuronal activity (Ikeda and Kaplan 1970; Stern and Ganetzky 1989; Yao and Wu 1995) responsible for behavioral abnormalities including leg shaking (Kaplan and Trout 1969) and abnormal locomotion in larvae (Wang et al. 1997). Moreover, K+ channel mutations have been shown to affect the habituation process of the jump-and-flight escape response in a defined circuit, and interestingly the effect of altering Hkbeta subunits is far more extreme than altering the alpha  subunits such as Sh and Slo (Engel and Wu 1998). In Drosophila, the in vivo function of the K+ channel alpha  subunits encoded by the Sh, eag, and slo genes has been well characterized in neuromuscular junctions (Ganetzky and Wu 1982; Jan et al. 1977), muscle fibers (Elkins et al. 1986; Salkoff 1983; Salkoff and Wyman 1981; Singh and Wu 1989; Wu and Haugland 1985; Wu et al. 1983; Zhong and Wu 1993), and neurons (Baker and Salkoff 1990; Saito and Wu 1991, 1993; Tanouye et al. 1981). Therefore it is desirable and also feasible to study the interaction between Hkbeta and specific alpha  subunits in excitable cells of the corresponding double mutants, such as Hk Sh.

We employed cultured Drosophila giant neurons (Wu et al. 1990), which enabled voltage- and current-clamp studies on the same cells (Saito and Wu 1991; Zhao and Wu 1997), to delineate the Hk phenotype in terms of neuronal excitability and firing patterns and to characterize the modulation of K+ current properties through the interactions of Hkbeta with alpha  subunits. Some of the results have previously been presented in abstract form (Yao and Wu 1995).


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Fly stocks

All fly stocks were maintained at 20-22°C on standard Drosophila media. The Canton S strain was used as wild-type control in all experiments. Three mutant alleles, Hk0, Hk1, and Hk4 were used. Among them, HkIE18, a deletion mutant designated as Hk0 in this paper, was kindly provided by Dr. B. Ganetzky; Hk1, induced by ethyl methanesulfonate, is likely a point mutation. Hk4 was isolated in an X-ray-irradiation screen (by R. Kreber in Ganetzky's lab) and has been characterized in behavioral studies (Wang et al. 1997). The Sh mutants, ShM and ShKS133 (Sh133), were from the collection of Dr. S. Benzer. The double mutants Hk1ShM and Hk1Sh133 were both generated in the authors' laboratory.

Cell culture

The procedure for culturing Drosophila giant neurons has been described previously (Saito and Wu 1991; Wu et al. 1990). Briefly, embryos at the early gastrulation stage were homogenized in modified Schneider medium (GIBCO, Grand Island, NJ) containing 200 ng/ml insulin (Sigma, St. Louis, MO), 20% fetal bovine serum (FBS), 50 µg/ml streptomycin, and 50 U/ml penicillin. Cells were washed two times in the above medium and resuspended in medium containing 2 µg/ml cytochalasin B (Sigma) and then plated on glass coverslips. Cultures were maintained in humidified chambers at room temperature (20-25°C) for 2-5 days before recordings.

Electrophysiology

Whole cell patch-clamp recording was described previously (Saito and Wu 1991; Zhao and Wu 1997). Recording electrodes were prepared from 75 µl glass micropipettes (VWR Scientific, Chicago, IL). The tip opening of the electrodes had a diameter of ~1.2 µm and an input resistance of 3-5 MOmega in bath solutions. The bath solution contained (in mM) 128 NaCl, 2 KCl, 4 MgCl2, 1.8 CaCl2, and 35.5 sucrose, buffered with 5 mM HEPES at pH 7.1-7.2. Patch pipettes were filled with a solution containing (in mM) 144 KCl, 1.0 MgCl2, 0.5 CaCl2, and 5.0 EGTA, buffered with 10 mM HEPES, pH 7.1-7.2. Recordings were performed primarily on the neuronal soma with diameters ranging from 10 to 25 µm, using a patch-clamp amplifier (Axopatch 1B, Axon Instruments, Foster City, CA). The seal resistance was usually >10 GOmega . Junction potentials were nulled before the establishment of the whole cell configuration. All recordings were made at room temperature.

An IBM 386 PC computer in conjunction with an A/D and a D/A converter and pClamp software (version 5.5.1, Axon Instruments) was used to generate the current and voltage commands and data acquisition. During current-clamp experiments, the membrane potential was maintained at or near the resting level through constant current injection. The majority of cells did not require current injection, whereas in some cells, the resting membrane potential varied slowly and required readjustments of the amplitude of current injected within a period of several minutes. Cells that showed significant drift (>15% in 2 min) were discarded. In voltage-clamp studies, the membrane was held at -80 mV. Voltage and current data were filtered at 1-2 kHz (4-pole Bessel filter) and digitized at 1-10 kHz. Recordings of spontaneous neuronal activity were stored in an FM tape recorder (Store 4, Lockheed Electronics, Plainfield, NJ) for later analysis. A series of four pulses from -80 to -75 mV were applied and the resultant currents were used to determine, and to compensate for, the series resistance and whole cell capacitance. Membrane currents as a result of leakage were subtracted digitally off-line. All data analysis was performed using pClamp 5.5, Excel 4.0 (Microsoft, Redmond, WA), AxoGraph 2.0 (Axon Instruments), Systat 5.2 (SYSTAT, Evanston, IL), and Superpaint 2.0 (Aldus, San Diego, CA) software.


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Spontaneous firing in Hk neurons

We performed whole cell current-clamp recordings from isolated giant neurons in culture. These multinucleated neurons, derived from cytokinesis-arrested embryonic neuroblasts, develop a variety of neuronal phenotypes, including characteristic arborization patterns (Wu et al. 1990), neurotransmitters (Huff et al. 1989), and ionic currents and firing properties (Saito and Wu 1991, 1993; Zhao and Wu 1997). The preserved morphological and functional diversity allowed an assessment of beta  subunit function in isolated CNS neurons of different functional categories. We studied neurons isolated from wild type and three alleles of Hk (Hk0, a null mutation, and Hk1 and Hk4, two hypomorph mutations, see METHODS).

