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1Department of Physiology and Biophysics C-240, 2Medical Scientist Training Program, University of Colorado Health Sciences Center, Denver, Colorado 80262
Submitted 17 May 2004; accepted in final form 4 August 2004
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ABSTRACT |
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INTRODUCTION |
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In embryonic Xenopus spinal neurons, delayed-rectifier potassium current IKv triples in density during the first 24 h of postmitotic differentiation (Spitzer and Ribera 1998
for review). A small, slowly activating IKv characterizes young neurons (
1-day embryo) and permits impulses to be of long duration and calcium dependent (Barish 1986; Lockery and Spitzer 1992; O'Dowd et al. 1988). Over the next day, a threefold increase in IKv density converts action potentials to the brief sodium-dependent spikes, characteristic of mature neurons that are beginning to form synapses. Subsequently, IKv density remains constant, despite continued differentiation of neurons (Ribera and Spitzer 1990). Thus even though spinal neurons first exhibit dramatic plasticity in electrical excitability, stable membrane properties appear by the time synapses begin to form.
Although the mechanisms that regulate plasticity and stability of IKv are not yet known, molecular determinants of IKv have been identified. Xenopus spinal neurons express Kv1.1, Kv1.2, Kv2.2, and Kv3.1 mRNAs (Burger and Ribera 1996; Gurantz et al. 2000; Ribera 1990; Ribera and Nguyen 1993). Dominant-negative and antisense approaches demonstrated that Kv1, Kv2, and Kv3 channels contribute to IKv (Blaine and Ribera 2001; Ribera 1996; Vincent et al. 2000). Further, dominant-negative strategies revealed that Kv2.2 subunits play an essential role in repolarization of the action potential (Blaine and Ribera 2001).
Transcriptional mechanisms appear to direct the rapid threefold increase in IKv density of differentiating spinal neurons (Blaine and Ribera 2001; Gurantz et al. 1996; Jones and Ribera 1994; Ribera 1996; Ribera and Spitzer 1989; Vincent et al. 2000). After the first 24 h of postmitotic differentiation (Ribera and Spitzer 1989), Xenopus spinal neurons normally maintain a constant IKv density. Much less is known about the mechanisms that establish the IKv set point in mature neurons.
Microinjection of RNA into Xenopus embryos is a standard method for perturbing the levels of a desired mRNA and by-passing normal transcriptional control mechanisms. Previous work demonstrated that microinjection of Kv1 mRNA led to increases in IKv density in both young and mature neurons (Jones and Ribera 1994). These data raised the possibility that transcriptional mechanisms were responsible not only for the rapid increase in IKv density in young neurons but also for establishment of the IKv set point in mature neurons. However, Kv2, and not Kv1, channels play the major role in action potential repolarization (Blaine and Ribera 2001). Thus we became interested in testing this possibility for the Kv2 potassium channel subfamily.
We show here, by in vivo overexpression of Kv2 subunits, that IKv density of young neurons was increased by RNA injection, regardless of Kv2 subunit type. However, in mature neurons, overexpression of Kv2.2 did not increase current density, in contrast to results obtained with Kv2.1. Chimeric Kv2 channels were generated by exchanging domains between Kv2.2 and the structurally related subunit, Kv2.1. All chimeric subunits were capable of inducing potassium current when expressed heterologously in oocytes. Similarly, in vivo in young neurons, chimeric subunits increased current density. However, in mature neurons, chimeric subunits that contained a unique Kv2.2 proximal carboxyl-terminus domain (proxC) did not increase current density when overexpressed. In contrast, chimeric subunits that lacked proxC did increase current density on overexpression. Therefore we propose that proxC domain of the Kv2.2 subunit mediates novel developmental and subunit-specific posttranslational regulation of IKv.
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METHODS |
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All studies were done with RNAs coding for Xenopus Kv2 channels. In vitro synthesis of RNAs coding for Xenopus Kv2 genes was as described previously (Blaine and Ribera 1998, 2001). In vitro fertilization and embryo injections were carried out using standard methods (Blaine and Ribera 2001). Briefly, each blastomere of a 2-cell stage Xenopus embryo was injected, either with RNA (e.g., encoding GFP and a Kv2 subunit) or rhodamine-conjugated dextran. GFP and Kv2 RNAs were coinjected at concentrations of 60–80 and 100–150 pg/nl, respectively. In situ hybridization was performed using a nonradioactive method (Harland 1991). RNA probes were synthesized in the presence of digoxigenin-labeled UTP and hybridized to whole-mount embryo preparations (Burger and Ribera 1996).
