| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Department of Physiology and Biophysics and 2Department of Pediatrics, University of Colorado Denver at Anschutz Medical Campus, Aurora, Colorado
Submitted 11 June 2008; accepted in final form 31 July 2008
 |
ABSTRACT |
|---|
|
-subunit genes and 2) the 24- and 48-h developmental period, during which IKv undergoes developmental regulation. At both 24 and 48 h, antisense methods produced efficient knock-down of both Kvβ2 protein and IKv. At both times, dominant negative suppression of Kv1 channels also eliminated IKv, indicating that Kv1 channels require Kvβ2 subunits to function in dorsal spinal neurons. Even though Kv1 channels determined the IKv values of both dorsal neuron types, comparisons of their IKv properties revealed important differences at both developmental stages. The latter results support the notion that different Kv1 -subunits and/or posttranslational modifications underlie the IKv values of the two dorsal neuron types. Overall, the results demonstrate that Kvβ2 subunits function in vivo as obligatory subunits of Kv1 channels in at least two neuron types and two different developmental stages. |
|
INTRODUCTION |
|---|
|
). Functional Kv channel protein complexes contain pore-forming () and auxiliary (e.g., β) subunits (Li et al. 2006; Pongs et al. 1999; Torres et al. 2007; Trimmer 1998). The pore-forming -subunits comprise members of several different Kv subfamilies (Kv1–12). When expressed in heterologous systems, Kv1, Kv2, Kv3, Kv4, Kv7, Kv10, Kv11, and Kv12 -subunits form functional channels even in the absence of auxiliary subunits (Coetzee et al. 1999; Gutman et al. 2003).
In vivo biochemical and immunohistochemical studies have revealed that the majority of Kv1 channel complexes contain auxiliary subunits that belong to the Kvβ gene family (Kvβ1–Kvβ3; Heinemann et al. 1995, 1996; Nakahira et al. 1996;Parcej et al. 1992; Rettig et al. 1994; Rhodes et al. 1995, 1996, 1997; Scott et al. 1994; Shamotienko et al. 1997; Shi et al. 1996). Kvβ subunits also interact with Kv4 and Kv12 -subunits (Chen et al. 1996, 2000; Wilson et al. 1998; Yang et al. 2001; but see Tang et al. 1998). Despite the widespread presence of Kvβ2 subunits in native Kv channel complexes, little is known about their physiological roles in vivo.
The limited available information about in vivo Kvβ2 subunit function has been obtained from studies of the Drosophila mutant, hyperkinetic (Hk; Chouinard et al. 1995; Ikeda and Kaplan 1970; Kaplan and Trout 1974), and a genetically engineered Kvβ2 mouse knock-out (McCormack et al. 2002). Drosophila has a single gene, Hk, that is orthologous to mammalian Kvβ1, Kvβ2, and Kvβ3 genes. Hk mutants display an abnormal motor phenotype consisting of rhythmic leg shaking. Interestingly, Kv1 (Shaker) currents have reduced amplitudes and altered kinetic properties (Wang and Wu 1996; Yao and Wu 1999). The Kvβ2 knock-out mouse displays seizures, cold swimming-induced tremors, and reduced life spans (McCormack et al. 2002). Despite work from heterologous systems suggesting chaperone-like functions for Kvβ2 genes on Kv1 complexes, the mouse knock-out provided no evidence for abnormal Kv1 channel biosynthesis or trafficking on elimination of Kvβ2 protein. These results highlight that the in vivo roles of Kvβ subunits are poorly understood.
Studies of Kvβ2 subunits expressed heterologously have provided information regarding their interactions and functional effects on -subunits. Xu et al. (1998) analyzed subunit stoichiometry and found four Kvβ2 subunits per channel; that is, equal numbers of Kvβ2 and Kv1 -subunits exist in a single Kv1 channel complex. In vitro, Kvβ2 subunits produce diverse functional consequences, including effects on voltage dependence of activation, activation and inactivation kinetics, channel surface membrane expression, and current density (Heinemann et al. 1996; Manganas and Trimmer 2000; Morales et al. 1996; Rettig et al. 1994). Because Kvβ2 subunits increase channel complex stability and cell surface insertion of Kv1 -subunits in heterologous systems, chaperone-like roles have been proposed for Kvβ2 subunits (Accili et al. 1997; Campomanes et al. 2002; Nagaya and Papazian 1997; Shi et al. 1996).
Because effects seen in heterologous systems do not always recapitulate mechanisms in vivo, we took advantage of unique features of Xenopus embryonic spinal cord neurons to test the in vivo function of Kvβ2 subunits. Voltage-gated potassium currents (IKv) have been studied in vitro as well as in vivo in spinal neurons of the Xenopus embryo (Desarmenien et al. 1993; O'Dowd et al. 1988; Pineda and Ribera 2008). The Xenopus embryo expresses Kvβ2 mRNA in spinal cord neurons during the same developmental period during which extensive regulation of voltage-gated potassium current occurs (Lazaroff et al. 1999). Further, the developmental changes in IKv consist of increases in current density and acceleration of activation kinetics (Barish et al. 1986; Lockery and Spitzer 1992; O'Dowd et al. 1988), potassium current properties that are modulated by Kvβ2 subunits when coexpressed heterologously with Kv1 -subunits. Moreover, dorsal spinal neurons express Kv1.1 -subunit and Kvβ2 transcripts, providing an experimentally accessible and relevant neuronal population for the study of the in vivo roles of Kvβ2 subunits (Lazaroff et al. 1999; Ribera and Nguyen 1993).