Figure 1 compares the spontaneous activities for wild-type and Hk neurons. An immediate hallmark of the Hk cells was a significant increase in the sustained activities of spontaneous spikes (maintained for minutes and often for the entire experiment period), which were rarely seen in wild-type neurons. Other spontaneous activities, including sporadic firing and bursts of spikes terminated by a plateau potential, were found in both wild-type and Hk cultures. The majority of wild-type neurons were quiescent, showing no activity at resting levels. The proportion of quiescent cells was reduced in Hk cultures, primarily due to an increase in persistent rhythmic firing. Among the three categories of spontaneous activity, nonrhythmic "sporadic" activity was observed in 6.3% of wild-type neurons versus 7.5, 8.6, and 6.6% of Hk0, Hk1, and Hk4 neurons, respectively; "plateau" potentials, developed from occasional bursts of spikes, were observed in 2.1% of wild-type neurons versus 2.5, 0.0, and 4.9% of Hk0, Hk1, and Hk4 neurons, respectively. Significantly, only 3.6% of wild-type cells, in contrast to 20.0, 15.7 and 13.4% of Hk0, Hk1, and Hk4 neurons, displayed persistent rhythmic firing (Fig. 1). Such cells in wild-type cultures might correspond to the endogenous pace-making cells expected to be present in the normal CNS. In addition, the variety of spontaneous activity patterns observed in the heterogeneous giant neuron culture system might reflect activities of the diverse cell types previously described in the nervous system of Drosophila (Fischbach and Dittrich 1989; Heisenberg and Wolf 1984) and may involve differential expression of and interplay between multiple channel proteins (see DISCUSSION).



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Fig. 1. Spontaneous activities in cultured wild-type and Hyperkinetic (Hk) neurons. A: 3 categories of spontaneous firing patterns. "Sporadic" refers to occasional spike activity or cluster of spikes lasting for seconds; "plateau" refers to bursts of spike activity that turned into sustained depolarization; "persistent" refers to long-lasting rhythmic spike activity that persisted for minutes and often for the entire period of the experiment (>10 min). Responses were recorded under whole cell current-clamp conditions from somata of isolated neurons. Resting membrane potential, as indicated, was measured at 0 current injection and represented the starting levels of the membrane potential. B: distribution of the 3 spontaneously active cell categories in wild type (WT) and 3 alleles of Hk mutants. Quiescent cells (not shown) accounted for the remaining fraction of the total population in both the wild-type and the Hk cultures. More cells displaying persistent rhythmic firing (), but not sporadic () or plateau () spontaneous firing, were observed in Hk mutant cultures. Hk0 is a deletion mutant, and its phenotype appeared more extreme than Hk1 and Hk4. In this and the following figures, numbers in the parentheses indicate the sample size. One-sample binomial tests (Rosner 1982) for the percentage differences between WT and Hk lines within the sporadic, plateau, and persistent categories indicate that only the percentage of cells displaying persistent firing was significantly different between each Hk alleles and WT (P < 0.01).

On the basis of the spike waveform, two patterns of persistent spontaneous firing can be recognized in Hk neurons (Fig. 2). Class I neurons (16.3, 12.8, and 9.7% in Hk0, Hk1, and Hk4, respectively) showed regular all-or-none action potentials with varying prepotential, spike duration, and hyperpolarizing afterpotential. Class II neurons (3.8, 2.9, and 3.7% in Hk0, Hk1, and Hk4, respectively), a smaller population of cells, were characterized by repetitive bursts of spikes riding on depolarizing potentials. It should be noted that Class II firing appears to be Hk-specific because it was not seen in wild type and in other mutant genotypes (see Sh and Hk Sh in the following text). Notably, the two classes of firing patterns could not be interconverted by simple current injections but in certain cases could be converted between each other by blocking Na+ channel or Ca2+ channels (data not shown), suggesting that Class I and II neurons are indeed physiologically different.



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Fig. 2. Patterns of persistent, rhythmic activity in Hk neurons. Right: spike waveforms for the 2 classes (I and II) are displayed at a faster time base.

The results suggest that the Hkbeta subunit is important in regulating the quiescent state of CNS neurons. Intriguingly, among the various cell types found in wild-type cultures, only the persistent rhythmic activity was enhanced by Hk mutations (Fig. 1), apparently at the expense of converting a subpopulation of quiescent cells. A further question would be whether the mutational effect, indicative of the expression of Hkbeta subunits, is restricted to this category of neurons only. Additional observations suggested that, although the robust spontaneous firing clearly identifies a category of neurons in which Hkbeta subunits play a distinct functional role, it is not the sole category affected by Hk mutations (see following text).

Increased action potential duration in Hk neurons

The functional role of beta  subunits in neuronal firing properties was examined further by step current injection in neurons with and without spontaneous activities. Three types of voltage responses, all-or-none, graded, and nonregenerative (Saito and Wu 1991), were observed for both wild-type and Hk neurons. Action potential duration was determined in neurons displaying all-or-none spikes. As shown in Fig. 3 and Table 1, action potentials were broadened in some Hk neurons. The broadening of action potentials was variable, to ~10-fold in duration, as exemplified in Fig. 3A. It is worth noting that the percentage of Hk neurons displaying spontaneous activities was not significantly different from the ensemble population of neurons for each Hk allele examined (Fig. 3B). Furthermore, some quiescent Hk cells also showed prolonged action potentials in response to current injection.



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Fig. 3. Broadening of action potentials in Hk neurons. A: sample current-clamp traces from 1 WT and 3 Hk0 neurons illustrating varying degrees of increase in action potential duration in Hk cells. Action potentials were evoked by current injection (50 pA). B: histograms comparing the distribution of action potential duration in WT and 3 alleles of Hk and in a population of Hk cells that showed spontaneous activity (Hk Per. spont.). Action potential duration was determined as the width at the voltage of the inflection point during take-off. Notice that a subpopulation of Hk neurons showed increased spike duration, which resulted in significant differences in the variance between Hk alleles and WT (P < 0.0001, F-tests).