Spinal neuron culture and electrophysiology
Isolated cell cultures were prepared as described previously (Blaine and Ribera 2001). A 10- to 15-min incubation in divalent cation solution, without enzymatic treatment, sufficed for cell dissociation (Spitzer and Lamborghini 1976). Neurons were studied within 27 h of plating. Neurons in culture were identified as either control (rhodamine+) or +Kv2 (GFP+).
Tight-seal, whole cell recordings (Hamill et al. 1981) were made from neurons in culture at either young (3–7 h) or mature (19–31 h) stages. Whole cell currents were recorded with an Axopatch 1-D amplifier in conjunction with pCLAMP6/8 computer programs. Recordings were obtained at room temperature only from neurons with short processes (<50 µm). Electrodes were pulled from borosilicate glass and had resistances ranging between 2 and 4 M
when filled with the standard pipette solution [(in mM) 100 KCl, 10 EGTA, and 10 HEPES, pH 7.4 with KOH; final [K+] = 114 mM).
Cell capacitance was used to determine membrane surface area (1 µF/cm2) for normalization of current amplitudes to densities. Recordings were excluded if the capacitative transient changed during a recording or if the input resistance was <0.5 G. The neuronal membrane was held at –80 mV and stepped for 60 ms to voltages ranging between –60 and +90 mV in 10-mV increments; in some experiments tail currents were elicited at –40 mV prior to returning the membrane potential to rest. Currents were filtered at 5 kHz and digitized at 25 kHz. Leak subtraction was achieved using a modified P/4 protocol (pCLAMP6). For IKv, the bath solution contained (in mM) 80 NaCl, 3 KCl, 5 MgCl2, 10 CoCl2, and 5 HEPES, pH 7.4, and 1 µM tetrodotoxin (Calbiochem). Steady-state IKv amplitudes were measured at the end of the pulse. ICa was recorded using a bath solution containing (in mM) 40 NaCl, 40 TEA, 3 KCl, 10 CaCl2, and 5 HEPES, pH 7.4, and 1 µM tetrodotoxin. Electrodes were filled with (in mM) 100 CsCl, 10 tetraethylammonium-Cl (TEA-CL), 10 EGTA, and 10 HEPES, pH 7.4 with CsOH.
Normalization of current-density values (+50 mV) was done for data obtained within a recording session to avoid problems associated with seasonal and clutch variations (O'Dowd et al. 1988; Vincent et al. 2000); we calculated the average control current density and then divided the current density values from each neuron by the average control value for that recording session. Conductance densities were obtained by dividing current densities by driving force or from tail currents. Similar G-V curves were obtained by either method, indicating that single-channel current-voltage relationships were linear over the voltage range examined. G-V data were fit with the Boltzmann equation (G = Gmax/{1 + exp[(V1/2 – V)/k]}) to obtain the maximal Gmax, V1/2, and slope factor (k). Current and conductance-density plots were not corrected for the voltage error introduced by the series resistance, which ranged between 3 and 9 M.
Action potentials were recorded in the current-clamp mode. The neuronal membrane potential was set at –80 mV by steady-state current injection and 2.5-ms depolarizing current injections were given to elicit an impulse. The bath solution contained (in mM) 125 NaCl, 3 KCl, 10 CaCl2, and 5 HEPES, pH 7.4. Analysis of action potentials entailed measurement of the following parameters: overshoot, amplitude, maximum rate of rise, maximum rate of decay, time to peak, time to half-decay from the peak. We define the duration of the action potential as the time to half-decay because of our focus on repolarization of the action potential.
Construction of chimeric and Green Fluorescent Protein (GFP)-tagged Kv2 subunits
Standard polymerase chain reaction (PCR) cassette mutagenesis was used to create Kv2 chimeras and the Kv2t truncation mutant (Blaine and Ribera 1998). We took advantage of endogenous restriction sites and SpeI and AflI sites engineered by silent mutations into the coding regions of Kv2.1 and Kv2.2, respectively. All mutants were sequenced to confirm that the desired mutations had been made without introduction of frame-shifts.
The GFP sequence was amplified using the polymerase chain reaction (PCR) from pEGFP (Clontech) as the template. The primer pairs used to amplify the GFP sequence added three glycines to the carboxyl-terminus of GFP and were specifically designed for fusion to the amino terminus of the Kv2.1 or Kv2.2 coding region to yield either pGFP-Kv2.1 or pGFP-Kv2.2. The resultant clones were initially screened by restriction digest. DNA sequencing then confirmed that the GFP tag had been inserted in frame. RNAs synthesized from pGFP-Kv2.1 or pGFP-Kv2.2 were injected into Xenopus oocytes and normal Kv2.1 or Kv2.2 currents were induced by the GFP-tagged subunits.
