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Journal of Physiology (2002), 541.3, pp. 673-683
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.016568
2 function in Xenopus embryonic myocytes |
ABSTRACT |
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Kv1 potassium channels consist of pore-formingsubunits as well as auxiliary
(Received 7 January 2002; accepted after revision 19 March 2002)
Corresponding author A. B. Ribera: Department of Physiology and Biophysics C-240, University of Colorado Health Sciences Center, Denver, CO 80262, USA. Email: angie.ribera{at}uchsc.edu
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INTRODUCTION |
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Pore-forming () and auxiliary (, 
, 
) subunits coassemble to form voltage-gated ion channels (Hille, 2001). In vertebrates, at least 10 Kv1 and four Kv subunit genes have been identified (for review, see Pongs et al. 1999; Trimmer, 1999). Because Kv1 subunits alone form functional channels, it is possible to examine their properties and structure-function relationships heterologously. In contrast, Kv subunits alone do not generate functional channels and analysis of function requires coexpression with Kv1 subunits. In heterologous systems, Kv subunits affect surface expression and kinetic properties of channel complexes (Trimmer, 1999). These functional roles of Kv subunits implicate them as candidate mediators of plasticity in excitable cells of the developing embryo as well as the adult.
Even though Kv1 subunits suffice for heterologous formation of functional channel complexes, the majority of native Kv1 channel complexes contain Kv subunits (Rhodes et al. 1996; Shamotienko et al. 1997). However, little information exists regarding the functional roles of vertebrate Kv subunits in vivo. Pharmacological suppression of individual potassium channel isotypes is hampered by the lack of specific blockers. Consequently, study of Drosophila Kv mutants (hyperkinetic; Hk) has provided important insights into the in vivo function of this subunit (Chouinard et al. 1995; Wang & Wu, 1996; Yao & Wu, 1999). However, vertebrates possess at least four Kv genes (Kv1, Kv2, Kv3, Kv4; Trimmer, 1999) in comparison to the single Drosophila Hk Kv gene. Mice have been engineered genetically to lack a functional Kv1.1 gene (Giese et al. 1998; Pongs et al. 1999). In hippocampal neurons of Kv1.1 knock-out mice, potassium currents display less inactivation; the amplitudes of after-hyperpolarizations are diminished and the durations of action potentials are prolonged. Thus, Kv1.1 subunits play essential roles in vivo in regulation of Kv1 channel function.
Biochemical and immunocytochemical studies reveal that Kv2 subunits are abundantly expressed in excitable cells and components of most mammalian brain Kv1 channel complexes (Rhodes et al. 1996; Shamotienko et al. 1997). These auxiliary subunits exist in a 1:1 ratio with subunits in heterologously expressed Kv1 channel complexes (Xu et al. 1998). Little information exists about the in vivo roles of the Kv2 subunit. Xenopus embryos express the Kv2 gene, implicating it in the well-known developmental regulation of voltage-dependent potassium current (IKv) (Ribera & Spitzer, 1991; for review, see Ribera & Spitzer, 1992; Lazaroff et al. 1999).
We reduced expression of the Kv2 gene in the developing Xenopus embryo using an antisense approach. We focused on myocytes because these excitable cells display robust expression of the Kv2 gene (Lazaroff et al. 1999). Elimination of Kv2 function reduced the whole-cell potassium current density of myocytes. Interestingly, dominant-negative suppression of Kv1 subunits decreased current density to a similar, not significantly different, extent. Moreover, no further reduction was produced by simultaneous suppression of Kv2 and Kv1 function. While Kv1 subunits suffice for functional expression of Kv1 potassium current in heterologous systems, our results raise the possibility that expression of native Kv1 channel complexes in vivo depends upon both and subunits.