                              
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Table 1. Comparison of membrane properties between wild-type and Hyperkinetic neurons

Alterations in K+ currents in Hk neurons

The effect of the beta  subunit on neuronal excitability as revealed in different alleles of Hk mutants suggests modifications of the underlying K+ currents in mutant neurons. Voltage-activated K+ currents were examined in saline containing TTX and Cd2+ to eliminate the inward Na+ and Ca2+ currents as well as outward Ca2+-activated K+ currents (Saito and Wu 1991). We studied K+ current properties in large samples of neurons from wild type and three alleles of Hk. The different mutant alleles showed consistent defects in K+ currents, although the deletion mutant, Hk0, was the most extreme. For simplicity, only Hk0 is illustrated in this section while results on the other two alleles are summarized in Table 1.

As shown in Fig. 4, the voltage-activated outward K+ currents were affected substantially in Hk0. Apparently both the peak and sustained currents were reduced by Hk mutations as indicated by current density measured at the peak and at 700 ms after the onset of the depolarization step (the end of the pulse). More significantly, the reduction in K+ currents was not uniform over all membrane potentials but was proportionally more pronounced at lower potentials near the spike threshold (Fig. 4, inset). In addition, the kinetics of K+ current activation also were affected in Hk neurons. The rise time of K+ currents, measured from the onset of the depolarization steps to the peak, was slower in Hk (Fig. 5, A and B). Notably, the effect was again more pronounced at lower voltages (Fig. 5A, compare between -20 and +20 mV; and Fig. 5B, top). The results suggest that Hkbeta subunits modulate multiple aspects of K+ channel activation especially at lower levels of depolarization, where spike initiation is critically controlled by K+ currents.



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Fig. 4. Reduced peak and sustained K+ currents in populations of neurons from the Hk0 allele. Current density (normalized to membrane capacitance in pA/pF) at the peak (A) and at the end of the 700-ms pulse (B) was reduced in the total populations of Hk0 neurons surveyed. Proportion of reduction in peak current in Hk was not uniform, being more prominent at lower membrane potentials, as shown in the inset. Peak current densities for Hk0 are normalized to the corresponding values for WT, which is shown as a horizontal line of 1.0. For voltage-clamp experiments described in this and the following figures, TTX and Cd2+ were introduced in saline to eliminate the inward Na+ and Ca2+ currents and the outward Ca2+-activated K+ currents. Remaining voltage-activated K+ currents were elicited by depolarization steps (700 or 950 ms in some experiments) from a holding potential of -80 mV to voltages between -60 and +20 mV in equal spacing. Data are presented as the means ± SE for the number of cells indicated in parentheses. For this and the following figures, error bars indicate SE (not shown if smaller than the symbol size).



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Fig. 5. Slower rise and decay of K+ currents in Hk neurons. A: sample currents from a Hk0 and a WT neuron elicited by depolarization to -20 and +20 mV from a holding potential of -80 mV. Note that differences were more prominent at -20 than +20 mV. Traces are normalized and superimposed for comparison between Hk and WT. B: time to peak (top) and the fast decay time constant tau 1 (bottom) as a function of membrane potential. Time to peak was measured from the onset of voltage commands to the peak of the current response. Decay time constants were determined by fitting the decay phase of current traces with the equation I = A1 exp(-t/tau 1) + A2 exp(-t/tau 2) + I0, where tau 1 and tau 2 are the fast and slow decay time constants for the two components A1 and A2, and I0 the baseline current. In some cells, a single exponential (tau 1) is sufficient to fit the decay (A2 = 0). Each data point represents mean ± SE for the total number of cells indicated in parentheses. In some cells, the measurements at certain voltages were not available because of the small current amplitude (mostly at -40 mV). C: distribution of the fast decay time constant at -20 mV in WT and Hk0. Reduction in the percentage of Hk cells displaying faster decay (tau 1 < 100 ms, region I) apparently correlated with an increase in a population showing slower decay (100 ms < tau 1 < 800 ms, region II).

Interestingly, the effects of beta  subunit mutations on K+ current inactivation again varied with membrane potential. The decay phase of the K+ currents in these cultured neurons could be fitted with one or two exponential plus a baseline current (Zhao et al. 1995). The time constant of the exponential decay (or of the faster component if double exponential approximation was required to fit the decay) was examined for indications of altered rates of inactivation. We found that the decay of K+ currents was slower in Hk neurons (Fig. 5).

Distribution of the time constant in Fig. 5C indicated that Hk mutations affect K+ currents with faster decay (tau 1 < 100 ms, region I). There was a decrease in the population of cells showing faster decay (region I) and a corresponding increase in the number of neurons displaying slower decay (region II but not in region III). The proportions of cells in regions I, II, and III are 49, 38, and 13% for wild type and 28, 57, and 15% for Hk0 [Fig. 5C; the percentage differences for regions I and II, but not III, are significant (P < 0.0001) under 1-sample binomial tests]. The preferential effects of Hk mutations on faster decaying currents are consistent with the idea that Hkbeta interacts mainly with the alpha  subunits of certain fast-inactivating channels. Among the K+ channel alpha  subunits expressed in Drosophila neurons, Sh subunits are thought to be associated preferentially with channels showing faster inactivation (Baker and Salkoff 1990). The slower decay of K+ currents observed at the whole cell level in Hk neurons may not simply represent a slower inactivation process because a convolution of a delay in channel opening, which has been demonstrated at the single-channel level in some Sh mutants (Zagotta et al. 1989), and a slowed transition from the open to inactivated state (Wang and Wu 1996) could lead to similar results.

The parameters for steady-state inactivation and recovery from inactivation also were determined in wild-type and mutant neurons. The voltage for 50% inactivation (Vh1/2) of the transient component was slightly shifted to positive potentials in Hk mutants (Table 1). Figure 6 shows that recovery of the transient component from inactivation was significantly slower in Hk0. The recovery process could be approximated by two exponential processes (cf. Wang and Wu 1996; Wu and Haugland 1985; Zhao et al. 1995) with time constants of 90 and 300 ms for wild type versus 170 and 700 ms for Hk0. The time required for 50% recovery from inactivation was 112.8 ms in wild-type neurons versus 206.7, 353.0, and 212.1 ms for Hk0, Hk1, and Hk4 neurons, respectively.