Confocal imaging of GFP-tagged Kv2
subunits in Xenopus nerve-muscle cultures
GFP-tagged Kv2 subunit coding mRNAs were injected into single blastomeres of two-cell-stage embryos and cultures were prepared as described in the preceding text. In these experiments, lineage tracers (GFP RNA, dextran) were not used. Live neurons were viewed with an Olympus confocal microscope (Tokyo Japan). GFP and Nomarski images were collected and merged using National Institutes of Health ImageJ and Adobe PhotoShop software.
Heterologous expression in Xenopus oocytes
Standard two-electrode voltage-clamp recordings were obtained with an Oocyte Clamp OC-725C (Warner Instruments, Hamden, CT) 48 h after RNA injection as described previously (Blaine and Ribera 1998). Data acquisition and analysis were accomplished with pCLAMP and Axograph software (Axon Instruments, Foster City, CA). 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 between 0.1 and 0.7 M. Currents were elicited by depolarizing the membrane for 60 or 200 ms in 10-mV increments to potentials ranging between –60 and +100 mV from a holding potential of –80 mV. Current amplitudes were averaged during 20 ms at the end of the activating pulse. For analysis of conductance voltage-relationships, tail currents were recorded by clamping the membrane potential at –40 mV prior to return to the holding potential (–80 mV; e.g., Fig. 6). Leak and capacitative transient currents were subtracted using the P/4 protocol of the Clampex program modified with 11 subpulses.
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Mean values ± SE are presented. Differences between means for single comparisons were analyzed using the Student's t-test (2-tail). For multiple comparisons, ANOVA tests were applied. P values 
0.05 indicated statistical significance.
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RESULTS |
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; Jones and Ribera 1994; Kintner 1988). Thus the injected RNAs persisted to the time of interest, at least in vivo in the embryo.
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; Lim et al. 2000).
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We recorded from control and +Kv2.2 young neurons 3–7 h after plating. At this time, the amplitudes of potassium currents recorded from +Kv2.2 neurons were twofold larger than those of control neurons (Fig. 3A). Cell size did not vary between control and +Kv2.2 neurons, indicating that the larger current amplitudes reflected increases in current density rather than cell membrane expansion (Table 1; Fig. 3B). Consistent with the increased potassium current amplitudes, maximum conductance density (Gmax) was twofold larger for +Kv2.2 (12 ± 2 pS/µm2, n = 11) than for control neurons (6 ± 1 pS/µm2, n = 10; P = 0.008 vs. +Kv2.2).
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). Further, biochemical data demonstrate that Kv2.1 and Kv2.2 subunits do not coassemble in vivo in neurons even though they can when expressed heterologously in oocytes (Blaine and Ribera 1998; Malin and Nerbonne 2002).
If Kv2.2 homotetramers were responsible for the increase in IKv conductance in young neurons, the resultant current might display new properties reflecting behavior characteristic of the overexpressed channels (i.e., Kv2.2 homotetramers). We found that IKv activated more slowly in +Kv2.2 neurons than in controls, as revealed by the prolonged time-to-half-maximum activation (t1/2; Fig. 3D). This finding is consistent with the slow activation kinetics of heterologously expressed Kv2.2 channels (Blaine and Ribera 1998; Burger and Ribera 1996).
Several measures indicated that nonspecific effects of RNA injection did not account for the effects on IKv. For example, other membrane properties (input resistance, capacitance, and resting membrane potentials) were unaffected (Table 1). Further, another voltage-gated current, calcium current (ICa), did not differ between control and +Kv2.2 young neurons (Fig. 3, E and F). Moreover, using a computer based model (Lockery and Spitzer 1992), we found that the duration of the action potential recorded from +Kv2.2 neurons agreed with that predicted by a twofold increase in Kv conductance (Fig. 4). Because action potential generation requires the activity of a diverse set of channels, the computer simulations support the conclusion that only effects on the targeted current, IKv, occurred on overexpression of Kv2.2 channels.
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To determine the consequences of Kv2.2 overexpression on IKv of mature neurons, we recorded from neurons 11–22 h later than when recordings from young neurons were obtained. Even though Kv2.2 overexpression increased IKv density in young neurons, we found the Kv2.2 RNA injection had no effect on the density of IKv in mature neurons (Fig. 5, A and B). This result contrasted with effects obtained previously on overexpression of Kv1.1 that led to increases in IKv density in both young and mature neurons (Jones and Ribera 1994).
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Overexpression of Kv2.1 channels affected both the density as well as the activation of IKv. The t1/2 of IKv in mature +Kv2.1 neurons was significantly greater than in control or +Kv2.2 neurons (Fig. 5A). The slower activation of IKv on overexpression of Kv2.1 RNA is consistent with the possibility that slowly activating Kv2.1 homotetramers contribute to the whole cell IKv. The results indicate that the inability of Kv2.2 overexpression to increase IKv density in mature neurons is not a general property of Kv2 subfamily members but rather a specific property of the Kv2.2 subunit.
