The contribution of Kv2.2-mediated currents decreases during the postnatal development of mouse dorsal root ganglion neurons
2016; Wiley; Volume: 4; Issue: 6 Linguagem: Inglês
10.14814/phy2.12731
ISSN2051-817X
AutoresGlenn Regnier, Elke Bocksteins, Gerda Van de Vijver, Dirk J. Snyders, Pierre‐Paul van Bogaert,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoPhysiological ReportsVolume 4, Issue 6 e12731 Original ResearchOpen Access The contribution of Kv2.2-mediated currents decreases during the postnatal development of mouse dorsal root ganglion neurons Glenn Regnier, Glenn Regnier Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, CDE, Antwerpen, BelgiumSearch for more papers by this authorElke Bocksteins, Elke Bocksteins Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, CDE, Antwerpen, BelgiumSearch for more papers by this authorGerda Van de Vijver, Gerda Van de Vijver Laboratory for Cardiovascular Research, Institute Born-Bunge, University of Antwerp, CDE, Antwerpen, BelgiumSearch for more papers by this authorDirk J. Snyders, Corresponding Author Dirk J. Snyders Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, CDE, Antwerpen, Belgium Correspondence Dirk J. Snyders, Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, CDE, Universiteitsplein 1, 2610 Antwerpen, Belgium. Tel: +32-3-265.23.35 Fax: +32-3-265.23.26 E-mail: dirk.snyders@uantwerpen.beSearch for more papers by this authorPierre-Paul van Bogaert, Pierre-Paul van Bogaert Laboratory for Cardiovascular Research, Institute Born-Bunge, University of Antwerp, CDE, Antwerpen, BelgiumSearch for more papers by this author Glenn Regnier, Glenn Regnier Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, CDE, Antwerpen, BelgiumSearch for more papers by this authorElke Bocksteins, Elke Bocksteins Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, CDE, Antwerpen, BelgiumSearch for more papers by this authorGerda Van de Vijver, Gerda Van de Vijver Laboratory for Cardiovascular Research, Institute Born-Bunge, University of Antwerp, CDE, Antwerpen, BelgiumSearch for more papers by this authorDirk J. Snyders, Corresponding Author Dirk J. Snyders Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, CDE, Antwerpen, Belgium Correspondence Dirk J. Snyders, Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, CDE, Universiteitsplein 1, 2610 Antwerpen, Belgium. Tel: +32-3-265.23.35 Fax: +32-3-265.23.26 E-mail: dirk.snyders@uantwerpen.beSearch for more papers by this authorPierre-Paul van Bogaert, Pierre-Paul van Bogaert Laboratory for Cardiovascular Research, Institute Born-Bunge, University of Antwerp, CDE, Antwerpen, BelgiumSearch for more papers by this author First published: 31 March 2016 https://doi.org/10.14814/phy2.12731Citations: 3 Funding Information This work was supported by the section cardiovascular research of the Sheid-van Bogaert foundation, the postdoctoral fellowships FWO-1291913N and FWO-1291916N to EB and the grants G.0449.11N & G.0443.12N to DJS from the Research Foundation – Flanders (FWO). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Delayed rectifier voltage-gated K+ (Kv) channels play an important role in the regulation of the electrophysiological properties of neurons. In mouse dorsal root ganglion (DRG) neurons, a large fraction of the delayed rectifier current is carried by both homotetrameric Kv2 channels and heterotetrameric channels consisting of Kv2 and silent Kv (KvS) subunits (i.e., Kv5-Kv6 and Kv8-Kv9). However, little is known about the contribution of Kv2-mediated currents during the postnatal development of DRG neurons. Here, we report that the Stromatoxin-1 (ScTx)-sensitive fraction of the total outward K+ current (IK) from mouse DRG neurons gradually decreased (~13%, P < 0.05) during the first month of postnatal development. Because ScTx inhibits both Kv2.1- and Kv2.2-mediated currents, this gradual decrease may reflect a decrease in currents containing either subunit. However, the fraction of Kv2.1 antibody-sensitive current that only reflects the Kv2.1-mediated currents remained constant during that same period. These results suggested that the fractional contribution of