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Fig. 6. Slower recovery from inactivation of transient currents in Hk mutant neurons. A: representative examples of superimposed traces showing the recovery of transient K+ currents in WT and Hk0. Recovery from inactivation was determined by 2 identical pulses to +20 mV from a holding potential of -80 mV for 250 ms with varying interpulse intervals. B: comparison of the recovery time course in WT and Hk neurons. Fraction of recovery (r) was calculated using r = (Ip2 - IS)/(Ip1 - IS) where Ip1 and IP2 are the peak current elicited by the 1st and 2nd pulse, respectively, and IS the steady-state current remaining at the end of the 1st pulse. Each data point represents Mean ± SEM for the number of cells indicated in parentheses. The curves represent double-exponential fit to the average data, r = r1[1 - exp(-t/tau 1)] + r2[1 - exp(-t/tau 2)], where r1, r2 and tau 1 and tau 2 stand for the proportions of the maximal attainable fast and slow components and their recovery time constants, respectively. Values for the curves shown are 0.6, 0.4, 90, and 300 ms for WT and 0.6, 0.4, 170, and 700 ms for Hk0, respectively. Note that Hk cells showing persistent spontaneous activities (Hk Per. Spont.; 2 Hk1 and 2 Hk0 neurons were combined) were no more extreme than the whole Hk populations.

Reduced sensitivity of Hk K+ channel to blockade by neurotoxins

It can be concluded that the cytoplasmic Hkbeta subunit is important in the regulation of the amplitude, voltage dependence, and kinetics of voltage-activated K+ currents. Such regulation may involve modulation of the channel conformation. We examined how mutations of the Hkbeta subunit would affect the interaction of pharmacological agents with membrane-spanning alpha  subunits for evidence of conformation changes. Sensitivity to 4-aminopyridine (4-AP), a routinely used A-type channel blocker (Hille 1992; Rudy 1988), was examined in both wild-type and Hk neurons. As in other Drosophila preparations, 4-AP inhibits inactivating currents in both muscle (Haugland 1987; Wang and Wu 1996) and neurons (Zhao et al. 1995). We used the difference between the peak and sustained components to indicate the amplitude of inactivating current. We found that Hk currents were less sensitive to 4-AP, as 0.5 mM 4-AP blocked ~52% of inactivating currents in wild type but only 21% in Hk0 and 11% in Hk1 (Fig. 7, A and D). The altered inhibition implies that functional beta  subunits are necessary to confer normal 4-AP sensitivity and supports the idea that Hkbeta subunits influence the conformation in the cytoplasmic pore region of certain alpha  subunits (Fig. 7D, inset) (cf. Wang and Wu 1996), in which the 4-AP binding site is thought to reside (Kirsch et al. 1993; McCormack et al. 1991; Yao and Tseng 1994).



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Fig. 7. Reduced sensitivity to K+ channel blockers in Hk neurons. A-C: reduced sensitivity to 4-aminopyridine (4-AP; A), alpha -dendrotoxin (DTX; B), and TEA (C). K+ currents were activated by depolarization to +20 mV from a holding potential of -80 mV. Transient currents (1) were preferentially inhibited (2) by bath applications of 0.5 mM 4-AP (A) and 100 nM alpha -DTX (B), whereas both transient and sustained currents were inhibited by 5 mM TEA (C). Note that in Hk cells, the transient currents were more resistant to the 4-AP and alpha -DTX, and only the sustained currents were inhibited by TEA in Hk. Effects of 4-AP, but not alpha -DTX, were reversed by wash (A3). D: comparison of 4-AP, alpha -DTX, and TEA effects on WT and Hk. Sensitivity of the inactivating currents (the difference between peak and sustained currents) to 3 drugs was altered significantly by Hk mutations. Fractional reduction was measured as 1 - [(Ip2 - Is2)/(Ip1 - Is1)], where Ip and Is are the peak and sustained currents, and subscripts 1 and 2 correspond to currents before and after treatment (shown in A, B, and C), respectively. Inset: sites on the alpha  subunit that are thought to be involved in 4-AP, alpha -DTX, and TEA binding as well as in beta  subunit association. * P < 0.05, ** P < 0.01, Student's t-test.

The K+ channel beta  subunit initially was identified by copurification with the alpha  subunits by using dendrotoxin (DTX) (Pacej and Dolly 1989; Scott et al. 1994). We examined how Hk mutations alter K+ current sensitivity to alpha -DTX, which inhibits certain fast-inactivating voltage-dependent K+ channels in vertebrate neurons (Dolly et al. 1984; Halliwell et al. 1986). We found that in Drosophila neurons the toxin inhibited preferentially the transient component, leaving the steady-state current unchanged (Fig. 7, B and D). At 100 nM, the suppression was 21% for wild type, 1% for Hk0 and Hk1, and 10% for Hk4. Unlike the 4-AP effect (Fig. 7A), the suppression of K+ currents by alpha -DTX was not reversible after washing, consistent with a tight association of alpha -DTX with the K+ channels. Because the binding sites of the toxin have been mapped to a region on the alpha  subunit near the outer mouth of the channel pore (Hurst et al. 1991) (Fig. 7D, inset), the data suggested that the cytoplasmic beta  subunit exerts transmembrane influence on channel conformation in the extracellular pore region as well.

We found that Hk mutations also affect the sensitivity of K+ currents to a broad-spectrum blocker, tetraethylammonium (TEA). A variety of K+ channels, both inactivating and noninactivating, have been shown to be inhibited by TEA (Hille 1992; Rudy 1988). Externally applied TEA blocks K+ channels by binding to the extracellular mouth region of the pore (Fig. 7D, inset) (Choi et al. 1991; Grissmer and Cahalan 1989). Our results showed that TEA significantly reduced both the peak and sustained currents in Drosophila neurons. At 5 mM, TEA inhibited 22% of the peak and 24% of the sustained currents in wild type. In contrast, the same concentration of TEA did not affect peak currents in Hk neurons but blocked sustained currents to an extent similar to that in wild type (19% for Hk0 and 15% for Hk1). Thus the net effect of TEA on Hk current was an apparent increase, rather than a decrease, of the inactivating current component (operationally defined as the difference between the peak and sustained currents). Apparently, the sensitivity of slower decaying currents to TEA was not significantly altered whereas the fast decaying components became clearly less sensitive in Hk mutant neurons.