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METHODS |
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RNA synthesis
The entire coding regions of Xenopus Kv1.2 (referred to here as Kv1), a dominant-negative Kv1 (referred to here as Kv1DN), and the Kv2 and Kv4 potassium channel genes were previously cloned into the pSP64T, pALTER, and pCS2+ expression vectors, respectively (Ribera & Nguyen, 1993; Ribera, 1996; Lazaroff et al. 1999). The Ultra-GFP vector (a kind gift of Dr M. Klymkowsky, University of Colorado Boulder) was used for synthesis of RNA encoding green fluorescent protein (GFP). The constructs were linearized with Pst I (Kv1), Xba I (Kv1DN), or Not I (Kv2, Kv4 and Ultra-GFP) and cRNA was synthesized by in vitro transcription with SP6 RNA polymerase (Promega, Madison, WI, USA) in the presence of ribonucleotide triphosphates (Pharmacia Biotech, Piscataway, NJ, USA) and cap analogue (Boehringer Mannheim, Indianapolis, IN, USA). Synthesis of antisense Kv2 (AS2) RNA was achieved by linearizing with HindIII followed by in vitro transcription with T7 RNA polymerase (Promega). RNA concentrations were determined spectrophotometrically and confirmed by electrophoresis in agarose-formaldehyde gels.
Oocyte injection, two-electrode voltage clamp recording and data analysis
Stage VI Xenopus oocytes were removed and defolliculated as described previously (Lazaroff et al. 1999). This procedure is in accordance with NIH guidelines and approved by the University of Colorado Health Sciences Center (UCHSC) Institutional Animal Care and Use Committee. Kv1 cRNA was injected either alone or with Kv2 (or Kv4) and/or AS2 at a ratio of 1:20 (Kv1:Kv2 [or Kv4], Kv1:AS2) or 1:20:20 (Kv1:Kv2 [or Kv4]:AS2). RNA, 50 nl (Kv1 RNA, 2.5 µg ml-1; Kv2, Kv4 and AS2, 50 µg ml-1) was injected into oocytes. Oocytes were incubated at 18 °C in Barth's solution (mM: 88 NaCl, 1 KCl, 0.41 CaCl2, 0.33 Ca(NO3)2, 2.4 NaHCO3, 0.82 MgSO4 and 5 NaHepes, pH 7.4). Standard two-electrode voltage-clamp recordings were obtained with an Oocyte Clamp OC-725C (Warner Instruments, Hamden, CT, USA) 48 h after RNA injection. Data acquisition and analysis were accomplished with the pCLAMP suite of programs and Axograph software (Axon Instruments, Union City, CA, USA). Currents were sampled at 100 µs and filtered at 5 kHz. The external bath consisted of Barth's solution. The electrode solution consisted of 3 M KCl and 10 mM Hepes, pH 7.4. Electrode resistances ranged from 0.1-0.7 M
. Currents were elicited by depolarizing the membrane in 10 mV increments to potentials ranging between -60 and +100 mV from a holding potential of -80 mV. The leak and capacitative transient currents were subtracted using the P/4 protocol of the Clampex program (pCLAMP 6) modified with 11 subpulses. Current amplitudes were measured 50-55 ms into a 60 ms pulse using Axograph software (Axon Instruments). The apparent rate-of-activation was assessed by measuring the time-to-half maximum current (t1/2) using Axograph software (Axon Instruments).
Embryo injections
Introduction of RNA into developing embryos was achieved by microinjection of one cell of a two-cell-stage embryo (Jones & Ribera, 1994; Ribera, 1996; Blaine & Ribera, 2001). Experimental protocols were approved by the UCHSC Institutional Animal Care and Use Committee. RNA, 5 nl, of Kv1DN or AS2 or Kv1DN + AS2 (each subunit RNA at 160-200 pg nl-1) along with GFP RNA (80 pg nl-1) as a lineage tracer was pressure injected (2-3 p.s.i. (0.14-0.20 Pa) for 2-3 s) using fine-drawn micropipettes (approximately 6 µm tip diameter; Narashige, Sea Cliff, NY, USA). The progeny of the other cell (control) were identified by injection of rhodamine-labelled dextran (20-30 mg ml-1; Molecular Probes, Eugene, OR, USA). In a subset of experiments, embryos were injected with 80-140 pg nl-1 AS2 RNA in order to determine an effective dose for the antisense treatment.