To test the in vivo roles of Kvβ2 subunits, we used two different antisense (AS) strategies to knock down Kvβ2 protein. Western blot analysis indicated that morpholino oligonucelotides (MOs) produced effective knock-down of Kvβ2 protein. We focused on two different populations of neurons in the dorsal spinal cord: Rohon–Beard (RB) primary mechanosensory neurons and immediately adjacent dorsal non-Rohon–Beard cells (non-RBs). In the absence of Kvβ2 protein, specific populations of dorsal spinal cord neurons that express the gene displayed a near total elimination of IKv. This was true for both dorsal neuron types at two different developmental stages. However, IKv properties differed substantially between RBs and non-RBs and as a function of development. With respect to Kvβ2 knock-down, the Kv1 dominant negative efficiently suppressed IKv channel in the two different neuron types regardless of developmental stage. Thus irrespective of developmental stage, Kvβ2 protein knock-down reduced IKv density in spinal neurons as effectively as did dominant negative suppression of Kv1 channel function. Taken together, the results support the view that Kvβ2 subunits play an essential role in Kv1 channel function. Moreover, because Kvβ2 knock-down effectively suppressed IKv in both 24- and 48-h neurons, the essential role of Kvβ2 in Kv1 channel function is constant and not developmentally regulated during a period when IKv properties undergo substantial changes.
|
|
METHODS |
|---|
|
All experimental procedures were approved by the Animal Care and Use Committees of the Center for Comparative Medicine at the University of Colorado Denver at the Anschutz Medical Campus. In vitro fertilization and RNA microinjection were performed as described previously (Jones and Ribera 1994). Embryos were staged on the basis of external morphology (Nieuwkoop and Faber 1967).
Working MO (1–150 pg/nl) or RNA (120–200 pg/nl) solutions were prepared by dilution of stock aliquots with RNAse-free water containing as a lineage tracer RNA encoding green fluorescent protein (GFP; 6 ng/nl, a kind gift of Dr. Michael Klymkowsky, University of Colorado, Boulder; Blaine and Ribera 2001). A total volume of 10 nl was injected into one cell of two-cell stage embryos using a gas-driven injection apparatus (2–3 psi for 2 s; PLI-100, Medical System, Greenvale, NY) with fine-drawn micropipettes (
1- to 2-µm tip diameter; Sutter P-87 Puller, Sutter Instruments, Novato, CA). The day after injection, embryos were examined with epifluorescent illumination and those expressing GFP within the neural tube were kept at room temperature until the desired developmental stage.
KNOCK-DOWN OF KVβ2. Kvβ2 function was knocked down by injection of either morpholino (MO) or antisense RNA. MOs were designed and synthesized by GeneTools (Philomath, OR). The Xenopus Kvβ2(Kvβ2MO) targeted the predicted translation start methionine and had the following sequence: 5'-AgT CTg Tgg TCg ATT CTg gAT ACAT-3'. The control Kvβ2MO (CtlMO) was designed by inverting the Kvβ2MO sequence: (5'-TAC ATA ggT CTT AgC Tgg TgT CTgA-3'). Aliquots of MO stock solutions were prepared by resuspending the oligonucleotides in RNAse-free water at a final concentration of 12.5 µg/µl (1.5 mM) and stored at –80°C. For both Kvβ2MO and antisense Kvβ2, dose–response curves were determined to assess specificity of the knock-down.
ANTISENSE KVβ2 RNA (ASβ2).
ASβ2 was synthesized as described previously (Lazaroff et al. 2002). Briefly, the plasmid containing Kvβ2 (pCS2+) was linearized with Hin dIII and cRNA was synthesized by in vitro transcription with T7 RNA polymerase (Promega, Madison, WI) in the presence of ribonucleotide triphosphates (Pharmacia Biotech, Piscataway, NY). As a control for the antisense, an irrelevant RNA (GFP) was used. We found no differences between IKv in neurons derived from uninjected or GFP-injected blastomeres at either 24 or 48 h (not shown). In addition, previous work (Lazaroff et al. 2002) demonstrated ASβ2 selectively eliminated effects of Kvβ2 but not Kvβ4 RNA injection into Xenopus oocytes.
DOMINANT NEGATIVE KV1 -SUBUNIT.
The Kv1 -subunit dominant negative (Kv1DN) was generated as described previously (Ribera 1996). cRNA was synthesized by linearizing the plasmid with Xba I and in vitro transcription with SP6 RNA polymerase in the presence of ribonucleotide triphosphates (Pharmacia Biotech) and cap analogue (Boehringer Manheim, Indianapolis, IN). RNA concentrations were determined spectrophotometrically (Nanodrop N-1000, NanoDrop Technologies, Wilmington, DE). RNA integrity was assessed by agarose-formaldehyde gel electrophoresis.
Protein extraction
St 34/35 Xenopus embryos were homogenized in MK lysis buffer (in mM: 50 Tris pH 8.0, 150 NaCl, 0.5% NP40, 0.5% Triton-X100, 1 EGTA, pH 7.4; Klymkowsky Lab On-line Methods; http://spot.colorado.edu/klym/) containing 1x protease inhibitor (Halt Protease Inhibitor Cocktail Kit; Pierce, Rockford, IL) or 2% SDS in 50 mM Tris (pH 7.5). Homogenates were centrifuged and embryo supernatants were treated to remove excess lipid with PHM-L Liposorb absorbent according to the manufacturer's instructions (Calbiochem, San Diego, CA). Protein extract aliquots were stored at –80°C until use.
Western blots
Whole embryo protein extracts (20 µg) were resolved using SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon P; Millipore, Billerica, MA) by wet electrotransfer (Towbin et al. 1992). Prior to incubation with antibody, membranes were blocked for 2 h in Tris-buffered saline (TBS; in mM: 136 NaCl, 2.6 KCl, 24.7 Tris; pH 7.4) with 5% nonfat evaporated milk and 0.1% Tween 20. Blots were then incubated overnight at 4°C in blocking buffer containing the primary antibody, either anti-Kvβ2 (1:50, Clone 17/70, NeuroMab, Davis, CA; http://www.neuromab.org; Bekele-Arcuri et al. 1996) or anti-Kv1.1 (1:50, clone K20/78, NeuroMab). After being rinsed in TBST (TBS containing 0.5% Tween 20), blots were incubated with secondary antibody. For standard Western blot analysis, a horseradish peroxidase-conjugated anti-mouse secondary antibody was used (1:2,000; Bio-Rad Laboratories, Hercules, CA). Blots were then incubated in a chemiluminescent substrate at room temperature for 1–5 min (Pierce) and imaged using a Kodak Image Station 440 CF and Molecular Imaging Software (Carestream Health, Rochester, NY). These experiments were repeated at least three times.