It should be noted that TEA, as well as 4-AP and alpha -DTX, did not seem to alter either the fast or the slow decay time constant required for fitting the decay process but merely changed the relative proportion of the two components (i.e., the same 2 time constants could be used to fit the current decay before and after drug treatment; data not shown). The altered pharmacological profiles of Hk mutant channels support the idea that Hkbeta subunits influence the function of K+ channels by modulating channel conformation, which is detectable by pharmacological probes to specific regions of the alpha  subunits.

Correlation of K+ current properties with firing patterns: effects of Hk on functionally distinct cell types

As stated earlier, the robust phenotype of persistent spontaneous activity can be used as an indicator for identifying neurons that are affected by Hk mutations. However, the extensive effects of Hk on K+ currents demonstrated by voltage-clamp analysis of the entire neuronal population imply that not only the cells capable of spontaneous firing are affected. To examine how Hkbeta subunits function in various cell types, we performed both current-and voltage-clamp experiments on the same neuron to correlate the underlying K+ current with different excitability properties. Current clamping was performed first in normal physiological saline and followed by voltage-clamp recording in solution containing TTX and Cd2+. As shown in Fig. 8, a great majority of Hk neurons examined in this manner could be divided into three types: cells with persistent rhythmic spontaneous activity (Per. spont.; cf. Fig. 1, persistent), cells that were quiescent but exhibited all-or-none action potentials in response to current injection (All-or-none), and quiescent cells unable to fire all-or-none action potentials (Graded). Because of the rare occurrence (<4%) of wild-type cells displaying rhythmic spontaneous firing, a satisfactory sample was not obtained to determine the current properties of this cell category in wild-type cultures. Furthermore, cells with spontaneous activities of sporadic spikes or plateau potentials constituted a small population in both Hk and wild-type cultures (Fig. 1B) and were excluded from the analysis.



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Fig. 8. Voltage- and current-clamp correlation from the same neurons with distinct excitability patterns. Cell populations were divided into 3 types: persistent spontaneous (Per. spont.) type includes cells with persistent rhythmic spontaneous activity; the all-or-none type consists of cells not capable of spontaneous firing but exhibiting all-or-none action potentials in response to current injection; the graded type includes quiescent cells unable to fire all-or-none action potentials. Current- and voltage-clamp recording from the same neuron correlated the firing properties with the underlying K+ currents. Note the slower decay of K+ currents in the spontaneously firing Hk cells. For this and the next figure, Hk neurons from different alleles were combined according to firing properties. Resting membrane potentials, obtained at 0 current injection, are indicated.

As shown in Fig. 9A, a reduction in current density was observed unexpectedly in all three types of Hk neurons. Furthermore the density for either the peak or the sustained K+ current was not significantly different among the three types of Hk neurons, with or without spontaneous activities (1-way ANOVA tests, see Fig. 9). Therefore Hk mutations appear to affect different cell types with distinct excitability properties. This result also suggests that simple reduction in K+ current amplitude alone is not sufficient to account for the abnormal spontaneous firing: additional explanations may include altered kinetic properties of K+ currents (see following text). Furthermore a complete account for the effect of Hk mutations on the firing pattern in a given neuron also should include voltage-clamp examinations of the underlying inward (e.g., Ca2+ and Na+) and nonvoltage-dependent K+ (e.g., Ca2+-activated K+) currents.



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Fig. 9. K+ currents were altered by Hk mutations in functionally different cell types. A: both the peak and sustained current densities were reduced in Hk cells (P < 0.05 in all 2-way ANOVA tests for WT vs. Hk, data nested according to the grouping of voltage-clamp properties, i.e., either the peak or sustained currents, or according to the grouping of current-clamp properties, i.e., either the all-or-none or graded type). However, K+ currents did not differ significantly among neuronal categories of distinct types of firing properties within WT (P > 0.05 for both the peak and sustained current densities, Student's t-test) and within Hk cultures (F test for 1-way ANOVA. For peak current density, F(2, 34) = 0.31, P > 0.05; for sustained current density, F(2, 34) = 2.01, P > 0.05). Bar graphs represent the mean ± SE of current density (Vm = +20 mV) for the number of cells indicated in parentheses. B: histograms comparing the decay time constant, tau 1, between WT and Hk neurons of different firing types. Hk cells showing persistent rhythmic spontaneous activity appeared to express K+ currents with slower decay kinetics (Vm = +20 mV).

As shown in the preceding text, spontaneously active and quiescent Hk neurons could not be distinguished by the extent of reduction in current density (Fig. 9A) nor by action potential broadening (Fig. 3). Furthermore, the slowed recovery process of K+ currents in Hk cells with (confirmed) spontaneous activity was no more severe than those of the overall Hk0 population (Fig. 6). Nevertheless, the decay time constant appeared to show more extreme alterations in Hk cells with persistent spontaneous activities. Figure 9B compares the distribution of decay time constant in the three cell types between wild type and Hk. There appear to be no obvious differences among cells of the graded and all-or-none types in both Hk and wild type. However, Hk neurons that displayed persistent spontaneous activity deviated from other types by showing an increased proportion of cells with slower K+ current decay (tau  > 100 ms, Fig. 9B). Even though Hk mutations affect several aspects of K+ currents in multiple cell categories, the demonstrable enhancement in deceleration of the current decay process associated with persistently active Hk cells suggests interaction between Hkbeta and a particular type of alpha  subunit, rendering robust rhythmic activities in a subpopulation of neurons.

Role of Sh in mediating Hk mutant phenotypes

Hkbeta has been shown to interact with Shalpha , which confers a fast-inactivating K+ current IA in Drosophila larval muscle (Wang and Wu 1996) as well as in a heterologous expression system (Chouinard et al. 1995). We analyzed the functional consequences of interaction between Hkbeta subunits and Shalpha subunits in cultured central neurons.