Whole mount in situ hybridization
The non-radioactive whole-mount detection method (Harland, 1991; Ferreiro et al. 1993) was used as described previously (Lazaroff et al. 1999). Sense control probes contained the entire Kv2 coding region and antisense probes contained 685 bp of the most 3' coding sequence. cRNA probes were synthesized in the presence of digoxigenin-labelled UTP (Boehringer Mannheim) and hybridized to whole-mount embryos. After removal of the probe, embryos were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer Mannheim). The alkaline phosphatase reaction product was developed in the presence of chromogenic substrate (Boehringer Mannheim). Whole mount embryos were cleared in Murray's solution (2:1 benzyl benzoate/benzyl alcohol) for imaging. Imaging was performed with a Zeiss Axiocam camera and software (Carl Zeiss Inc., Thornwood, NY, USA) and the final figure was prepared using Adobe Photoshop 5.5 Software (Adobe Systems Inc., San Jose, CA, USA).
Cell culture preparation
Xenopus embryos were produced by standard in vitro fertilization techniques and staged according to Nieuwkoop & Faber (1994). Neural and somitic tissue were removed from neural tube stage embryos, dissociated by exposure to divalent cation-free solution, and cultures were prepared using standard methods (Spitzer & Lamborghini, 1976; Ribera & Spitzer, 1989; Blaine & Ribera, 2001). Culture media consisted of (mM): 116 NaCl, 0.67 KCl, 1.3 MgSO4, 9.4 CaCl2, 0.6 Ca(NO3)2, 4.6 Tris, pH 7.8.
Electrophysiological methods and analyses
Xenopus nerve-muscle cultures were studied 18-24 h after plating. Myocytes were identified on the basis of their morphology and were viewed with epifluorescence to identify them as either GFP (AS2 or Kv1DN or AS2 + Kv1DN injected) or rhodamine (internal control) positive cells. Whole cell gigaohm recording methods (Hamill et al. 1981) were used to record IKv and action potentials in myocytes. An Axopatch-1D amplifier (Axon Instruments) was used in conjunction with a Digidata 1320A interface (Axon Instruments), pCLAMP computer programs (Axon Instruments) and a PC computer for data acquisition. Electrodes were pulled from borosilicate glass (Drummond Scientific, Broomall, PA, USA) and had resistances ranging between 2.0 and 2.5 M when filled with pipette solution (100 mM KCl, 10 mM EGTA, 10 mM Hepes, pH 7.4 for recording of potassium currents and action potentials; 100 CsCl, 10 TEA-Cl, 10 EGTA, 10 Hepes, pH 7.4 with CsOH for recording of sodium currents). The external bath solution for potassium current recordings contained (mM): 80 NaCl, 3 KCl, 5 MgCl2, 10 CoCl2, 5 Hepes, pH 7.4. Sodium currents were blocked by the addition of 1 µM tetrodotoxin (Calbiochem, La Jolla, CA, USA). For the recording of sodium currents, bath solutions contained (mM): 80 NaCl, 3 KCl, 10 CoCl2, 40 TEA-Cl, 5 Hepes, pH 7.4; and for recording of action potentials: 125 NaCl, 3 KCl, 10 CaCl2, 5 Hepes, pH 7.4.