Kv2.1/2.2 chimerae form functional channels when expressed heterologously
Because injected Kv2.2 RNA and translated protein persisted to stages of interest (Figs. 1 and 2), the underlying mechanisms did not involve an unusually rapid Kv2.2-specific turnover. Further, overexpression of a dominant-negative Kv2.2 subunit was found previously to reduce IKv density in both young and mature neurons (Blaine and Ribera 2001); the wild-type and dominant-negative Kv2.2 subunits differ by only two residues in the pore region. The latter result suggests that protein also Kv2.2 also persisted to stages of interest. These considerations pointed to a posttranslational mechanism as the basis for the different regulation of Kv2.1 versus Kv2.2 in mature neurons.
The primary amino acid sequences of Kv2.1 and Kv2.2 predict proteins that are similar in the amino-terminal and membrane spanning domains but differ substantially across the large cytoplasmic carboxyl-tail region. Kv2 truncation mutants that lack substantial amounts of the carboxyl-tail maintain the ability to form functional Kv channels in heterologous systems (Pak et al. 1991). These data indicate that functional channel formation can occur in the absence of carboxyl-tail regions. However, given the high divergence of this region between Kv2.1 and Kv2.2, carboxyl-tail domains may determine subunit-specific channel properties, such as the novel regulation that we observe for Kv2.2 channels in mature neurons.
To test whether the Kv2.2 carboxyl-tail region mediated channel regulation in mature neurons, we exchanged regions of the Kv2.1 and Kv2.2 carboxyl-tail sequences and constructed five chimeric Kv2 subunits (Fig. 6). We divided the large cytoplasmic carboxyl-tail region into proximal (proxC) and distal halves. One truncation Kv2.2 mutant (Kv2.2t) was also created that retained proxC but not the distal half of the carboxyl-tail. (Portions of coding region as well as the small amounts of endogenous 3' untranslated sequence contained within the Kv2 constructs were exchanged; see Fig. 6A).
Consistent with previous work showing that Kv2 truncation mutants form functional channels (Pak et al. 1991), the Kv2 chimerae and truncation mutant induced potassium current when expressed heterologously in oocytes (Fig. 6B). These data also suggest that construction of Kv2 chimerae did not introduce major changes in subunit structure.
In heterologous systems, the biophysical properties of wild-type Kv2.1 and Kv2.2 heteromers differ slightly with respect to steady-state activation properties and kinetics of activation (Blaine and Ribera 1998). When expressed heterologously in oocytes, the chimeric channels displayed biophysical properties that reflected those of the Kv2 subunit that donated the transmembrane region of the subunit (Fig. 6). For example, Kv2.2 channels had voltage-dependent properties that were shifted to positive voltages versus those of Kv2.1 channels (Fig. 6C). Kv2G chimeric channels activated similarly to Kv2.1, the donor of Kv2G's transmembrane region. Further, Kv2H channels activated similarly to Kv2.2, the donor of Kv2H's transmembrane region. Moreover, the kinetics of activation of chimeric channels were predicted by the identity of donor of the transmembrane region (Fig. 6D). Chimeric channels with the transmembrane domain of Kv2.1 (KvF, Kv2G) activated more rapidly than did channels with Kv2.2's transmembrane domain (Kv2E, Kv2H, Kv2I, Kv2t; Fig. 6D).
On the basis of steady-state properties and activation properties of heterologously expressed channels, carboxyl-tail regions of Kv2 channels were not the primary determinants of biophysical properties of Kv2 channel function. These findings indicated that the functional role of these domains was not evident in heterologous systems and require analysis in a neuronal context.
In vivo overexpression reveals a subunit specific role for proxC
The one truncation and six chimeric Kv2 subunits were overexpressed in the developing embryo and IKv was recorded from young and mature neurons. As for overexpression of wild-type subunits (Fig. 5), we present data from test neurons as a proportion of that obtained from internal control neurons, which were set to a value of 1. Thus values >1 indicate that Kv2 overexpression increased IKv density in test neurons.
We generated Kv2 chimerae by exchanging coding as well as noncoding 3'UTR regions of Kv2.1 and Kv2.2 (Fig. 6A). The in vivo overexpression results indicated that 3'UTR sequences did not influence functional overexpression of channel subunits (Fig. 7A). For example, overexpression of either Kv2H or Kv2I resulted in an increase in IKv density as did overexpression of Kv2.1. However, the RNAs coding for Kv2H and Kv2I contained the 3'UTR sequence of Kv2.2, whereas the RNA coding for Kv2.1 contained the 3'UTR of Kv2.1. Conversely, overexpression of Kv2G did not produce an increase in IKv density even though the RNA contained the 3'UTR of Kv2.1.