For quantitative measurements, blots were incubated with an Alexa 647-conjugated anti-mouse secondary antibody (1:2,000; Invitrogen, Carlsbad, CA) for 2 h at room temperature (20–22°C) and then scanned using a Typhoon 9400 multimode imager (GE Healthcare; Little Chalfont, Buckinghamshire, UK). Gels were analyzed using ImageQuant Densitometer software (Molecular Dynamics, GE Healthcare, Pittsburgh, PA). An image of a representative assay is shown as well as average data for the total of three experiments.
Semiintact preparations of Xenopus embryos
St 22/23 and St 35/36 Xenopus embryos were dissected using slight modifications of methods previously described for semiintact preparations of zebrafish embryos (Pineda et al. 2005; Ribera and Nüsslein-Volhard 1998). Briefly, in the presence of Ringer solution (in mM: 145.0 NaCl, 3.0 KCl, 1.8 CaCl2, 10.0 HEPES; pH 7.2) containing 0.02% Tricaine (ethyl 3-aminobenzoate methanesulfonate salt; Sigma–Aldrich, St. Louis, MO), the yolky endoderm was removed and embryos were mounted ventral-side down onto glass coverslips using Vetbond Tissue Adhesive (3M Animal Care Products, St. Paul, MN). Embryos were then killed in the presence of anesthesia by transection at the level of the hindbrain. Removing the skin and dorsal fin fold exposed the spinal cord. Tricaine was removed by washing the preparation with 
40 ml of recording solution over the course of 15 min. Preparations were viewed with differential interference contrast optics on an Axioskop FS2 microscope (Carl Zeiss MicroImaging, Hamburg, Germany) at a magnification of x640. RB cells were identified on the basis of: 1) superficial location at the dorsal surface of the spinal cord, 2) large soma diameter (20 µm), and 3) position relative to the midline (Baccaglini and Spitzer 1977). On the basis of the absence or presence of GFP, cells were identified as internal control (GFP–) or MO, AS, or Kv1DN (GFP+) neurons, as done previously (Blaine and Ribera 2001).
Electrophysiological methods
Conventional whole cell patch-clamp techniques (Hamill et al. 1981) were used in voltage-clamp mode. Experiments were conducted at room temperature (20–22°C) using an Axopatch 200B amplifier and a Digidata 1440A analog to digital interface in conjunction with the pClamp 10.0 software recording package (Molecular Devices, Sunnyvale, CA).
Unpolished electrodes were fabricated from borosilicate glass (Microcaps; Drummond Scientific, Broomall, PA) with tip resistances ranging between 2.0 and 3.5 M
when filled with pipette solution (in mM: 100 KCl, 10 mM EGTA, 10 HEPES; pH 7.4 with NaOH). After establishment of the whole cell configuration, monoexponential capacitative transients were indicative of adequate spatial control of membrane voltage. Additional criteria were used to assess the quality of the recordings: 1) membrane resistance >120 M, 2) holding current <250 pA, and 3) stable access resistance of <12 M.
For recording of the outward potassium currents, the bath solution contained (in mM): 80 NaCl, 3 KCl, 5 MgCl2, 10 CoCl2, 5 HEPES, and 0.003 tetrodotoxin; pH 7.4 with NaOH. Currents were elicited by 60-ms depolarizing voltage steps to test potentials ranging between –60 and +100 mV in 10-mV increments from a holding potential of –80 mV; tail currents were recorded at –40 mV after the activating steps. Series resistance was routinely compensated by 75–85% with a lag of 10 µs. Currents were filtered at 5 kHz and digitized at 25 kHz. Passive leak and capacitative transients were subtracted on-line using the P/8 algorithm of the software.
Data analysis
Data analysis was accomplished using AxoGraph 10 (Axograph Scientific, Sidney, Australia), Excel (Microsoft, Redmond, WA), and Origin (OriginLab, Northampton, MA) software. Variability in cell size was accounted for by dividing current amplitudes by the membrane capacitance, which serves as an indicator of membrane surface area (1 pF/cm2; Marty and Neher 1983). Thus current data are presented as current densities (pA/µm2). For current density–voltage (I–V) plots, steady-state currents were measured by averaging values during a 10-ms interval at the end of each pulse (from 45 to 55 ms). Conductance–voltage (G–V) relationships were constructed by dividing current density by driving force, using the calculated potassium equilibrium potential of –88.3 mV. The Boltzmann equation (G = Gmax/{1 = exp[(V1/2 – V)/k]}, where Gmax is the maximal conductance, V1/2 is the voltage of half-maximal activation, and k is the slope factor) was fitted to the data. G–V plots were not corrected for the small voltage errors (<4 mV) introduced by the uncompensated fraction of the series resistance.
Data presentation
Results are presented as means ± SE. Statistical analysis was performed using GraphPad InStat software (GraphPad Software, San Diego, CA). Statistical comparisons were done using the Student's t-test or ANOVA, for comparisons of two or multiple groups, respectively. ANOVA analysis was followed by Bonferroni correction to consider multiple comparisons. A threshold of P 
0.05 was used to determine statistical significance.
|
|
RESULTS |
|---|
|
-subunits and Kvβ2 subunits (Lazaroff et al. 1999; Ribera and Nguyen 1993). To investigate the functional role of Kvβ2 subunits in vivo, we developed a semiintact preparation that allowed us to identify two different types of dorsal spinal cord neurons. Primary sensory RB and neighboring dorsal non-RB neurons were identified during an experiment on the basis of position and soma diameter (Fig. 1A). RBs had larger soma membrane area, as assessed by cell capacitance, than that of non-RB neurons (Fig. 1B). The difference in cell size between RBs and non-RBs permitted their reliable identification in situ.