Potassium currents were elicited in myocytes by depolarizing the membrane in 10 mV increments to potentials ranging between -30 and +120 mV from a holding potential of -40 mV for 80 ms. For sodium currents, the membrane potential was held at -40 mV, stepped to -100 mV for 80 ms to remove inactivation, and then depolarized to potentials ranging between -30 and +120 mV in 10 mV increments for 20 ms. Leak subtraction was accomplished using a modified P/4 protocol with eight subpulses. Action potentials were recorded with whole-cell electrodes by switching to current clamp. A steady level of current was injected to hold the membrane potential near -80 mV and impulses were elicited by injecting brief depolarizing current pulses. Recordings were sampled at 40 µs and filtered at 10 kHz. The duration of the action potential was calculated as the time between peak and 80 % repolarization.
Data were analysed using pCLAMP8 and Axograph software (Axon Instruments). Cell capacitance was used to evaluate the membrane surface area (2 µF cm-2; DeCino & Kidokoro, 1985) for normalization of current amplitude to density. Cell capacitance was determined from the capacitative current transient recorded after break-in in separate trials in response to 20 mV depolarizing pulses sampled at 13 µs. Current amplitudes were averaged between 70 and 75 ms after starting the test pulse. The apparent rate-of-activation was assessed by measuring t1/2. To determine the maximal conductance density of a myocyte, we divided the maximal current density recorded from the cell by the driving force, with a calculated potassium equilibrium potential of -86 mV.
Data presentation
Data are presented as means ± S.E.M. Statistical analyses involved multiple comparisons, and analysis of variance (ANOVA) was performed. Comparisons that were significantly different at the 95 % or greater level are indicated. If statistical significance is not indicated, it was less than 95 %.
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RESULTS |
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Antisense Kv2 RNA suppresses the Kv2 function in a heterologous system
To determine whether AS2 RNA could suppress selectively the function of Kv2 subunits, we first examined its effects in a heterologous expression system, the Xenopus oocyte. Injection of only Kv1 RNA induced expression of a sustained delayed-rectifier type potassium current (Fig. 1A). Coinjection of Kv2 with Kv1 RNA led to the previously reported increase in current amplitude for these Xenopus subunits (Lazaroff et al. 1999). However, upon coinjection of AS2 RNA with Kv1 and Kv2 RNAs, the effect of sense Kv2 RNA was suppressed. Current amplitudes now resembled those found upon expression of Kv1 subunits alone (Fig. 1A and B). The effects of antisense suppression were observed throughout the voltage range examined. The effect of AS2 RNA required the presence of sense Kv2 RNA because no effect on Kv1 currents were observed in the absence of sense RNA.
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Figure 1. AS A, representative voltage-dependent potassium current recordings from Xenopus oocytes injected with Kv1 | ||
Antisense Kv2 RNA selectively eliminates the Kv2 but not Kv4 function
Previous work has shown that Xenopus embryos express two closely related but different Kv genes, Kv2 and Kv4. At the amino acid and nucleotide levels, Kv2 and Kv4 are 71 and 58 % identical, respectively (Lazaroff et al. 1999). Further, the effects of Kv2 and Kv4 on Kv1 channel function differ: Kv2 coexpression increases current amplitudes and accelerates activation kinetics while Kv4 coexpression accelerates the rates of both current activation and inactivation and consequently produces modest reductions in steady-state amplitudes. We examined the specificity of AS2 treatment by determining whether AS2 could suppress the effects of coexpression of sense Kv4 RNA on current activation and inactivation kinetics. In contrast to the inhibitory action of AS2 RNA on sense Kv2 coexpression, AS2 did not suppress the effects of coexpression of sense Kv4 with Kv1 subunits (Fig. 1C and D). Specifically, coexpression of Kv4 subunits still led to the normal increase in the apparent rates of current activation and inactivation despite the presence of AS2 RNA. These data indicate that AS2 RNA spares the function of the closely related gene, Kv4, and specifically inhibits the function of the Kv2 gene.
In vivo effects of antisense Kv2 subunits on endogenous Kv2 transcript levels
The results described above suggested that AS2 RNA selectively eliminated the function of the Kv2 gene, at least in a heterologous expression system. We next examined the effects of AS2 treatment in vivo on the levels of endogenous Kv2 transcripts. Previous studies demonstrated that the endogenous Kv2 gene is expressed in excitable tissues of the developing embryo (Lazaroff et al. 1999).