Spontaneous nerve activities were compared for wild type, Hk (Hk0 and Hk1) and Sh (ShM) single mutants and Hk Sh (Hk1 ShM and Hk1 Sh133) double mutants. ShM is a null allele (Iverson and Rudy 1990; Zhao et al. 1995) and Sh133 an antimorphic allele (Haugland and Wu 1987; Tanouye et al. 1986). Each single mutant and double mutant exhibited higher excitability with characteristic spontaneous activities as described earlier (Fig. 1). As shown in Fig. 10A, the total percentage of spontaneously active ShM neurons was similar to that seen in Hk1 and Hk0 cultures. However, the proportion of ShM neurons with persistent rhythmic firing was significantly lower than that of Hk neurons, attaining only a level comparable to that of wild-type neurons. Correspondingly, sporadic or plateau activities were increased in ShM cultures.



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Fig. 10. Role of Shalpha and Hkbeta subunits in mediating spontaneous activities in different cell categories. A: more persistent rhythmic firing was observed in Hk mutants and more sporadic/plateau firing was seen in Sh neurons. However, Hk Sh double-mutant neurons resembled Sh rather than Hk neurons in their firing properties. Data for WT and Hk0 and Hk1 were replotted and rearranged from Fig. 1 for ease of comparison with Sh and Hk Sh phenotypes. Although the percentage of neurons displaying sporadic or plateau activities was significantly greater in Sh and Hk Sh than WT cultures (P < 0.05), the percentage of persistently active neurons was not statistically different among these genotypes (P > 0.05). Percentage of neurons within both the persistent and the sporadic/plateau categories was significantly different between Sh (ShM or Hk ShM) and Hk (Hk0 and Hk1) mutants (P < 0.05) but not different between ShM and Hk Sh (P > 0.05). However, compared with WT cultures, Hk cultures showed an increase in the persistent category to an extent similar to the excess of the sporadic/plateau category in Sh and Hk Sh cultures. After subtracting the WT value from the percentage of the persistent category in Hk0 and Hk1 cultures, the adjusted values were not significantly different from the percentage of the sporadic/plateau category in Sh or Sh Hk cultures after subtraction of the corresponding WT value (P > 0.05). All statistical tests were one-sample binomial tests (cf. Fig. 1, Rosner 1982). B: hypothetical subdivisions, based on analysis of data presented in Figs. 1 and A, of cultured CNS neurons into 4 functionally different categories of neurons that are differentially regulated by the Shalpha and Hkbeta subunits. Category a neurons may be related to native pace-making cells in the CNS, and the rhythmic activities in these cells are not affected by Sh mutations. Category b represents cells with spontaneous sporadic and plateau activity, which is apparently unaffected by Hk mutations. Category c includes cells that are quiescent in wild type but appear to be converted by Hk mutations to fire persistent rhythmic spikes and by Sh mutations to display spontaneous sporadic spikes or plateau activities. Expression of this particular Hk phenotype requires intact Shalpha subunits because Hk Sh double-mutant neurons assume Sh-like, but not Hk-like, activity. Category d represents cells that remain quiescent in Hk, Sh, Hk Sh cultures. Role of the Sh gene cannot be determined, but the Hk gene may play some roles in cells of category d as suggested by data shown in Figs. 8 and 9. See DISCUSSION for detail. C: suggestive correlation between the categories of wild-type neurons described here and the previously characterized firing patterns in response to current injection. Tonic, adaptive, and delayed firing patterns of all-or-none spikes, as well as nonspiking graded potentials, were seen in neurons of categories c and d. Category a neurons were exclusively of the tonic type. Category b consists of neurons displaying tonic or adaptive spikes. Levels of current injection were 10-30 pA for samples shown in categories a and b and 30-150 pA for samples shown in categories c and d.

We then examined Hk Sh double-mutant neurons to see if mutating Hk in a Sh mutant background would result in additional abnormalities attributable to Hk. Interestingly, double-mutant neurons showed defects similar to ShM, but distinct from Hk, single-mutant neurons. Mostly transient clusters of spontaneous spikes and plateau potentials, but rarely prolonged rhythmic spike trains, were seen in Hk Sh neurons (Fig. 10A).

The data also raised the possibility that mutations of the Sh gene in Hk Sh double-mutant neurons convert a major portion of those cells expected to display persistent rhythmic firing (when only Hk is mutated) into those displaying sporadic or plateau activities (see Hk data in Fig. 10A, replotted from Fig. 1). In other words, intact Sh products seem to be required to enable Hk mutations to induce persistent rhythmic activities in this population of neurons. It is also interesting to note that, as shown in Fig. 10A, a small percentage of Sh and Hk Sh neurons, in which the Sh gene is disrupted, exhibited persistent rhythmic firing, suggesting a mechanism independent of Shalpha subunits. Significantly, a similar proportion of such cells also was observed in wild-type cultures (Figs. 1 and 10A). Therefore these cells are likely the endogenous pace-making neurons in the fly nervous system, which may involve non-Sh channels such as Shal, Shab, and Shaw (Butler et al. 1989).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This paper presents a comprehensive study of the function of ion channel auxiliary subunits in native neurons, in which we examined the role of Hkbeta subunits in regulation of neuronal K+ currents and activity patterns, which may be important in determining neuronal circuit function, such as activity-dependent plasticity (Byrne and Kandel 1996; Engel and Wu 1998; Getting 1989; Turrigiano et al. 1994, 1995). Discrete patterns of spontaneous activity were categorized in cultured Drosophila neurons. Hk mutations enhanced neuronal excitability by lengthening action potential duration and increasing the proportion of cells firing persistent rhythmic spikes. Voltage-clamp analysis suggested extensive alterations in properties of voltage-gated K+ currents, including density, voltage dependence, and kinetics. Alterations in both activation and inactivation processes were more pronounced at subthreshold voltage levels, suggesting a role in the regulation of the quiescent state in certain categories of neurons. Altered pharmacological profiles in Hk neurons suggested that the principal effector domain of beta  subunits may include both the intra- and extracellular pore regions of K+ channels, the previously proposed acceptor sites of 4-AP, alpha -DTX, and TEA.