Kv2.2-mediated currents relative to IK decreased with postnatal age. Semiquantitative RT-PCR analysis indicated that this decrease can be attributed to developmental changes in Kv2.2 expression as the mRNA levels of the Kv2.2 subunit decreased gradually between 1 and 4 weeks of age. In addition, we observed age-dependent fluctuations in the mRNA levels of the Kv6.3, Kv8.1, Kv9.1, and Kv9.3 subunits. These results support an important role of both Kv2 and KvS subunits in the postnatal maturation of DRG neurons. Introduction Neuronal function depends heavily on the spatial and temporal expression of voltage-gated K+ (Kv) channels which contribute to neuronal excitability by regulating the membrane potential, action potential waveform and firing frequency, transmitter release, and synaptic strength (Hille 2001). Kv channels are integral transmembrane proteins consisting of four α-subunits that form a central ion conducting pore through which K+ ions flow according to their electrochemical gradient. Each α-subunit consists of six transmembrane segments (S1-S6) and a cytoplasmic NH2- and COOH-terminus (Long et al. 2005). Based on sequence homology, eight closely Shaker-related subfamilies can be distinguished: Kv1-Kv6 and Kv8-Kv9 (Gutman et al. 2005). All members of the Kv1-Kv4 subfamilies form functional homotetrameric channels and the diversity within these subfamilies is further increased by both the formation of heterotetrameric channels and the interaction with auxiliary β-subunits (Rhodes et al. 1995; Xu et al. 1995). On the other hand, members of the Kv5, Kv6, Kv8, and Kv9 subfamilies do not form functional channels due to retention in the endoplasmic reticulum (ER) and were therefore designated silent Kv (KvS) subunits (for review see (Bocksteins and Snyders 2012)). Co-assembly of these KvS subunits with members of the Kv2 subfamily relieves this ER retention leading to heterotetrameric Kv2/KvS channel complexes with biophysical properties that differ from the homotetrameric Kv2 channels. These differences include shifts in the voltage dependence of activation and inactivation, changes in gating kinetics, and alteration of the current density. Due to the molecular diversity of Kv channel complexes, it is very challenging to determine the molecular composition of native Kv currents in neurons. In rodent dorsal root ganglion (DRG) neurons, at least three different Kv currents have been distinguished: the M-current (IM), the transient outward current (IA), and the delayed rectifier current (IDR) (Akasu and Tokimasa 1992; Gold et al. 1996; Fedulova et al. 1998). The M-current is a non-inactivating K+ current generated by channels composed of Kv7.2-Kv7.5 subunits; the major component is carried by heterotetrameric Kv7.2/Kv7.3 channels and homotetrameric Kv7.2 channels (Passmore et al. 2003). IA is a very fast activating and inactivating current generated by Kv1.4, Kv3.4, Kv4.1, Kv4.2, and/or Kv4.3 subunits. Depending on the subpopulation of DRG neurons, one or several of these subunits contribute to IA (Rasband et al. 2001; Winkelman et al. 2005; Chien et al. 2007; Phuket and Covarrubias 2009). The major component (≈60%) of IDR is carried by both homotetrameric Kv2 and heterotetrameric Kv2/KvS channels, although subunits of the Kv1 and Kv3 subfamilies also contribute to IDR (Beekwilder et al. 2003; Utsunomiya et al. 2008; Bocksteins et al. 2009, 2012). The functional diversity of different neuronal subtypes does not only emanate from the molecular composition of different channel complexes but also from the variation in expression of K+ currents during different developmental stages (for review see (Ribera and Spitzer 1992)). In the case of the Kv2 subfamily, changes in spatiotemporal expression and cellular abundance have been demonstrated during the development of different neuronal cell types (Maletic-Savatic et al. 1995; Gurantz et al. 1996; Antonucci et al. 2001; Guan et al. 2011; Sanchez-Ponce et al. 2012). For example, in rat neocortical pyramidal neurons, the density of Kv2-mediated currents increased with postnatal age (Guan et al. 2011) while in embryonic Xenopus spinal neurons, a