AS2 RNA was injected into single blastomeres of two-cell stage embryos. The uninjected blastomeres served as an internal control. Endogenous Kv2 transcripts were detected by whole-mount in situ hybridization (Lazaroff et al. 1999). If injection of AS2 RNA led to a reduction in expression of the endogenous Kv2 gene, the level of endogenous Kv2 transcripts would be reduced in the injected versus the non-injected control sides of the embryo. In fact, the hybridization signal (purple) was markedly reduced on the injected side of the embryo in contrast to the normal expression pattern observed on the control non-injected side (Fig. 2C). In contrast, injection of RNA encoding an irrelevant RNA (GFP) had no effect on the hybridization signal (Fig. 2E). Further, in situ hybridization of control uninjected embryos produced results similar to those obtained from embryos injected with GFP RNA (Fig. 2A). In all cases, hybridization with the control sense probe did not produce a hybridization signal (Fig. 2B, D and F). These data indicate that AS2, but not irrelevant RNA treatment, reduced in vivo expression of Kv2 mRNA.
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Figure 2. AS Two-cell stage embryos were injected on one side with either AS | ||
In vivo effects of Kv2 subunits on voltage-dependent potassium current
We next examined the functional consequences of elimination of Kv2 gene function in vivo by recording potassium currents from embryonic myocytes. Xenopus embryonic myocytes express both Kv2 and Kv1 genes (Lazaroff et al. 1999; Fry et al. 2001). In addition, myocytes also express Kv2 genes (Burger & Ribera, 1996). Thus, a combination of Kv1 and non-Kv1 channels will contribute to the myocyte IKv, and only a portion of the potassium current should be vulnerable to suppression of Kv1 currents achieved by elimination of either Kv1 and/or Kv2 function. Another portion of the current will be resistant and serve as a control for non-specific or toxic effects of antisense (Woolf et al. 1992).
We introduced AS2 RNA into embryonic myocytes by injecting single blastomeres of two-cell stage Xenopus embryos. The cell injected with antisense RNA was coinjected with RNA encoding green fluorescent protein (GFP); the other cell was injected with rhodamine-conjugated dextran and served as an internal control. We compared IKv recorded from rhodamine (internal controls) to that of GFP (AS2-positive) fluorescent myoctyes. In all GFP-positive cells examined, some potassium current persisted, indicating that not all of it was sensitive to AS2 treatment.
We injected different concentrations of AS2 RNA (80-200 pg nl-1) and examined the effects on potassium current density. Increasing the dose of AS2 RNA from 80 to 160 pg nl-1 led to greater reductions in the density of IKv, as expected if the AS2 RNA were specifically targeting endogenous Kv2 RNA (Fig. 3). In additional experiments examining the duration of the action potential (see below), we found that increasing the dose of AS2 above 160 pg nl-1 did not produce greater effects, consistent with saturation. While this dose of AS2 RNA reduced the density of IKv, it did not affect another voltage-dependent current, sodium current (Table 1; data not shown), assuring specificity of the antisense treatment. In subsequent experiments, we used AS2 RNA at doses ranging between 160 and 200 pg nl-1 and pooled the data.
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Figure 3. AS A, elimination of Kv | ||
Examination of IKv in GFP-positive myocytes indicated that AS2 RNA reduced the density of IKv by a factor of approximately 1/5 (Fig. 3A and B). As found in the Xenopus oocyte, effects were evident across the full extent of the voltage range examined (Fig. 3B).
We next compared the effect of AS2 treatment to that of overexpression of Kv1DN subunits. We have previously demonstrated that this mutant subunit leads to efficient suppression of Kv1 currents in vivo (Ribera, 1996). In myocytes, overexpression of Kv1DN RNA led to a similar significantly different reduction versus control in the whole-cell IKv, density (Fig. 3A and B). However, the extent of reduction in current density was not significantly different from that produced by AS2 treatment.