A combination of genetic and neurophysiological analyses enabled us to identify a distinct category of neurons in which the effects of Hk and Sh mutations were not additive, and the mutational consequence of Hkbeta is conferred through intact Shalpha subunits to generate persistent rhythmic firing in this category of neurons. Our results also suggested that Hk mutations affect additional categories of neurons as well, possibly through interaction with non-Shalpha subunits. Recent molecular and genetic studies have linked mammalian epileptic diseases to several Na+ and Ca2+ channel auxiliary subunits (Burgess et al. 1997; Letts et al. 1998; Wallace et al. 1998). It will be of interest to explore whether K+ channel beta  subunits are also candidates for neurological disorders, such as epilepsy.

Regulation of firing patterns by Hkbeta subunits

A general picture appears to emerge from the analysis of spontaneous activities in Sh, Hk, and Hk Sh mutant neurons. It suggests a model that according to the differential roles of Shalpha and Hkbeta subunits, Drosophila neurons can be subdivided into four or more categories (Fig. 10). In this model based on mutant phenotypes, "category a" represents a small population of neurons (~4%, Fig. 10) that display pace making-like activity (Class I, Fig. 2) in wild-type, Sh and Hk Sh cultures. As mentioned in RESULTS, these presumably are associated with native pace-making cells, similar to those documented in other preparations (Adams and Benson 1985; Kramer and Zucker 1985; McCormick and Prince 1988; Smith and Thompson 1987). Because a similar proportion of cells was observed in wild-type as in Sh and Hk Sh cultures, rhythmic activities in this category of cells do not depend on intact Shalpha subunits. "Category b" consists of a slightly larger population in wild-type neurons (~8%, Fig. 10) in which sporadic and plateau activities can be seen. Note that since a similar percentage of Hk neurons also showed the same spontaneous activities, Hkbeta may not be required for generating such activities. Our preliminary results indicated that K+ current properties were not significantly different in this category of cells between wild-type and Hk cultures.

"Category c," as schematized in Fig. 10, represents a larger population of neurons (~15%, Fig. 10), which are normally quiescent in wild-type cultures. The suggestion for this category is based on the interesting observation that the persistent rhythmic neurons in Hk0 and Hk1 cultures in excess of those seen in wild-type cultures were roughly the same as the increased proportion of neurons with sporadic and plateau activities in Sh and Hk Sh cultures as compared with wild type (Fig. 10, A and B). It can be hypothesized that this category of quiescent cells in wild-type cultures is converted to display distinct firing patterns by either Sh or Hk mutations. Significantly, the phenotypes of Hk Sh double-mutant neurons resembled those of Sh neurons rather than those of Hk neurons, suggesting that the effects of Hk and Sh mutations are not additive and that Sh is epistatic to Hk (see Stern and Ganetzky 1989 for a similar epistatic effect at larval neuromuscular junctions). The notion that the consequences of Hkbeta subunits are mediated by Sh-related alpha  subunits in this category is supported by the lack of detectable differences in either voltage dependence, inactivation, or recovery kinetics of K+ currents between Sh and Sh Hk neurons (data not shown). It is interesting to note that roughly the same percentage of neurons was proposed previously to express Sh products in dissociated pupal (15%) (Baker and Salkoff 1990) and embryonic (13%) (Tsunoda and Salkoff 1995) neuronal preparations.

"Category d" includes the remaining cell population. Cells in category d are quiescent in all genotypes (~73%, Fig. 10). At the present time, we cannot determine how the Sh and Hk products function in these cells, although the reduction in K+ currents in quiescent Hk neurons (Figs. 8 and 9) suggests that the Hkbeta subunit does play a role in at least some of the category d cells.

Our results are consistent with earlier in situ intracellular studies that showed that neurons in the motor regions of adult Hk thoracic ganglia spontaneously fire persistent rhythmic action potentials not seen in wild-type preparations (Ikeda and Kaplan 1970). Most interestingly, a major portion of these neurons were reported to display typical pace-making potentials (type II) (Ikeda and Kaplan 1970, 1974) and our Class I firing pattern (Fig. 2) appears remarkably similar to such spontaneous activity revealed by intracellular recording. This agreement lends support to the idea that basic in vivo neuronal properties may be preserved in the giant neuron culture system, which provides a more accessible preparation for genetic analysis of physiological functions in different categories of neurons.

In addition to analyses based on spontaneous activity presented above, the firing patterns in response to current injection can provide further information about the functional diversity of these cell categories. Previous work has demonstrated that cultured giant neurons can be characterized functionally by distinct firing patterns, including adaptive, tonic, delayed, and interrupted spike trains, as observed in current-clamp experiments (Zhao and Wu 1997). We noticed a suggestive correlation between the categories of neurons in wild-type cultures described here and the previously characterized firing patterns (Fig. 10C). Category a neurons were exclusively of the tonic type at low levels of current injection. Category b consisted mostly of neurons firing tonic and adaptive spike trains during current clamp. The population of "quiescent" neurons in wild-type cultures, which includes categories c and d, was a mixture of subpopulations exhibiting the different firing patterns previously described, i.e., adaptive, tonic, delayed and interrupted spike trains, plus graded membrane oscillations (cf. Zhao and Wu 1997).

Regulation of K+ channels by the Hkbeta subunit

Our data suggest that the primary role of the Hkbeta subunit is to modulate voltage-gated K+ channels. A preliminary voltage-clamp survey has not detected any appreciable differences in voltage-activated Ca2+ and Na+ currents between Hk and WT neurons (unpublished results). Moreover, a previous study (Wang and Wu 1996) on Drosophila muscle also detected no differences in either inward Ca2+ currents or outward Ca2+-activated K+ currents in Hk alleles. Nevertheless subtle developmental adjustments of Ca2+ or Na+ currents in some Hk neurons cannot be ruled out; this may influence the expression of mutant firing patterns.