|
We tested the contribution of the Kvβ2 auxiliary subunit to IKv of RB and non-RB dorsal neurons by using both morpholino and standard antisense RNA strategies to knock down the Kvβ2 protein in the Xenopus embryo. In 24-h embryos injected unilaterally with 1 ng Kvβ2MO and GFP RNA, we recorded from internal control GFP– RB neurons and GFP+ neurons (see METHODS). GFP– RB neurons displayed substantial IKv (Fig. 2A). In contrast, in GFP+ RB neurons, we found effective suppression of IKv (Fig. 2A). Similar effects of Kvβ2MO on IKv were observed in non-RB neurons (Fig. 2B). We also determined the dose–response relationship for the Kvβ2 morpholino (Kvβ2MO) on IKv density (Fig. 2C). Morpholino injection led to a steep dose-dependent decrease in IKv amplitude in both RB and non-RB cells. For injected doses <0.02 ng, the Kvβ2MO blocked between 20 and 30% of the recorded total IKv amplitude. Using a dose 0.05 ng resulted in a surprising, almost complete knock-down of IKv. In contrast, injection of the CtlMO at a dose of 1 ng had no effect on IKv density in either RB or non-RB neurons (Fig. 2D).
|
-subunits; however, they are not required for formation of functional Kv1 channels (Coetzee et al. 1999; Gutman et al. 2003). Given this precedent, we were surprised by the complete knock-down of IKv produced by Kvβ2MO and concerned about nonspecific effects. To confirm that the Kvβ2MO acted as expected by knocking down Kvβ2 protein, we used Western blot analysis to determine Kvβ2 protein levels in uninjected, CtlMO- and Kvβ2MO-injected embryos (Fig. 3). We used a dose of MO that led to complete knock-down of IKv (1.0 ng; Fig. 2C). In uninjected embryos, the levels of Kvβ2 protein increased slightly, but significantly, between 24 and 48 h (Fig. 3, A and B; P < 0.01). Furthermore, embryos injected with CtlMO had Kvβ2 protein levels similar to those of uninjected embryos, suggesting that the injection itself or MOs in general did not produce nonspecific effects on IKv. In contrast, embryos injected with Kvβ2MO had significantly reduced levels of Kvβ2 protein at both 24 and 48 h (P 0.001). Kvβ2MO had no effect on levels of the internal control, β-tubulin (Fig. 3B). These results suggest that the Kvβ2MO led to a specific knock-down of Kvβ2 protein.
|
). As an alternative to MO-mediated knock-down of Kvβ2 protein, we used traditional RNA antisense (AS) methods to block translation of Kvβ2 subunits. We previously used this method to knock down Kvβ2 subunits and found that it effectively eliminated endogenous Kvβ2 mRNA in Xenopus embryos and Kv1 currents in embryonic myocytes (Lazaroff et al. 2002).
As found for the Kvβ2MO, antisense Kvβ2 (ASβ2) led to an efficient knock-down of IKv in a dose-dependent manner in both RB and non-RB neurons (Fig. 4). In 24-h embryos injected unilaterally with 1 ng ASβ2, internal control RB neurons displayed substantial IKv. In contrast, in GFP+ RB we found effective suppression of IKv (Fig. 4A). Similar effects of ASβ2 on IKv were observed in non-RB neurons (Fig. 4B). In comparison to the Kvβ2MO dose–response curve, the ASβ2 dose–response curve was less steep (Fig. 4C). However, at doses of ASβ2 0.25 ng, IKv was effectively eliminated in both RB and non-RB neurons. Therefore two different strategies for blocking Kvβ2 protein translation produced similar and efficient reductions in IKv density.
|
|
|
|
). As expected, if the morpholino targeted Kvβ2 mRNA specifically, Kvβ2MO injection had no effect on IKv density of ventral neurons (Fig. 7).
|
Dominant negative suppression of Kv1 function also abolished IKv in RB and non-RB dorsal cells
The preceding data suggest that Kvβ2 subunits are required for the formation of functional Kv1 potassium channel complexes in dorsal spinal neurons. In heterologous systems, Kvβ2 subunits associate with Kv1, Kv4, and Kv12 -subunits. Whereas expression of Kv4 or Kv12 -subunits in Xenopus embryos has not been reported, evidence for Kv1 -subunit expression in the spinal cord exists. Kv1.2 -subunit mRNA is expressed in the dorsal ectoderm at the time of neural induction (Ribera 1990). Kv1.1 -subunit transcripts localize to dorsal regions of the Xenopus spinal cord, similarly to Kvβ2 mRNA (Lazaroff et al. 1999; Ribera and Nguyen 1993). On this basis, Kv1 -subunits are good candidates for targets of Kvβ2 association and modulation.
Overexpression of a dominant negative Kv1 -subunit (Kv1DN) has served previously as an efficient method for blocking the contribution of the Kv1 subunit containing channels to IKv (Lazaroff et al. 2002; Ribera 1996). We used this approach to block functional Kv1 channels in situ. We found that dominant negative suppression of Kv1 -subunit-containing channels produced a nearly total reduction in IKv amplitude in both RB and non-RB neurons (Fig. 8), suggesting that Kv1 channels underlie the IKv of dorsal spinal neurons. Moreover, dominant negative suppression of Kv1 channels reduced IKv amplitudes as effectively as did Kvβ2MO or ASβ2, supporting the view that both β- and -subunits are obligate members of functional Kv1 channel complexes.
|
On the basis of previous studies of spinal neurons developing in culture, the IKv values of RB and non-RB neurons should undergo dramatic changes in density and activation kinetics between 24 and 48 h (Barish 1986; O'Dowd et al. 1988). To test this prediction, we determined how IKv changed in vivo between 24 and 48 h in RB and non-RB neurons.
RB IKv density increased about fivefold between 24 and 48 h (Fig. 9, A and B; 0.20 ± 0.06 to 0.90 ± 0.10 pA/µm2, respectively, at +20 mV; P < 0.0001). In contrast, during the same period, the rate of activation, evaluated as the time to half-maximum current (t1/2), did not change (Fig. 9C; 2.50 ± 0.23 vs. 2.48 ± 0.28 ms for 24 and 48 h, respectively, at +20 mV). Compared with the average developmental changes noted for IKv of spinal neurons in culture (e.g., O'Dowd et al. 1988), RB IKv in vivo showed larger increases in current density but no decrease in t1/2.
|
|
Between 24 and 48 h, non-RB IKv density increased about fourfold from 0.30 ± 0.10 to 1.10 ± 0.20 pA/µm2 (Fig. 10, A and B; +20 mV; P < 0.0001). However, no statistically significant differences were found between RB and non-RB IKv densities when compared at the same developmental stage (cf. Figs. 9, A and B and 10, A and B).
|
; O'Dowd et al. 1988). Consistent with the changes in current density, non-RB conductance density increased significantly about threefold between 24 and 48 h (Fig. 10D; 0.55 ± 0.10 to 1.60 ± 0.30 pS/µm2, respectively, P < 0.03). Contrary to RB IKv, however, non-RB IKv showed a negative shift in the V1/2 for steady-state activation between 24 and 48 h (Fig. 10E; Table 2), an effect that would lead to increased current amplitudes at more depolarized potentials. In addition, voltage sensitivity, as assessed by the slope factor k, decreased between 24 and 48 h (Table 2; P < 0.001). These results indicate that potassium conductance increased and altered its voltage-dependent properties of activation between 24 and 48 h.