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Despite their chimeric nature, Kv2G and Kv2H were overexpressed in young neurons (Fig. 7B). Further, they resulted in similar increases in current density. Moreover, the extent of current density increase produced by overexpression of the chimeric subunits was similar to that found for wild-type Kv2.1 and Kv2.2 subunits in young neurons (Figs. 3 and 5).
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DISCUSSION |
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; Jones and Ribera 1994; Ribera and Spitzer 1989; Vincent et al. 2000). For example, blockade of transcription during a critical period prevented the developmental upregulation of IKv in spinal neurons (Ribera and Spitzer 1989). Further, elevations of Kv1.1 channel RNA led to increased IKv density in both young and mature neurons (Jones and Ribera 1994). Conversely, antisense suppression of Kv3.1 channels decreased IKv density in mature neurons. In contrast, here, we provide evidence that nontranscriptional mechanisms may regulate IKv density, at least in mature neurons. The mechanism under study targeted one potassium channel subunit specifically, Kv2.2. Unlike for Kv1.1 and Kv2.1, injection of excess Kv2.2 RNA into the developing embryo did not increase IKv density in mature neurons. The simple possibility that Kv2.2 RNA or protein turns over rapidly is not supported by our data (Figs. 1 and 2). Further, we found no evidence to support the possibility that increases in expression of Kv2.2 channel expression in mature neurons were accompanied by decreases in another population that contributes to IKv. Instead, our data indicate that a cytoplasmic domain in the proximal carboxyl-terminus of the Kv2.2 subunit, proxC, determines whether or not IKv density will increase on overexpression of Kv2 subunits in mature neurons. The proxC domain is found in other vertebrate Kv2.2 subunits but not any other protein. Thus identification of the proxC domain has revealed a novel motif involved in developmental regulation of ion channel function.
Why would Kv2.2 channels be targeted for this novel regulation? It is likely that regulation is tailored to the specific role a channel plays in neuronal function. Even though Kv2.2 and Kv2.1 channels share many properties, they clearly play different roles in neurons. Kv2.1 channels localize to somatic and dendritic regions of principal and inhibitory neurons and play critical roles in regulating excitability, especially during high-frequency stimulation (Du et al. 2000; Lim et al. 2000). In mammalian superior cervical ganglion neurons, both Kv2.1 and Kv2.2 contribute to action potential repolarization (Malin and Nerbonne 2002). However, Kv2.2 channel function also influences the resting membrane potential and the threshold for action potential generation. Consequently, Kv2.2 channels have a greater impact on excitability in this neuronal population. Similarly, our previous work indicated that Kv2.2 was a major channel type required for action potential repolarization in embryonic Xenopus spinal neurons (Blaine and Ribera 2001). Thus in several systems, Kv2.2 channel function plays a major role in determining a neuron's excitability level. This level, or set point, might require minute-to-minute regulation in mature neurons, a goal that could more easily be achieved by posttranslational versus transcriptional mechanisms.
Mechanisms that maintain neuronal activity set points have been characterized as either activity dependent (Desai et al.1999; Golowasch et al. 1999; LeMasson et al. 1993; Turrigiano et al. 1994, 1995) or activity independent (MacLean et al. 2003). In the former, an abnormal change in a neuron's activity leads to compensatory mechanisms that alter channel function so that a neuron's firing is returned to the appropriate range (for review, Davis and Bezprozvanny 2001; Turrigiano and Nelson 2004). In the latter, a neuron's firing properties remain stable because expression of one channel is coupled to expression of one of opposing function (IA and IH) (MacLean et al. 2003). Despite the differences in these two models, they share in common that the regulated variable is the neuron's activity level. The mechanism we describe for regulation of IKv differs fundamentally because the regulated variable appears to be the number of channels of a particular class, in this case Kv2.2.
If the mechanisms that determine the IKv set point of mature neurons were activity dependent, one might expect that manipulations that perturbed activity in similar ways would also have the same effects on IKv density of mature neurons. For example, overexpression of either Kv1.1 or Kv2.2 subunits increases IKv density of young neurons and leads to premature shortening of the duration of the action potential (Jones and Ribera 1994; this study). However, overexpression of Kv1.1 increases IKv density of mature neurons, whereas overexpression of Kv2.2 subunits has no effect. Moreover, Xenopus spinal neurons can be grown in culture under conditions that would perturb activity, such as the presence of tetrodotoxin or the absence of extracellular calcium has no effect on IKv density of mature neurons (Bixby and Spitzer 1984; Desarmenien and Spitzer 1991). However, growth in such activity suppressing conditions has no effect on IKv density of mature neurons.