Kv2.2-specific upregulation was demonstrated during maturation (Gurantz et al. 1996). However, it is not known if the contribution of Kv2-mediated currents to IK in DRG neurons is influenced by postnatal age. Therefore, we analyzed the Kv2-containing currents and characterized the expression of Kv2 and their modulatory KvS subunits in mouse DRG neurons during the first month of postnatal development. Material and Methods Animals and cell culture Dorsal root ganglion neurons were obtained from P7 ± 1, P14 ± 1, P21 ± 1, and P28 ± 1 old C57BL/6 male mice. Experiments were conducted in agreement with the European Communities Council Directive on the protection of animals used for experimental and other scientific purposes (2010/63/EU). DRG neurons were isolated as described previously (Schnizler et al. 2008). Briefly, DRGs were dissected from the spinal cord and dissociated by consecutive enzymatic treatment with 2 mg/mL collagenase A (Merck Millipore, Billerica, MA) and 1 mg/mL pronase (Merck Millipore). After enzymatic dissociation, DRG neurons were further dissociated using flame-polished Pasteur pipettes of decreasing diameters and plated on glass-bottom dishes coated with poly-D-lysine (MatTek Corp., Ashland, MA). Cells were grown in 50:50 DMEM/TNB medium (ThermoFisher Scientific, Waltham, MA/Merck Millipore) supplemented with 2.5% horse serum (ThermoFisher Scientific), 2.5% fetal bovine serum (ThermoFisher Scientific), 100 U/mL penicillin/streptomycin, 1.25% lipid-protein complex (Merck Millipore), 1 mmol/L l-glutamine and 0.25 μg/mL nerve growth factor (Sigma-Aldrich, Saint Louis, MO), and maintained at 37°C in a humidified atmosphere of 5% CO2. Electrophysiological and RT-PCR analyses were performed 3 days after plating. Electrophysiology Whole-cell patch clamp current recordings were performed on DRG neurons (30–60 pF) at room temperature (20–22°C) with an Axoclamp-2A amplifier (Molecular Devices, Sunnyvale, CA) in the two-electrode voltage clamp configuration and were sampled with a TL-1 labmaster (Molecular Devices). Patch pipettes with a resistance of 3–5 MΩ were pulled from 1.7 mm glass capillaries with a Brown Flaming P-87 horizontal pipette puller and heat-polished. DRG neurons were superfused continuously with an extracellular solution, containing (in mmol/L): 140 N-methyl d-glucamine, 5 KCl, 1 MgCl2, 1.8 CaCl2, 10 glucose, and 5 HEPES with the pH adjusted to 7.4 with HCl. Pipettes were filled with an intracellular solution, containing (in mmol/L): 140 KCl, 10 HEPES, 5 EGTA, 5 NaCl, 3 MgATP, 1 MgCl2, 1 CaCl2, and 0.1 cAMP with the pH adjusted to 7.4 with KOH. Outward K+ currents were elicited by 500 msec depolarizing pulses between −60 and +60 mV from a holding potential of −70 mV, followed by a 1 sec pulse at −40 mV. Cell capacitance was obtained from the current evoked by a 30 msec step from −60 to −65 mV. Stromatoxin-1 (ScTx)-sensitive currents were obtained by subtracting the currents obtained after application of 300 nmol/L ScTx (Alomone Labs, Jerusalem, Israel) (dissolved in the extracellular solution) from the currents obtained before ScTx application. For the anti-Kv2.1 current recordings, patch pipettes were dipped in normal intracellular solution and back filled with the anti-Kv2.1-containing solution obtained by dissolving 10 μg/mL Kv2.1 antibody (Alomone Labs) in the intracellular solution. Steady-state reduction of the total outward K+ current was reached 15–20 min after patch rupture. The specificity of this reduction (i.e., due to Kv2.1 antibody block and not due to time artifacts) was confirmed previously (Bocksteins et al. 2009). The anti-Kv2.1-sensitive currents were obtained by subtracting the currents obtained after steady-state Kv2.1 block from the currents obtained immediately after patch rupture. RT-PCR analysis Total RNA was isolated from the DRG cultures as previously described (Bocksteins et al. 2012). Briefly, RNA was isolated using the TriZol (ThermoFisher Scientific) reagent, samples were treated with deoxyribonuclease I (ThermoFisher Scientific) to exclude genomic DNA contamination and cDNA was obtained using the Superscript III RT-PCR system (ThermoFisher Scientific) according to the manufacturer's guidelines. Expression of the Kv2 and KvS subunits was assessed using gene-specific primers that spanned intron boundaries (except for the intronless Kv5.1) (Table 1). Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was used as a loading control to perform the semiquantitative RT-PCR analysis. Co-amplification of the target gene and G3PDH was performed in a reaction mixture containing 1× Colorless GoTaq Flexi buffer, 3 mmol/L MgCl2, 0.4 mmol/L dNTP mix, 2.5 U GoTaq G2 Flexi DNA Polymerase (Progema, Madison, WI), and 0.5 μmol/L of each forward and reverse primer. The cDNA samples were amplified for 35 cycles, separated on a 1% agarose gel, and stained with SYBR Safe gel stain (ThermoFisher Scientific) for densitometric analysis. For each PCR analysis, one positive control (reaction that contains the target subunit cDNA) and two negative controls (reaction without cDNA or without reverse transcriptase) were performed. We ensured that the amplification of each gene was still within the exponential phase of the PCR reaction after 35 cycles by comparing the densitometric values with these obtained after 33 and 37 cycles of amplification. Gels were scanned with the LumiImager system using the LumiAnalyst software (Roche Diagnostics, Basel, Switzerland) and the data were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD). Each PCR product was sequenced to exclude nonspecific amplification. Table 1. List of primer pairs used in semiquantitative RT-PCR experiments Primer pair sequence G3PDH 5′-ACGGGAAGCTCACTGGCATG-3′ 5′-GGGAGTTGCTGTTGAAGTCG-3′ Kv2.1 5′-TCGACAACACGTGCTGTGCT-3′ 5′-GGCCAACTTCAGGATGCGC-3′ Kv2.2 5′-TTGATAACACCTGCTGCCCG-3′ 5′-TGGCGAGTTTCAGTATCCTGA-3′ Kv5.1 5′-CTGGTGGGCTATCATCACCA-3′ 5′-CGGGTCCTATGATGCTTCTC-3′ Kv6.1 5′-GTCCGTTCTGTTTGTCACCG-3′ 5′-GGATCAGCACCCGTTCTTGT-3′ Kv6.2 5′-GGCTCTTCGCCTACGTCTC-3′ 5′-CATCACGCGTGCTGTCCTC-3′ Kv6.3 5′-GTGGTGTTCGTGATCGTGTC-3′ 5′-CTTGAGAGTCAAGCCCAGTG-3′ Kv6.4 5′-GCCAGGAGTTCTTCTTCGAC-3′ 5′-CATCAGGAGACCAAACTCTC-3′ Kv8.1 5′-TCTGCGCATGCTGAAACTGG-3′ 5′-AGTACTTGCTCTCTCCCTGC-3′ Kv8.2 5′-CTTCCGAATCCTCAAGCTGG-3′ 5′-GTTGACCTTTCCTCGTTCCC-3′ Kv9.1 5′-AGGTAGTGCAAGTGTTCCGC-3′ 5′-AAGTCCTCAAACTCGCGCTG-3′ Kv9.2 5′-TTCTCAAGCTGGCCAGACAC-3′ 5′-TGACCGAAGGGACCTCTTTC-3′ Kv9.3 5′-TGTAGGGCTTCGGTCTCTTG-3′ 5′-AGTACGGTAGCTCATGGCAC-3′ Data analysis Densitometric analysis of the bands obtained after electrophoresis was done using the ImageJ software. The signal intensity of the target band was normalized to the intensity of the G3PDH signal which was represented as relative densitometric value (RDV). All values are presented as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by Tukey's test in case a significant difference was present among the test groups. Trend analysis of age-dependent changes was performed using linear regression and represented by the regression coefficient ± SE. P-values < 0.05 were considered to be significant. Results The fractional contribution of the ScTx-sensitive current gradually decreases during postnatal development while that of the Kv2.1 antibody-sensitive current remains unchanged To study the potential changes in the Kv2-mediated currents during the postnatal development of mouse DRG neurons, we determined the fractional contribution of the ScTx-sensitive and anti-Kv2.1-sensitive currents relative to the total outward K+ current (IK) in these neurons obtained from mice at different postnatal ages. We used this dual approach to determine the fractional contribution of the Kv2.1- and Kv2.2-mediated currents relative to IK in developing DRG neurons since ScTx inhibits both Kv2.1- and Kv2.2-containing channels (Escoubas et al. 2002) while Kv2.1 antibodies only block Kv2.1-containing channels (Murakoshi and Trimmer 1999; Guan et al. 2007; Bocksteins et al. 2009). It has previously been demonstrated that the ScTx-induced Kv2 inhibition is less efficient at higher potentials (>0 mV) (Escoubas et al. 2002) and that the fractional contribution of Kv2-mediated currents relative to the whole-cell outward K+ current is higher after 500 msec (Bocksteins et al. 2012). Therefore, we determined the fractional