As predicted on the basis of study of spinal neurons in culture (Barish 1986; O'Dowd et al. 1988), the IKv values of RBs and non-RBs showed substantial developmental regulation between 24 and 48 h of embryonic development (Figs. 9 and 10). However, the changes in each neuron type differed. The IKv of RB neurons increased in fivefold in density without any apparent changes in activation kinetics. In non-RB neurons, IKv also increased in density, about fourfold. In contrast to RBs, IKv of non-RB neurons also showed an apparent increase in activation kinetics, as assessed by a decrease in t1/2.
Kv1 -subunits underlie developmental changes in IKv of both RB and non-RB neurons
Knock-down of either Kvβ2 or dominant negative suppression of Kv1 channels effectively eliminated the IKv values of RB and non-RB neurons at 24 h (Figs. 2, 4, and 8). Previous results suggest that developmental changes in IKv in some spinal neurons developing in culture rely on Kv1 channels (Gurantz et al. 1996; Ribera 1996). Accordingly, we next determined whether Kv1 channels account not only for the initial IKv recorded at 24 h from RB and non-RB neurons, but also for the different developmental changes in IKv in each neuron type.
Similar to results obtained at 24 h (Fig. 2), Kvβ2MO injection led to efficient elimination of IKv at 48 h (Fig. 11A). Kvβ2MO also led to efficient knock-down of IKv in non-RB neurons at 48 h (Fig. 11B). However, inward current densities did not differ between uninjected, CtlMO, or Kvβ2MO neurons at 48 h (Fig. 11C). Moreover, injection of ASβ2 produced results for RB (Fig. 11D) and non-RB (Fig. 11E) that were similar to those obtained by injection of Kvβ2MO. These results indicate that at both 24 and 48 h, knock-down of Kvβ2 subunits eliminated IKv.
|
|
|
DISCUSSION |
|---|
|
-subunit. Our results point to a yet more fundamental role in vivo, whereby Kv1 channel function has an essential requirement for the Kvβ2 subunit in dorsal spinal neurons. Knock-down of Kvβ2 protein, by either MO or RNA antisense methods, produced a nearly total reduction in IKv amplitude in dorsal spinal cord neurons. Further, partial knock-down of Kvβ2 protein reduced only the amplitude but not the properties (e.g., t1/2, V1/2) of IKv, consistent with normal function of Kvβ2 subunits in channels underlying the residual current (Fig. 3, Table 1). Moreover, Kvβ2 knock-down produced effects similar to those of dominant negative suppression of Kv1 channel complexes, as expected if Kvβ2 subunits function as obligatory components of Kv1 channels.
In heterologous systems, Kvβ2 subunits modulate properties of potassium currents (Accili et al. 1997; England et al. 1995a,b; Heinemann et al. 1994, 1995, 1996; Majumder et al. 1995; Morales et al. 1996; Rettig et al. 1994; Shi et al. 1996) that are developmentally regulated in embryonic spinal neurons, e.g., current density and activation kinetics (Barish 1986; Desarmenien et al. 1993; O'Dowd et al. 1988). However, we found that at both 24 and 48 h, Kvβ2 knock-down led to efficient elimination of IKv. These findings indicate that rather than play a role in developmental regulation of Kv1 channels, Kvβ2 subunits are always required for Kv1 channel function in two different dorsal spinal cord neurons.
The essential in vivo requirement for Kvβ2 subunits differs from conclusions obtained from study of heterologously expressed subunits. When expressed in Xenopus oocytes or cell lines (e.g., HEK293), Kvβ2 subunits can increase the surface and functional density of Kv1 channels. However, in heterologous systems, Kv1 channel formation occurs in the absence of the Kvβ2 subunit. It is possible that when Kv1 -subunits are expressed at physiological levels, the role of the Kvβ2 subunit becomes essential. In contrast, under the aphysiological and high expression levels associated with heterologous systems, the essential requirement may be masked. Further, we focused on two dorsal neuron types that are known to express Kvβ2 subunits but not other Kvβ subunits. It is likely that, for neurons that express multiple Kvβ subunits, another Kvβ may replace Kvβ2 when the latter is eliminated. Thus the essential role of Kvβ2 subunits may be evident only when it is absent in cells that express no other Kvβ subunit.
Heterologous system results would predict a reduction in Kv1 -subunit protein levels, maturation, and/or surface expression in the absence of Kvβ2. However, McCormack et al. (2002) found that the Kv1.2 -subunit underwent normal glycosylation to its mature form in a mouse Kvβ2 knock-out. In addition, Kv1.2 -subunit protein levels, as assessed by Western blot, were unchanged by knock-out of Kvβ2. Similarly, our results did not show any apparent changes in Kv1.1 protein expression as determined by Western blot analysis in ASβ2- or Kvβ2MO-injected embryos (Fig. 5). In the murine model, immunofluorescent examination of Kv1.1 and Kv1.2 -subunits in brain sections also showed no differences between wild-type and knock-out with respect to either localization or amount (McCormack et al. 2002).
The knock-out mice were also examined for behavioral deficits but not for defects that would be evident at the level of cellular electrophysiology. Our results suggest that the increased seizure incidence and shorter life spans of Kvβ2 knock-out mice might be a direct consequence of severe loss of Kv1 channel function. Consistent with this view, Kv1.1 -subunit knock-out mice also have seizures (Smart et al. 1998; Wenzel et al. 2007). Similarly, Kv1.2 -subunt knock-out mice have seizures and reduced life spans (Brew et al. 2007).