The kinetics of IKv is also developmentally regulated (O'Dowd et al. 1988), and perturbations that could have altered neuronal activity do alter kinetic properties of IKv (e.g., absence of extracellular calcium; Desarmenien and Spitzer 1991). In addition, our previous work has shown that activity of embryonic Xenopus spinal myocytes regulates the voltage sensitivity (V1/2) of presynaptic IKv via an NT-3-dependent mechanism (Nick and Ribera 2000). Thus existing data support a role for activity in regulating kinetics and voltage-dependent properties but not density of IKv.
A correlate of our model is that IKv density will not correspond directly to levels of Kv2.2 RNA or total protein. With respect to RNA levels, a lack of correspondence between Kv2 mRNA and IKv density has been observed for neurons of the ascidian (Ono et al. 1999). With respect to protein, Hwang et al. (1993) used Kv2.2-specific antibodies to examine protein expression and localization in rat brain. Interestingly, Kv2.2 immunoreactivity is present diffusely in neuronal cell bodies, consistent with an abundant intracellular location that would not contribute to Kv whole cell current.
We found that injection of Kv2.2 RNA could increase IKv density in young but not mature neurons. ProxC might allow only a certain number of functional Kv2.2 subunits in a neuron, regardless of developmental stage. Because the number of endogenous functional Kv2.2 subunits is small in young neurons, it is possible to increase their number at this stage but not later when the number of endogenous channels has reached the limit. Alternatively, the temporal restriction on proxC function could be achieved by developmentally regulated posttranslational modifications, such as phosphorylation, that restrict proxC function to mature stages. Interestingly, the primary sequence of proxC does predict consensus sites for protein kinase C, casein kinase 2 and tyrosine phosphorylation. Another possibility is that proxC interacts with a protein partner that is developmentally regulated. In heterologous systems, Kv2.2 has been shown to interact with two accessory proteins [potassium channel accessory protein, KChAP (Wible et al. 1998); mKv
3.2, originally designated as mKv4] (Fink et al. 1996) and thereby increase channel surface expression. Genes homologous to either KChAP or mKv3.2 have not yet been identified in Xenopus. In addition, members of the Kv6–Kv9 subfamilies have been shown to coassemble with Kv2 subunits (Hugnot et al. 1996; Kramer et al. 1998; Ottschytsch et al. 2002; Post et al. 1996; Richardson and Kaczmarek 2000; Salinas et al. 1997a, b; Sano et al. 2002; Zhu et al. 1999). Kv6–Kv9 subunits reduce current density when coexpressed with Kv2 subunits and thereby act as inhibitory subunits of Kv2 channels. However, the reductions in current density produced by coassembly of Kv6–Kv9 with Kv2 subunits are voltage dependent. We did not observe this type of voltage dependency for IKv of either control or test Xenopus spinal neurons. Moreover, Kv6–Kv9 as well as KChAP subunits do not show a specific interaction with Kv2.2 and also multimerize with Kv2.1 subunits. Thus known modes of regulation of Kv2 channel function do not account for the developmental- and subunit-specific control of IKv density that we report for embryonic spinal neurons.
In sum, subunit-specific mechanisms regulate IKv density in mature but not young neurons. The underlying mechanism requires the proxC cytoplasmic domain. Kv2.2 channels play the dominant role in action potential repolarization of spinal neurons (Blaine and Ribera 2001). Thus it might be critical and strategic that their function be regulated by a posttranslational mechanism that can more rapidly modulate channel function than might other levels of regulation.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: A. B. Ribera, Dept. of Physiology and Biophysics, 0837, UCHSC, Aurora, CO 80045 (E-mail: angie.ribera{at}uchsc.edu).
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REFERENCES |
|---|
|
Bixby JL and Spitzer NC. Early differentiation of vertebrate spinal neurons in the absence of voltage-dependent Ca2+ and Na+ influx. Dev Biol 106: 89–96, 1984.[CrossRef][Web of Science][Medline]
Blaine JT and Ribera AB. Heteromultimeric potassium channels formed by members of the Kv2 subfamily. J Neurosci 18: 9585–9593, 1998.
Blaine JT and Ribera AB. Kv2 channels form delayed-rectifier potassium channels in situ. J Neurosci 21: 1473–1480, 2001.
Burger C and Ribera AB. Xenopus spinal neurons express Kv2 potassium channel transcripts during embryonic development. J Neurosci 16: 1412–1421, 1996.