contribution of the ScTx- and anti-Kv2.1-sensitive currents relative to IK in the DRG neurons of different developmental stages by normalizing their current density to the total outward K+ current density at the end of a 500 msec depolarizing pulse to 0 mV. The ScTx- and anti-Kv2.1-sensitive currents were obtained as described in Material and Methods, and the current density was determined by dividing the recorded current amplitude at 0 mV after 500 msec by the cell capacitance. For the different development stages, we isolated DRG neurons from P7 ± 1, P14 ± 1, P21 ± 1, and P28 ± 1 mice that were considered as 1, 2, 3, and 4 weeks old mice, respectively. Only DRG neurons with a cell capacitance in the 30–60 pF range were selected for analysis; as a result, the mean whole-cell capacitance of the analyzed DRG neurons did not change significantly during postnatal development (Fig. 1A). However, the current density of the outward K+ current rose gradually (regression coefficient = 30 ± 9 pA/pF/week, P < 0.05) with age reaching significance (P < 0.05) between 1 week (270 ± 15 pA/pF) and 4 weeks (370 ± 23 pA/pF) (Fig. 1B–C). Figure 1Open in figure viewerPowerPoint Postnatal development of the whole cell outward K+ current in mouse dorsal root ganglion (DRG) neurons. (A) Average cell capacitance of the recorded DRG neurons obtained from 1, 2, 3, and 4 weeks old mice. The numbers above each bar indicate the number of recorded cells. (B) Representative current recordings of the whole cell outward K+ current in DRG neurons obtained from 1 week (gray) and 4 weeks (black) old mice elicited by a 500 msec depolarizing pulse to 0 mV from a holding potential of −70 mV. (C) Postnatal development of the whole-cell outward K+ current density at the end of a 500 msec pulse at 0 mV. The current density at 4 weeks was significantly higher than the current density at 1 week, indicated with an asterisk (P < 0.05). The numbers above each bar indicate the number of cells analyzed. Typical current recordings of DRG neurons obtained from 1 and 4 weeks old mice before and after application of 300 nmol/L ScTx are shown in Figure 2A. Extracellular application of 300 nmol/L ScTx reduced IK significantly as previously described (Bocksteins et al. 2012). The absolute current density of the ScTx-sensitive current did not change in DRG neurons of the different age groups (regression coefficient: 0.26 ± 6.39 pA/pF/week, P = 0.97) (Fig. 2B): the ScTx-sensitive current density was 143 ± 15, 144 ± 13, 136 ± 10, and 146 ± 17 pA/pF in DRG neurons of 1, 2, 3, and 4 weeks old mice, respectively. However, the total outward K+ current density rose significantly (Fig. 1C) and therefore the fractional contribution of the ScTx-sensitive current relative to IK (FCScTx) decreased gradually (regression coefficient = −0.044 ± 0.012 FCScTx/week, P < 0.05) with age reaching significance (P < 0.05) at 4 weeks, compared to 1 and 2 weeks old mice (Fig. 2C): ScTx inhibited 52 ± 4% and 49 ± 3% of the outward K+ current at week 1 and week 2 respectively, while only inhibiting 39 ± 3% at week 4. These results demonstrated that the fractional contribution of Kv2-containing channels relative to IK in DRG neurons decreased during postnatal development, but these data did not discriminate between Kv2.1- and Kv2.2-containing channels. Therefore, we determined the fraction of anti-Kv2.1-sensitive currents. Figure 2Open in figure viewerPowerPoint Postnatal development of the ScTx-sensitive current in dorsal root ganglion (DRG) neurons at 0 mV. (A) Representative current recordings of the total outward K+ (left), ScTx-insensitive (right) and ScTx-sensitive (bottom) currents in DRG neurons obtained from 1 week (gray) and 4 weeks (black) old mice elicited by a 500 msec depolarizing pulse to 0 mV from a holding potential of −70 mV. The ScTx-sensitive current was obtained by subtracting the current after application of 300 nmol/L ScTx (i.e., ScTx-insensitive current) from the total outward K+ current. The scale bar applies to all current recordings. (B) Current densities of the ScTx-sensitive component in the different age groups. The ScTx-sensitive current