Our results are consistent with those obtained by study of knock-out mice. We found that Kvβ2 subunits were not required for maturation of Kv1.1 -subunit-containing channels in the endoplasmic reticulum and Golgi because no increase in the immature form was detected after knock-down of Kvβ2 (Fig. 5). On the basis of our data, we conclude that Kvβ2 subunits affect trafficking of Kv1 channels to the surface membrane or the function of Kv1 channels in the surface membrane.
The finding that either Kvβ2 knock-down or dominant negative suppression of Kv1 channel function eliminated IKv leads to two additional conclusions. Our results provide evidence for Kvβ2 participation in Kv1 but not other Kv channels (e.g., Kv4, Kv12; Wilson et al. 1998; Yang et al. 2001). Previously, we found that Kvβ2 knock-down and Kv1 dominant negative suppression reduced IKv to the same extent in embryonic myocytes (Lazaroff et al. 2002). Although the identity of the current that persisted on Kvβ2 knock-down and Kv1 dominant negative suppression was not determined, Kv2 channels are likely candidates because myocytes express the gene (Burger and Ribera 1996). Because neither previous studies nor the present study examined cell types that are known to express either Kv4 or Kv12 -subunits, our results do not exclude the possibility that Kvβ2 subunits might affect function of Kv4 or Kv12 channels in vivo.
Second, recent work has indicated that the IKv properties of dorsal (type I) and ventral (type II) spinal cord neurons differ with respect to biophysical and pharmacological properties (Pineda and Ribera 2008). We interpret these data as evidence for Kv1 channel complexes serving as the molecular determinants of IKv in dorsal type I but not ventral type II spinal neurons. Interestingly, even though Kv1 channels underlie the IKvs of two different dorsal spinal neuron types, our results demonstrated functional differences between the IKv values of RB and non-RB neurons. The developmental increases in potassium conductance were similar in RBs and non-RBs (five- and fourfold, respectively; Figs. 9 and 10). However, the developmental changes in activation kinetics differed (Figs. 9 and 10). Between 24 and 48 h, the t1/2 did not change for RB IKv but did for that of non-RBs. From study of spinal neurons in culture, a decrease, rather than no change, in t1/2 was expected (O'Dowd et al. 1988).
The simplest interpretation of these data is that 1) RBs and non-RBs express different Kv1 -subunit isotypes and 2) Kvβ2 subunits are obligate members of a diverse range of Kv1 channel complexes at the developmental stages relevant to our studies. An alternative, more complex, interpretation is that RBs and non-RBs express the same Kv1 -subunit isotype but then apply different posttranslational mechanisms to sculpt functional properties of the native current during development. The latter possibility is especially intriguing given the findings of Desarmenien and Spitzer (1991). These authors found that the developmental change in activation kinetics of IKv was partially accounted for by a calcium/protein kinase C–dependent mechanism. Desarmenien and Spitzer (1991) studied spinal neurons in a culture of unknown identity. Our data suggest that the partial dependence on calcium and protein kinase C was a consequence of the necessary population analysis of the heterogeneous spinal cord neuron types that exist in culture, rather than study of specific cell types that is possible in the in situ preparations. For example, we found that the IKv of non-RBs, but not RBs, showed a developmental change in activation kinetics. In addition, it is possible that the calcium/protein kinase C mechanism could target some potassium channel subunit isotypes but not others. The in situ preparation that we have developed will allow future testing of these possibilities.
Overall, the results indicate that the "auxiliary" Kvβ2 subunit plays an essential role for Kv1 channel function in dorsal spinal neurons. In addition, Kv1 channel function shows the same absolute requirement for Kvβ2 subunits at both 24 and 48 h. Thus Kvβ2 subunits do not mediate the changes in IKv that occur during this period. Rather, developmental changes in Kv1 -subunit isoform expression or posttranslational modification underlie maturation of IKv in dorsal spinal neurons.
|
|
GRANTS |
|---|
|
|
|
ACKNOWLEDGMENTS |
|---|
|
|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: A. B. Ribera, Department of Physiology and Biophysics MS8307, 12800 East 19th Avenue, University of Colorado Denver at AMC, Aurora, CO 80045 (E-mail: angie.ribera{at}uchsc.edu)
|
|
REFERENCES |
|---|
|
Baccaglini PI, Spitzer NC. Developmental changes in the inward current of the action potential of Rohon–Beard neurones. J Physiol 271: 93–117, 1977.
Barish ME. Differentiation of voltage-gated potassium current and modulation of excitability in cultured amphibian spinal neurones. J Physiol 375: 229–250, 1986.
Bekele-Arcuri Z, Matos MF, Manganas L, Strassle BW, Monaghan MM, Rhodes KJ, Trimmer JS. Generation and characterization of subtype-specific monoclonal antibodies to K+ channel - and β-subunit polypeptides. Neuropharmacology 35: 851–865, 1996.[CrossRef][Web of Science][Medline]
Blaine JT, Ribera AB. Kv2 channels form delayed-rectifier potassium channels in situ. J Neurosci 21: 1473–1480, 2001.
Brew HM, Gittelman JX, Silverstein RS, Hanks TD, Demas VP, Robinson LC, Robbins CA, McKee-Johnson J, Chiu SY, Messing A, Tempel BL. Seizures and reduced life span in mice lacking the potassium channel subunit Kv1.2, but hypoexcitability and enlarged Kv1 currents in auditory neurons. J Neurophysiol 98: 1501–1525, 2007.
Burger C, Ribera AB. Xenopus spinal neurons express Kv2 potassium channel transcripts during embryonic development. J Neurosci 16: 1412–1421, 1996.
Campomanes CR, Carroll KI, Manganas LN, Hershberger ME, Gong B, Antonucci DE, Rhodes KJ, Trimmer JS. Kv beta subunit oxidoreductase activity and Kv1 potassium channel trafficking. J Biol Chem 277: 8298–8305, 2002.