Davis GW and Bezprozvanny I. Maintaining the stability of neural function: a homeostatic hypothesis. Annu Rev Physiol 63: 847–869, 2001.[CrossRef][Web of Science][Medline]
Desai NS, Rutherford LC, and Turrigiano GG. Plasticity in the intrinsic excitability of cortical pyramidal neurons. Nat Neurosci 2: 515–520, 1999.[CrossRef][Web of Science][Medline]
Desarmenien M and Spitzer NC. Role of calcium and protein kinase C in development of the delayed rectifier potassium current in Xenopus spinal neurons. Neuron 7: 797–805, 1991.[CrossRef][Web of Science][Medline]
Du J, Haak LL, Phillips-Tansey E, Russell JT, and McBain CJ. Frequency-dependent regulation of rat hippocampal somato-dendritic excitability by the K+ channel subunit Kv2.1. J Physiol 522: 19–31, 2000.
Fink M, Duprat F, Lesage F, Heurteaux C, Romey G, Barhanin J, and Lazdunski M. A new K+ channel beta subunit to specifically enhance Kv2.2 (CDRK) expression. J Biol Chem 271: 26341–2638, 1996.
Golowasch J, Abbott LF, and Marder E. Activity-dependent regulation of potassium currents in an identified neuron of the stomatogastric ganglion of the crab Cancer borealis. J Neurosci 19: RC33, 1999.
Gurantz D, Lautermilch NJ, Watt SD, and Spitzer NC. Sustained upregulation in embryonic spinal neurons of a Kv3.1 potassium channel gene encoding a delayed rectifier current. J Neurobiol 42: 347–356, 2000.[CrossRef][Web of Science][Medline]
Gurantz D, Ribera AB, and Spitzer NC. Temporal regulation of Shaker- and Shab-like potassium channel gene expression in single embryonic spinal neurons during K+ current development. J Neurosci 16: 3287–3295, 1996.
Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers 391: 85–100, 1981.[CrossRef]
Harland RM. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol 36: 685–695, 1991.[Medline]
Harland R and Mischer L. Stability of RNA in developing Xenopus embryos and identification of a destabilizing sequence in TFIIIA messenger RNA. Development 102: 837–852, 1988.
Henderson LP and Spitzer NC. Autonomous early differentiation of neurons and muscle cells in single cell cultures. Dev Biol 113: 381–387, 1986.[CrossRef][Web of Science][Medline]
Hugnot JP, Salinas M, LeSage F, Guillemare E, De Weile J, Heurteaux C, Mattei MG, and Lazdunski M. Kv8.1, a new neuronal potassium channel with specific inhibitory properties towards Shab and Shaw channels. EMBO J 15: 3322–3331, 1996.[Web of Science][Medline]
Hwang PM, Fotuhi M, Bredt DS, Cunningham AM, and Snyder SH. Contrasting immunohistochemical localizations in rat brain of two novel K+ channels of the Shab subfamily. J Neurosci 13: 1569–1576, 1993.[Abstract]
Jones SM and Ribera AB. Overexpression of a potassium channel gene perturbs neural differentiation. J Neurosci 14: 2789–2799, 1994.[Abstract]
Kintner C. Effects of altered expression of the neural cell adhesion molecule, N-CAM, on early neural development in Xenopus embryos. Neuron 1: 545–555, 1988.[CrossRef][Web of Science][Medline]
Kramer JW, Post MA, Brown AM, and Kirsch GE. Modulation of potassium channel gating by coexpression of Kv2.1 with regulatory Kv5.1 or Kv6.1 -subunits. Am J Physiol Cell Physiol 274: C1501–C1510, 1998.
LeMasson G, Marder E, and Abbott LF. Activity-dependent regulation of conductances in model neurons. Science 259: 1915–1917, 1993.
Lim ST, Antonucci DE, Scannevin RH, and Trimmer JS. A novel targeting signal for proximal clustering of the Kv2.1 K+ channel in hippocampal neurons. Neuron 25: 385–397, 2000.[CrossRef][Web of Science][Medline]
Lockery SR and Spitzer NC. Reconstruction of action potential development from whole cell currents of differentiating spinal neurons. J Neurosci 12: 2268–2287, 1992.[Abstract]
Malin SA and Nerbonne JM. Delayed rectifier K+ currents, IK, are encoded by Kv2 alpha-subunits and regulate tonic firing in mammalian sympathetic neurons. J Neurosci 22: 10094–10105, 2002.
MacLean JN, Zhang Y, Johnson BR, and Harris-Warrick RM. Activity-independent homeostasis in rhythmically active neurons. Neuron 37: 109–120, 2003.[CrossRef][Web of Science][Medline]
Nick TA and Ribera AB. Synaptic activity modulates presynaptic excitability. Nat Neurosci 3: 142–149, 2000.[CrossRef][Web of Science][Medline]
O'Dowd DK, Ribera AB, and Spitzer NC. Development of voltage-dependent calcium, sodium and potassium currents in Xenopus spinal neurons. J Neurosci 8: 792–805, 1988.[Abstract]
Ono F, Katsuyama Y, Nakajo K, and Okamura Y. Subfamily-specific post-transcriptional mechanism underlies K+ channel expression in a developing neuronal blastomere. J Neurosci 19: 6874–6886, 1999.