density did not change during postnatal development. (C) Fraction of the ScTx-sensitive current at the different developmental stages obtained by normalizing the current density of the ScTx-sensitive current to the current density of the total outward K+ current at the end of the 500 msec pulse at 0 mV. The fraction of the ScTx-sensitive current relative to IK in DRG neurons from 1 and 2 weeks old mice was significantly larger compared to the same fraction at 4 weeks (*P < 0.05). The numbers above each bar indicate the number of cells analyzed. Representative current recordings of the anti-Kv2.1-sensitive currents obtained from 1 and 4 weeks old mice are shown in Figure 3A. Intracellular diffusion of Kv2.1 antibodies reduced IK significantly as previously described (Bocksteins et al. 2009). The current density of the anti-Kv2.1-sensitive current rose (regression coefficient: 7.7 ± 5.1 pA/pF/week, P = 0.14), although not significantly, from 70 ± 9 pA/pF at 1 week to 95 ± 14 pA/pF at 4 weeks (Fig. 3B). However, the fractional contribution of the anti-Kv2.1-sensitive current relative to IK remained similar in DRG neurons obtained from the different age groups (Fig. 3C): the Kv2.1 antibody blocked 27 ± 3%, 26 ± 3%, 28 ± 3%, and 28 ± 3% of the total outward K+ current of the DRG neurons from 1, 2, 3, and 4 weeks old mice, respectively. These results together indicated that the fractional contribution of Kv2.1-mediated currents relative to IK remained similar, whereas the fractional contribution of Kv2.2-mediated currents relative to IK decreased with postnatal age. Figure 3Open in figure viewerPowerPoint Postnatal development of anti-Kv2.1-sensitive current in dorsal root ganglion (DRG) neurons at 0 mV. (A) Representative current recordings of the total outward K+ (left), anti-Kv2.1-insensitive (right) and anti-Kv2.1-sensitive (bottom) currents in DRG neurons obtained from 1 week (gray) and 4 weeks (black) old mice elicited by a 500 msec depolarizing pulse to 0 mV from a holding potential of −70 mV. The anti-Kv2.1 sensitive current was obtained by subtracting the current after intracellular diffusion of Kv2.1 antibodies (i.e., anti-Kv2.1-insensitive current) from the total outward K+ current. The scale bar applies to all current recordings. (B) Current densities of the anti-Kv2.1-sensitive component in the different age groups. The anti-Kv2.1-sensitive current density rose gradually, although not significantly, during postnatal development. (C) The fraction of the anti-Kv2.1-sensitive current relative to IK at the different developmental stages obtained as described in the Results section remained unchanged. The numbers above each bar indicate the number of cells analyzed. Since only a fraction of the Kv2.1 and Kv2.2 channels are already in an open state at 0 mV, the above results could be due to an age-dependent depolarizing shift in the voltage dependence of activation of the Kv2-containing currents. Therefore, we determined the fraction of ScTx-sensitive and Kv2.1 antibody-sensitive current at higher depolarizing potentials (+20 and +40 mV) (Fig. 4). The fraction of the anti-Kv2.1-sensitive current remained similar at the different postnatal ages, both at +20 mV (Fig. 4A) and +40 mV (Fig. 4B), whereas the ScTx-sensitive fraction of IK reduced with age at both potentials. At +20 mV, the fractional contribution of the ScTx-sensitive current relative to IK decreased significantly (regression coefficient = −0.036 ± 0.013 FCScTx/week, P < 0.05): ScTx inhibited 47 ± 5% at 1 week which is significantly different (P < 0.05) from the inhibition at 4 weeks (34 ± 3%) (Fig. 4A). At +40 mV, the fractional contribution relative to IK decreased gradually (regression coefficient = −0.035 ± 0.019 FCScTx/week, P = 0.075), but not significantly, from 41 ± 3% at 1 week to 31 ± 3% at 4 weeks (Fig. 4B). Although the fractional contribution of the ScTx-sensitive current relative to IK at +20 and +40 mV is lower compared to the fractional contribution at 0 mV due to the incomplete inhibition of the Kv2-containing currents by ScTx at higher potentials (Escoubas et al. 2002), a comparable decrease with postnatal