Chen ML, Hoshi T, Wu CF. Heteromultimeric interactions among K+ channel subunits from Shaker and eag families in Xenopus oocytes. Neuron 17: 535–542, 1996.[CrossRef][Web of Science][Medline]
Chen ML, Hoshi T, Wu CF. Sh and eag K+ channel subunit interaction in frog oocytes depends on level and time of expression. Biophys J 79: 1358–1368, 2000.[Web of Science][Medline]
Chouinard SW, Wilson GF, Schlimgen AK, Ganetzky B. A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus. Proc Natl Acad Sci USA 92: 6763–6767, 1995.
Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Miera E, Rudy B. Molecular diversity of K+ channels. Ann NY Acad Sci 868: 233–285, 1999.[CrossRef][Web of Science][Medline]
Desarmenien MG, Clendening B, Spitzer NC. In vivo development of voltage-dependent ionic currents in embryonic Xenopus spinal neurons. J Neurosci 13: 2575–2581, 1993.[Abstract]
Desarmenien MG, 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]
Eisen JS, Smith JC. Controlling morpholino experiments: don't stop making antisense. Development 135: 1735–1743, 2008.
England SK, Uebele VN, Kodali J, Bennet PB, Tamkun MM. A novel K+ channel beta subunit (hKv beta 1.3) is produced via alternative mRNA splicing. J Biol Chem 270: 28531–28534, 1995a.
England SK, Uebele VN, Shear H, Kodali J, Bennet PB, Tamkun MM. Characterization of a voltage gated K+ channel beta subunit expressed in human heart. Proc Natl Acad Sci USA 92: 6309–6313, 1995b.
Gurantz D, Ribera AB, Spitzer NC. Temporal regulation of Shaker and Shab-like potassium potassium channel gene expression in single embryonic spinal neurons during K+ current development. J Neurosci 16: 3287–3295, 1996.
Gutman GA, Chandy KG, Adelman JP, Aiyar J, Bayliss DA, Clapham DE, Covarriubias M, Desir GV, Furuichi K, Ganetzky B, Garcia ML, Grissmer S, Jan LY, Karschin A, Kim D, Kuperschmidt S, Kurachi Y, Lazdunski M, Lesage F, Lester HA, McKinnon D, Nichols CG, O'Kelly I, Robbins J, Robertson GA, Rudy B, Sanguinetti M, Seino S, Stuehmer W, Tamkun MM, Vandenberg CA, Wei A, Wulff H, Wymore RS. International Union of Pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels. Pharmacol Rev 55: 583–586, 2003.
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch 391: 85–100, 1981.[CrossRef][Web of Science][Medline]
Heinemann S, Rettig J, Scott V, Parcej DN, Lorra C, Dolly J, Pongs O. The inactivation behaviour of voltage-gated K-channels may be determined by association of alpha- and beta-subunits. J Physiol (Paris) 88: 173–180, 1994.[CrossRef][Web of Science][Medline]
Heinemann SH, Rettig J, Graack HR, Pongs O. Functional characterization of Kv channel β-subunits from rat brain. J Physiol 493: 625–633, 1996.
Heinemann SH, Rettig J, Wunder F, Pongs O. Molecular and functional characterization of a rat brain Kvβ3 potassium channel subunit. FEBS Lett 377: 383–389, 1995.[CrossRef][Web of Science][Medline]
Hille B. Ion Channels of Excitable Membranes. Sunderland, MA: Sinauer, 2001.
Ikeda K, Kaplan WD. Patterned neural activity of a mutant Drosophila melanogaster. Proc Natl Acad Sci USA 66: 765–772, 1970.
Jones SM, Ribera AB. Overexpression of a potassium channel gene perturbs neural differentiation. J Neurosci 14: 2789–2799, 1994.[Abstract]
Kaplan WD, Trout WE. Genetic manipulation of an abnormal jump response in Drosophila. Genetics 77: 721–739, 1974.
Lazaroff MA, Hofmann AD, Ribera AB. Xenopus embryonic spinal neurons express potassium channel Kvβ subunits. J Neurosci 19: 10706–10715, 1999.
Lazaroff MA, Taylor AD, Ribera AB. In vivo analysis of Kvβ2 function in Xenopus embryonic myocytes. J Physiol 541: 673–683, 2002.
Li Y, Um SY, McDonald TV. Voltage-gated potassium channels: regulation by accessory subunits. Neuroscientist 12: 199–210, 2006.
Lockery SR, Spitzer NC. Reconstruction of action potential development from whole-cell currents of differentiating spinal neurons. J Neurosci 12: 2268–2287, 1992.[Abstract]
Majumder K, De Biasi M, Wang Z, Wible BA. Molecular cloning and functional expression of a novel potassium channel beta-subunit from human atrium. FEBS Lett 361: 13–16, 1995.[CrossRef][Web of Science][Medline]
Manganas LN, Trimmer JS. Subunit composition determines Kv1 potassium channel surface expression. J Biol Chem 275: 29685–29693, 2000.
Marty A, Neher E. Tight-seal whole-cell recording. In: Single Channel Recording, edited by Sakmann B, Neher E. New York: Plenum Press, 1983, p. 107–122.
McCormack K, Connor JX, Zhou L, Ho LL, Ganetzky B, Chiu SY, Messing A. Genetic analysis of the mammalian K+ channel beta subunit Kvβ2 (Kcnab2). J Biol Chem 277: 13219–13228, 2002.
Morales MJ, Wee JO, Wang S, Strauss HC, Rasmusson RL. The N-terminal domain of a K+ channel beta subunit increases the rate of C-type inactivation from the cytoplasmic side of the channel. Proc Natl Acad Sci USA 93: 15119–15123, 1996.
Nagaya N, Papazian DM. Potassium channel alpha and beta subunits assemble in the endoplasmic reticulum. J Biol Chem 272: 3022–3027, 1997.
Nakahira K, Shi G, Rhodes KJ, Trimmer JS. Selective interaction of voltage-gated K+ channel beta-subunits with alpha-subunits. J Biol Chem 271: 7084–7089, 1996.
Nieuwkoop PD, Faber J. Normal Table of Xenopus laevis (Daudin). A Systematical and Chronological Survey of the Development from the Fertilized Egg Till the End of Metamorphosis. Amsterdam: North-Holland, 1967.