Ottschytsch N, Raes A, Van Hoorick D, and Snyders DJ. Obligatory heterotetramerization of three previously uncharacterized Kv channel -subunits identified in the human genome. Proc Natl Acad Sci USA 99: 7986–7991, 2002.
Pak MD, Covarrubias M, Ratcliffe A, and Salkoff L. A mouse brain homolog of the Drosophila Shab K+ channel with conserved delayed-rectifier properties. J Neurosci 11: 869–880, 1991.[Abstract]
Post MA, Kirsch GE, and Brown AM. Kv2.1 and electrically silent Kv6.1 potassium channel subunits combine and express a novel current. FEBS Lett 399: 177–182, 1996.[CrossRef][Web of Science][Medline]
Ribera AB. A potassium channel gene is expressed at neural induction. Neuron 5: 691–701, 1990.[CrossRef][Web of Science][Medline]
Ribera AB. Homogeneous development of electrical excitability via heterogeneous ion channel expression. J Neurosci 16: 1123–1130, 1996.
Ribera AB and Nguyen DA. Primary sensory neurons express a Shaker-like potassium channel gene. J Neurosci 13: 4988–4996, 1993.[Abstract]
Ribera AB and Spitzer NC. A critical period of transcription required for differentiation of the action potential of spinal neurons. Neuron 2: 1055–1062, 1989.[CrossRef][Web of Science][Medline]
Ribera AB and Spitzer NC. Differentiation of IKA in amphibian spinal neurons. J Neurosci 10: 1886–1891, 1990.[Abstract]
Richardson FC and Kaczmarek LC. Modification of delayed rectifier potassium currents by the Kv9.1 potassium channel subunit. Hear Res 147: 21–30, 2000.[CrossRef][Web of Science][Medline]
Salinas M, de Weille J, Guillemare E, Lazdunski M, and Hugnot J-P. Modes of regulation of Shab K+ channel activity by the Kv8.1 subunit. J Biol Chem 272: 8774–8780, 1997a.
Salinas M, Duprat F, Heurteaux C, Hugnot J-P, and Lazdunski M. New modulatory subunits for mammalian Shab K+ channels. J Biol Chem 272: 24371–24379, 1997b.
Sano Y, Mochizuki S, Miyake A, Kitada C, Inamura K, Yokei H, Nozawa K, Matsushime H, and Furuichi K. Molecular cloning and characterization of Kv6.3, a novel modulatory subunit for voltage-gated K+ channel Kv2.1. FEBS Lett 512: 230–234, 2002.[CrossRef][Web of Science][Medline]
Spitzer NC and Lamborghini JE. The development of the action potential mechanism of amphibian spinal neurons isolated in cell culture. Proc Natl Acad Sci USA 73: 1641–1645, 1976.
Spitzer NC and Ribera AB. Development of electrical excitability in embryonic neurons: mechanisms and roles. J Neurobiol 37: 9585–9593, 1998.
Turrigiano GG and Nelson SB. Homeostatic plasticity in the developing nervous system. Nat Rev Neurosci 5: 97–107, 2004.[CrossRef][Web of Science][Medline]
Turrigiano G, Abbott LF, and Marder E. Activity-dependent changes in the intrinsic properties of cultured neurons. Science 264: 974–977, 1994.
Turrigiano G, LeMasson G, and Marder E. Selective regulation of current densities underlies spontaneous changes in the activity of cultured neurons. J Neurosci 15: 3640–3652, 1995.[Abstract]
Vincent A, Lautermilch NJ, and Spitzer NC. Antisense suppression of potassium channel expression demonstrates its role in maturation of the action potential. J Neurosci 20: 6087–6094, 2000.
Wible BA, Yang Q, Kuryshev YA, Accili EA, and Brown AM. Cloning and expression of a novel K+ channel regulatory protein, KChAP. J Biol Chem 273: 11745–11751, 1998.
Zhu XR, Netzer R, Bohlke K, Liu Q, and Pongs O. Structural and functional characterization of Kv6.2 a new gamma-subunit of voltage-gated potassium channel. Rec Channels 6: 337–350, 1999.
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) resembles that of Kv2.2 (n = 14; 
). In contrast, the G-V curve of Kv2G (n = 8; 
) resembles that of Kv2.1 (n = 9; 
). For the 4 curves, error bars were smaller than the symbols. D: activation kinetics of chimeric channels depended on the identity of the Kv2 donor of the transmembrane but not the cytoplasmic carboxyl-tail region of the subunit. Kv2.1, Kv2F, and Kv2G channels (
) neurons was twofold larger than that of control neurons (n = 10; 