age could be observed at all analyzed potentials. These results suggested that the observed developmental changes are not due to age-dependent shifts in the voltage dependence of the Kv2.1- and/or Kv2.2-containing current. Figure 4Open in figure viewerPowerPoint Postnatal development of the ScTx- and anti-Kv2.1-sensitive current at +20 and +40 mV. The fraction of ScTx-sensitive current (left) and anti-Kv2.1-sensitive current (right) relative to IK of the different postnatal age groups at the end of a 500 msec depolarizing pulse at +20 mV (A) and +40 mV (B). (A) At +20 mV, the fraction of ScTx-sensitive current in dorsal root ganglion neurons from 1 week old mice was significantly larger compared to that from 4 week old mice (*P < 0.05), whereas the fraction of the anti-Kv2.1-sensitive current remained unchanged at the different developmental stages. (B) The fraction of ScTx-sensitive current reduced gradually (although not significantly) with postnatal age at +40 mV, while the fraction of anti-Kv2.1-sensitive current at the same potential remained constant during the same period. The numbers above each bar plot indicate the number of cells analyzed. The Kv2.2 mRNA level decreases during postnatal development while the Kv2.1 mRNA level remains similar To investigate whether the observed ScTx-sensitive and anti-Kv2.1-sensitive current densities can be attributed to developmental changes in Kv2.1 and/or Kv2.2 expression, we examined the Kv2.1 and Kv2.2 mRNA levels in our DRG cultures obtained at the different developmental stages using semiquantitative RT-PCR analysis, as described in Material and Methods. After 35 amplification cycles, the intensity of the Kv signal was normalized to the intensity of the G3PDH signal and plotted as RDV. This semiquantitative RT-PCR approach revealed that the level of Kv2.2 mRNA decreased gradually (regression coefficient = −0.082 ± 0.022 RDV/week, P < 0.05) with age, reaching significance (P < 0.05) after 3 weeks: the RDV value declined from 0.57 ± 0.03 at 1 week to 0.32 ± 0.06 and 0.35 ± 0.05 at 3 and 4 weeks, respectively (Fig. 5). On the other hand, the Kv2.1 mRNA level remained similar: the lowest RDV value was detected at 1 week (RDV = 0.70 ± 0.05 RDV/week), while the highest RDV value was detected at 4 weeks (RDV = 0.77 ± 0.11) (Fig. 5). These data indicated that the age-dependent decrease of the Kv2.2-mediated current was a result of a developmental decrease of Kv2.2 expression. Figure 5Open in figure viewerPowerPoint Postnatal development of the Kv2.1 and Kv2.2 mRNA levels in dorsal root ganglion (DRG) cultures. Electrophoretic analysis of the RT-PCR products obtained from cultured DRG neurons of 1 week, 2 weeks, 3 weeks, and 4 weeks old mice. The relative densitometric values (RDVs) (presented in panel C) were determined by normalizing the densitometric value of the 450 bp fragment, which corresponds to Kv2.1 (A) and Kv2.2 (B), to the densitometric value of G3PDH, which corresponds to the 250 bp fragment in both panels. (C) The RDV of Kv2.2 (white) decreased significantly determined in DRG cultures from 3 and 4 weeks old mice compared to the RDV determined in DRG cultures from 1-week-old mice (*P < 0.05). The RDV of Kv2.1 (black) remained unaltered in the different age groups. The numbers above every bar plot indicate the number of samples analyzed. Developmental changes in KvS mRNA levels It has been demonstrated that different KvS (i.e., Kv5, Kv6, Kv8 and Kv9) subunits are expressed in small mouse cultured DRG neurons (Bocksteins et al. 2009), and both Kv2.1 and Kv2.2 subunits assemble with those KvS subunits into heterotetrameric channels that possess unique biophysical properties (for review see (Bocksteins and Snyders 2012)). Furthermore, at least heterotetrameric Kv2.1/Kv9.3 and Kv2.1/Kv6.3 channels have been shown to be inhibited by ScTx (Escoubas et al. 2002; Moreno-Dominguez et al. 2009). In addition, we have demonstrated that Kv2.1/Kv9.3 currents are also blocked by Kv2.1 antibodies (Bocksteins et al. 2009). Therefore, it would be interesting to examine whether the observed developmental
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