Elimination of either Kv2 or Kv1 subunit function reduced IKv density. These data suggest that elimination of either subunit in vivo in embryonic myocytes suffices to suppress Kv1 channel function. If they are both required for function of the same channel complex, their effects should not be additive. However, if they contribute to different channel complexes or could replace each other, their effects would be additive (e.g. Brenner et al. 2001; Espinosa et al. 2001). To test the possibility that Kv2 and Kv1 subunits contribute to the same Kv channel complexes, we coinjected both AS2 and Kv1DN RNAs. Simultaneous elimination of both Kv1 and Kv2 subunit function produced no further reduction in IKv density over that observed after either antisense treatment or Kv1DN overexpression alone (Fig. 3A and B).
The data indicate that the effects of eliminating both subunits were not additive and are consistent with the possibility that Kv1 channel function in embryonic amphibian myocytes relies on contributions from both Kv1 and Kv2 subunits. Alternatively, different compensatory mechanisms could be induced when Kv1 rather than Kv2 subunits are eliminated. If so, one might expect other properties of IKv to differ when Kv1 rather than Kv2 subunits are eliminated. Indeed, elimination Kv2 subunits alone or in combination with suppression of Kv1 function produced an increase in the apparent t1/2 for current activation (Fig. 3C). This effect was not observed when Kv1 function alone was eliminated (Fig. 3C). In contrast, elimination of either or both subunits led to similar hyperpolarizing shifts in the conductance-voltage relationship and ~10 mV increases in the values of V1/2 (Fig. 4B and C). Further, AS2 treatment and/or Kv1DN RNA overexpression reduced Gmax to similar extents (Fig. 4A), consistent with the effects on current density. Thus, similar effects on steady-state properties of activation were produced when the function of either or both subunits was suppressed. All conditions led to a reduction in Gmax and a hyperpolarizing shift in V1/2 versus controls.
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Figure 4. AS A, elimination of Kv | ||
In summary, elimination of either Kv2 or Kv1 subunit function produced similar effects on current and conductance density as well as the value of V1/2. In contrast, elimination of Kv1 function alone did not alter the apparent t1/2 for current activation, although suppression of Kv2 subunit function alone or in combination with Kv1 elimination did (Fig. 3C).
In vivo effects of Kv2 and Kv1 subunits on action potential properties
Our results indicate that elimination of Kv2 and/or Kv1 subunits reduced the amplitude and maximal conductance of myocyte IKv to similar extents. To examine the functional roles of the Kv channel complexes targeted by these manipulations, we recorded action potentials from myocytes that contained AS2 or Kv1DN or AS2 + Kv1DN RNAs.
Overexpression of Kv1DN RNA prolonged the duration of the impulse approximately threefold (Fig. 5). No further increase in the duration of the action potential was produced by combining Kv1DN overexpression with AS2 treatment. AS2 treatment alone produced a modest increase in action potential duration that was not statistically different from control when multiple comparisons (ANOVA) were performed. Thus, with respect to impulse duration, elimination of Kv1 subunits alone had greater effects than did elimination of Kv2 subunits alone. However, eliminating both subunits did not produce any greater effects than did eliminating Kv1 subunits alone.
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Figure 5. Both AS A, representative action potentials recorded from control (left, top), AS | ||
Resting membrane potentials, action potential overshoots and amplitudes, times to peak and the maximum rates of rise and fall of the impulse did not differ between control and AS2 or Kv1DN or AS2 + Kv1DN myocytes (Table 1). However, there was a small increase (~15 %) in the amount of current required to elicit an action potential (rheobase current) in AS2 and Kv1DN cells but not for cells exposed to both AS2 treatment and Kv1DN overexpression. Thus, elimination of either subunit alone increases the amount of current required to trigger an action potential.