Because the giant neuron culture system potentially contains a variety of cell types expressing different K+ channel alpha  subunits that are encoded by Sh and other related genes (Butler et al. 1989), as mentioned in INTRODUCTION, multimeric assemblies of these subunits may generate channels with considerable overlap in voltage dependence and kinetic properties. Further diversity is possible because additional K+ channel beta  subunits may be encoded by genes other than Hk. Although our studies provide some insight into the in vivo role of beta  subunits, it will be technically more difficult to pinpoint the molecular mechanisms and specific subunit interactions in native neurons than in simple heterologous expression systems. Nevertheless, most of our results on beta -subunit modulation of K+ current properties are consistent with specific information obtained in heterologous expression studies using defined molecular lesions and subunit interactions.

The reduction in both peak and sustained voltage-activated K+ currents in Hk neurons is consistent with the role of Hkbeta in enhancing the magnitude of the Sh currents heterologously expressed (Chouinard et al. 1995). The Sh mutations are likely to affect transient as well as sustained K+ currents in cultured Drosophila neurons (Saito et al. 1993) and both types of currents can be mediated by different Sh splicing variants in the Xenopus oocyte (Iverson and Rudy 1990; Timple et al. 1988). The reduced current density may be due to a decrease in either single-channel conductance or surface expression, as beta  subunits have been shown to regulate the surface expression of alpha  subunits in mammalian cell lines (Shi et al. 1996). Further work at the single-channel level will be required to distinguish these two possibilities in Hk neurons.

Besides Shalpha subunits, K+ channel alpha  subunits of other subfamilies, encoded by the Shal, Shab, and Shaw genes, exist in Drosophila (Butler et al. 1989; Tsunoda and Salkoff 1995). Our results showed that Hk mutations affect K+ currents not only in cells that show the persistent spontaneous activity conferred by Sh channels but also in quiescent cells of other functional categories (Figs. 8-10). Thus potential interactions between Hkbeta and non-Sh alpha  subunits also may exist in certain native neurons.

Our results confirmed that the normal role of beta  subunits is to accelerate the time course of activation (Chouinard et al. 1995) and inactivation, as demonstrated in heterologous expression studies (Chouinard et al. 1995; England et al. 1995; Majumder et al. 1995; McCormack et al. 1995; Morales et al. 1995; Rettig et al. 1994; Uebele et al. 1996). In one such study (Chouinard et al. 1995), a more pronounced effect was found at low voltage levels, similar to our findings in native neurons. Results obtained from the oocyte expression system also suggest that beta  subunits influence both the N- and C-type inactivation of Sh channels (Chouinard et al. 1995; Morales et al. 1996). In the present study, complete isolation of identified inactivating current is difficult, preventing an analysis of C-type inactivation in separation of N-type inactivation. In addition to comparable effects on kinetic properties, altered voltage dependence of activation and inactivation of K+ currents in Hk neurons is in general agreement with the results in expression systems (Chouinard et al. 1995; England et al. 1995; Uebele et al. 1996), although more pronounced defects at lower voltages were seen in native neurons (this report) and muscles (Wang and Wu 1996).

Although the binding of the beta  subunit has been shown to involve N-terminal domains of the alpha  subunit (Rettig et al. 1994), the putative acceptor domain for beta  subunit action has been suggested to reside in the internal pore region of alpha  subunits (Chouinard et al. 1995; Wang and Wu 1996; Yu et al. 1996). The altered pharmacological profile in Hk neurons demonstrated by the use of TEA (cf. Chouinard et al. 1995), and 4-AP and alpha -DTX (which have not been used in heterologous expression studies) lends further support to this idea. Whereas 4-AP, an A-channel blocker, is known to bind to the cytoplasmic face (Kirsch et al. 1993; McCormack et al. 1991; Yao and Tseng 1994), alpha -DTX and TEA are thought to bind to the outer mouth of the pore region (Choi et al. 1991; Grissmer and Cahalan 1989; Hurst et al. 1991) of the alpha  subunits. Therefore the interaction of beta  and alpha  subunits on the cytoplasmic side can lead to a transmembrane change in the external mouth region.

Despite the consistency detailed in the preceding text, one critical difference between observations derived from native neurons and heterologous expression systems is the rate of recovery of transient K+ currents after inactivation. Coexpression of normal Hkbeta with Shalpha in Xenopus oocytes slowed down recovery of Sh currents (Chouinard et al. 1995), whereas in native neurons (this report) and muscles (Wang and Wu 1996), data indicate that the wild-type Hkbeta subunits accelerate recovery from inactivation (Fig. 6). Previous experiments on neuromuscular junctions of Hk mutant larvae have shown that high-frequency nerve stimulation results in repetitive nerve firing and increased postsynaptic response (Stern and Ganetzky 1989). This can be explained more readily by our data from native neurons, as slower recovery of K+ currents from inactivation would accumulate depolarization over time and cause repetitive firing at Hk nerve terminals. Thus functional Hkbeta subunits might be important in activity-dependent processes, such as those previously described in identified neurons of other species (Marcus and Carew 1991; Turrigiano et al. 1994, 1995; Wilson and Kaczmarek 1993) during development or physiological activity.

In conclusion, Hkbeta subunits may be involved in the regulation of the quiescent state and the fine tuning of firing patterns in neurons. Our results reveal the intricacy in beta -subunit regulation of nerve excitability: this auxiliary subunit may function as a switch to determine the excitability state of neurons and as a consequence, complex repertoires in neuronal firing can be achieved by beta  subunit-mediated modulation, rather than through elimination or addition, of specific types of K+ channels. Such regulation may contribute critically to the behavior of neuronal circuits (Getting 1989), and it may not be surprising to see that mutations affecting Hkbeta subunits can impose effects on habituation of an identified neural circuit more severely than mutations affecting Shalpha subunits (Engel and Wu 1998).


    ACKNOWLEDGMENTS

We thank Dr. B. Ganetzky for providing the HkIE18 stock, Drs. M. Daily, D. Eberl, S. England, and C. Rodesch and P. Taft for comments on the manuscript, and Drs. T. Hoshi, A. Ueda, and J. W. Wang for helpful discussions.

This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-18500 and NS-26528.


    FOOTNOTES

Address reprint requests to C.-F. Wu.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 16 September 1998; accepted in final form 25 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society