O'Dowd DK, Ribera AB, Spitzer NC. Development of voltage-dependent calcium, sodium, and potassium currents in Xenopus spinal neurons. J Neurosci 8: 792–805, 1988.[Abstract]
Parcej DN, Scott VE, Dolly JO. Oligomeric properties of alpha-dendrotoxin-sensitive potassium ion channels purified from bovine brain. Biochemistry 31: 11084–11088, 1992.[CrossRef][Web of Science][Medline]
Pineda RH, Heiser RA, Ribera AB. Molecular determinants of INa in vivo in embryonic zebrafish sensory neurons. J Neurophysiol 93: 3582–3593, 2005.
Pineda RH, Ribera AB. Dorsal-ventral gradient for neuronal plasticity in the embryonic spinal cord. J Neurosci 28: 3824–3834, 2008.
Pongs O, Leicher T, Berger M, Roeper J, Bahring R, Wray D, Giese KP, Silva AJ, Storm JF. Functional and molecular aspects of voltage-gated K+ channel beta subunits. Ann NY Acad Sci 868: 344–355, 1999.[CrossRef][Web of Science][Medline]
Rettig J, Heinemann SH, Wunder F, Lorra C, Parcej DN, Dolly JO, Pongs O. Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit. Nature 369: 289–294, 1994.[CrossRef][Web of Science][Medline]
Rhodes KJ, Keilbaugh SA, Barrezueta NX, Lopez KL, Trimmer JS. Association and colocalization of K+ channel alpha- and beta-subunit polypeptides in rat brain. J Neurosci 15: 5360–5371, 1995.[Abstract]
Rhodes KJ, Monaghan MM, Barrezueta NX, Nawoschik S, Bekele-Arcuri Z, Matos MF, Nakahira K, Schechter LE, Trimmer JS. Voltage-gated K+ channel beta subunits: expression and distribution of Kvβ1 and Kvβ2 in adult rat brain. J Neurosci 16: 4846–4860, 1996.
Rhodes KJ, Strassle BW, Monaghan MM, Bekele-Arcuri Z, Matos MF, Trimmer JS. Association and colocalization of the Kvβ1 and Kvβ2 beta-subunits with Kv1 alpha-subunits in mammalian brain K+ channel complexes. J Neurosci 17: 8246–8258, 1997.
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, Nguyen DA. Primary sensory neurons express a Shaker-like potassium channel gene. J Neurosci 13: 4988–4996, 1993.[Abstract]
Ribera AB, Nusslein-Volhard C. Zebrafish touch-insensitive mutants reveal an essential role for the developmental regulation of sodium current. J Neurosci 18: 9181–9191, 1998.
Scott VE, Muniz ZM, Sewing S, Lichtinghagen R, Parcej DN, Pongs O, Dolly JO. Antibodies specific for distinct Kv subunits unveil a heterooligomeric basis for subtypes of alpha-dendrotoxin-sensitive K+ channels in bovine brain. Biochemistry 33: 1617–1623, 1994.[CrossRef][Web of Science][Medline]
Shamotienko OG, Parcej DN, Dolly JO. Subunit combinations defined for K+ channel Kv1 subtypes in synaptic membranes from bovine brain. Biochemistry 36: 8195–8201, 1997.[CrossRef][Web of Science][Medline]
Shi G, Nakahira K, Hammond S, Rhodes KJ, Schechter LE, Trimmer JS. Beta subunits promote K+ channel surface expression through effects early in biosynthesis. Neuron 16: 843–852, 1996.[CrossRef][Web of Science][Medline]
Smart SL, Lopantsev V, Zhang CL, Robbins CA, Wang H, Chiu SY, Schwartzkroin PA, Messing A, Tempel BL. Deletion of the Kv1.1 potassium channel causes epilepsy in mice. Neuron 20: 809–819, 1998.[CrossRef][Web of Science][Medline]
Tang CY, Schulteis CT, Jimenez RM, Papazian DM. Shaker and ether-a-go-go K+ channel subunits fail to coassemble in Xenopus oocytes. Biophys J 75: 1263–1270, 1998.[Web of Science][Medline]
Torres YP, Morera FJ, Carvacho I, Latorre R. A marriage of convenience: beta-subunits and voltage-dependent K+ channels. J Biol Chem 282: 24485–24489, 2007.
Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Biotechnology 24: 145–149, 1992.[Medline]
Trimmer JS. Regulation of ion channel expression by cytoplasmic subunits. Curr Opin Neurobiol 8: 370–374, 1998.[CrossRef][Web of Science][Medline]
Wang JW, Wu CF. In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels. Biophys J 71: 3167–3176, 1996.[Web of Science][Medline]
Wenzel HJ, Vacher H, Clark E, Trimmer JS, Lee AL, Sapolsky RM, Tempel BL, Schwartzkroin PA. Structural consequences of Kcna1 gene deletion and transfer in the mouse hippocampus. Epilepsia 48: 2023–2046, 2007.[CrossRef][Web of Science][Medline]
Wilson GF, Wang Z, Chouinard SW, Griffith LC, Ganetzky B. Interaction of the K channel beta subunit, Hyperkinetic, with eag family members. J Biol Chem 273: 6389–6394, 1998.
Xu J, Yu W, Wright JM, Raab RW, Li M. Distinct functional stoichiometry of potassium channel beta subunits. Proc Natl Acad Sci USA 95: 1846–1851, 1998.
Yang EK, Alvira MR, Levitan ES, Takimoto K. Kvβ subunits increase expression of Kv4.3 channels by interacting with their C termini. J Biol Chem 276: 4839–4844, 2001.
Yao WD, Wu CF. Auxiliary Hyperkinetic beta subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons. J Neurophysiol 81: 2472–2484, 1999.
This article has been cited by other articles:
|
|
 |
|
  J. Golowasch, G. Thomas, A. L. Taylor, A. Patel, A. Pineda, C. Khalil, and F. Nadim Membrane Capacitance Measurements Revisited: Dependence of Capacitance Value on Measurement Method in Nonisopotential Neurons J Neurophysiol, October 1, 2009; 102(4): 2161 - 2175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Moreno and A. B. Ribera Zebrafish Motor Neuron Subtypes Differ Electrically Prior to Axonal Outgrowth J Neurophysiol, October 1, 2009; 102(4): 2477 - 2484. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
TOP
ABSTRACT
INTRODUCTION