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DISCUSSION |
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Despite numerous studies examining functional properties of Kv2 subunits in heterologous systems, very little is known about the functional roles of Kv2 subunits in vivo. Our data indicate that elimination of either Kv1 or Kv2 subunits in vivo had similar effects on the whole cell IKv density and Gmax. The data further suggest that these subunits contribute to the same Kv channel complexes, because the effects of elimination of Kv2 and Kv1 subunit function on myocyte IKv and Gmax were not additive. Moreover, the absence of additive effects raises the possibility that embryonic myocytes in vivo display a stronger requirement for Kv2 subunits than would be expected from studies in heterologous systems.
We used an antisense approach to eliminate Kv2 subunits in the developing embryo. Antisense has been used in several systems to reduce expression of individual channel subunits for both voltage-gated and ligand-gated ion channels (Listerud et al. 1991; Yu et al. 1993; Roy et al. 1996; Feng et al. 1997; Vincent et al. 2000). Recent reports indicate that this approach is also effective in the Xenopus embryo system to study the roles of developmentally regulated genes including ion channels (Kil et al. 1996; Retaux et al. 1996; Carl et al. 1999; Vincent et al. 2000). Vincent et al. (2000) used antisense methods to knock-down the function of Kv3.1 subunits in Xenopus embryonic neurons. Similar to AS2 treatment, Kv3.1 antisense treatment led to a partial reduction of IKv. In addition, antisense Kv3.1 RNA affected potassium current selectively without inducing changes in other membrane currents or properties.
To examine the specificity of the antisense approach in our studies, we first demonstrated that antisense Kv2 RNA selectively eliminated the function of Kv2 but not the closely related Kv4 subunit when expressed heterologously in oocytes (Fig. 1). Next, we found that injection of AS2 but not GFP RNA reduced in vivo expression of the endogenous Kv2 gene (Fig. 2). Further, the reduction was specific to the injected half of the embryo and the uninjected side (internal control) demonstrated normal expression of the Kv2 gene (Fig. 2). Further, we found that the effects of antisense Kv2 RNA on the density of IKv were dose dependent; saturating effects were achieved for RNA concentrations of 160 pg nl-1. Moreover, AS2 treatment did not affect other membrane currents or properties (Table 1).
Because the primary sequence and structure of the Kv2 subunit resemble those of aldo-ketoreductases, the Kv2 subunit could potentially couple the redox state to function of a variety of channels via an enzymatic mechanism (McCormack & McCormack, 1994; Chouinard et al. 1995; Gulbis et al. 1999). Indeed, several lines of evidence indicate that Kv2 subunits interact with non-Kv1 subunits (e.g. Wilson et al. 1998; Yao & Wu, 1999). In flies, the eag subunit HK has been reported to interact with Kv1 subunits under some (Chen et al. 1996, 2000) but not all conditions (Tang et al. 1998). Results from electrophysiological study of neurons cultured from Drosophila mutants that lack Kv (Hk), Kv1 (Shaker) or both subunits (Hk/Shaker) raised the possibility that the fly Kv subunit interacts with non-Kv1 subunits (Yao & Wu, 1999). Further, heterologously expressed fly Kv and eag subunits interact in vitro (Wilson et al. 1998). However, preliminary examination of the expression pattern of a Xenopus eag orthologue failed to reveal expression of this subunit mRNA in embryonic myocytes (A. D. Taylor & A. B. Ribera, unpublished observations).
In vertebrates, Kv4.2 and Kv4.3 subunits physically interact in vitro and in vivo, respectively, with Kv2 subunits (Nakahira et al. 1996; Yang et al. 2001). A Kv4.3 gene has been cloned in Xenopus, but it is not expressed in embryonic amphibian myocytes (Lautermilch & Spitzer, 1997). Taken together with our data, these considerations suggest that, in Xenopus embryonic myocytes, Kv2 interacts with Kv1 but not other Kv subunits.
In addition to effects due to direct physical interaction with specific subunits, elimination of Kv2 or Kv1 subunits might lead to compensatory effects on expression of function of other ion channel complexes. Is it possible that, in our studies, AS2 treatment and overexpression of Kv1DN subunits induced different compensatory mechanisms? For example in mammalian cardiac myocytes, overexpression of a dominant-negative Kv4 subunit induces expression of a rapidly activating and inactivating potassium current in addition to suppressing Kv4 potassium channel function (Barry et al. 1998; Xu et al. 1999). The induced, transient current results from upregulation of Kv1.4 channels (Guo et al. 2000). Further, in the murine heart, upregulation of Kv2.1 channels occurs when Kv1.1 replaced the Kv1.5 gene (London et al. 2001).
In the results presented here, AS2 treatment and overexpression of Kv1DN subunits, alone or in combination with each other, had similar effects on IKv density, Gmax and V1/2. The reductions in IKv density and Gmax are expected on the basis of elimination of potassium channel function. The hyperpolarizing shift in V1/2 may reflect that Xenopus Kv1 channels activate at less depolarized potentials than do Kv2 channels, whose mRNA is expressed in embryonic myocytes (Burger & Ribera, 1996). Elimination of lower threshold currents would shift V1/2 to more positive potentials. On the bases of these indicators, AS2 treatment and overexpression of Kv1DN subunits, alone or in combination with each other, did not appear to induce different compensatory changes in other potassium currents.
Examination of action potential properties revealed differences in the effects achieved by elimination of Kv2 versus Kv1 subunit function. The duration of the action potential was prolonged to a greater extent by Kv1DN overexpression alone than by AS2 treatment alone. In contrast, the apparent t1/2 of current activation was unaffected by Kv1DN overexpression alone but increased by AS2 treatment with or without Kv1DN overexpression (Fig. 3). This effect of AS2 treatment may reflect the fact that Kv1 channels activate more rapidly than other channels expressed in myocytes, e.g. Kv2 (Burger & Ribera, 1996). However, elimination of Kv1 subunits alone, which would also be expected to increase the contribution of Kv2 channels to the whole-cell IKv, did not change the apparent rate of current activation. In mammalian cardiac myocytes, overexpression of dominant-negative Kv4 subunits induced the appearance of a rapidly activating current, which would lead to a decrease in the apparent t1/2 (Barry et al. 1998; Xu et al. 1999). Perhaps, overexpression of Kv1DN similarly led to induction of a rapidly activating current in myocytes. The induced, rapidly activating current in mammalian cardiac cells also displayed rapid inactivation (Barry et al. 1998; Xu et al. 1999). However, overexpression of dominant-negative Kv1 subunits in Xenopus embryonic myocytes did not induce more inactivating current.
Overall, the results obtained here suggest that Kv1 channel complexes in embryonic Xenopus myocytes depend strongly on Kv2 subunits for functional expression, a result not predicted by work in heterologous systems (Lazaroff et al. 1999). The high expression levels that are possible in heterologous systems may be aphysiological and thereby prevent a complete evaluation of the in vivo roles of auxiliary subunits. Indeed, recent work has demonstrated that the auxiliary 1.1 subunit plays an essential role in regulating the calcium sensitivity of calcium-dependent BK potassium channels (Brenner et al. 2000; Pluger et al. 2000).
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Acknowledgements
We thank M. Klymkowsky (Department of Molecular, Cellular and Developmental Biology, UC Boulder) for the Ultra-GFP vector, R. Heiser, A. Linares and Dr K. Svoboda for discussion, and Dr K. Svoboda for gracious assistance with figure preparation. The work was supported by grants AHA CWFW-07-98 and SCRF1910-02 awards (M.A.L.) and NIH NS25217 (A.B.R.).
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and 
designate conditions without sense Kv
and 
indicate conditions with sense Kv
) did not further change t1/2 values, indicating that AS