XB-ART-18543J Neurosci February 15, 1996; 16 (4): 1412-21.
Xenopus spinal neurons express Kv2 potassium channel transcripts during embryonic development.
Developmentally regulated delayed rectifier potassium currents determine the waveform of the action potential in all Xenopus embryonic primary spinal neurons. To examine this developmental program at the molecular level, we have isolated Xenopus Kv2 potassium channel genes Kv2.1 and Kv2.2. Both genes induce functional heterologous expression of delayed rectifier potassium currents. Transcripts from both Kv2 genes are present in developing embryos; however, only Kv2.2 mRNA is detectable in embryonic spinal neurons. Notably, Kv2.2 transcripts localize to ventral spinal neurons, whereas previously described Kv1.1 transcripts are found in dorsal spinal neurons. Thus, spinal neuron subtypes express distinct potassium channel genes, yet they temporally coordinate functional expression of delayed rectifier potassium currents.
PubMed ID: 8778292
Article link: J Neurosci
Genes referenced: kcna1 kcna2 kcnb1 kcnb2 ncam1 tbx2
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|Figure 1. Nucleotide and deduced amino acid sequences of the coding regions ofxenopus Kv2.1 and Kv2.2 cDNAs. Amino acid and nucleotide positions are indicated in the right-hand column. The putative transmembrane domains (SI-S6) and the pore region (P) are overlined. A putative glycosylation site between S3 and S4 is indicated by a star. Consensus CAMP phosphorylation sites are indicated by filled circles above the amino acid position. The Xenopus Kv2.1 and Kv2.2 nucleotide sequences have been assigned Genbank accession numbers U20342 and U20343, respectively. A, Nucleotide and amino acid sequence of XShabY (Xenopus Kv2.1). Dashes appear under the region spanned by the pXb4 PCR product. pXb4 nucleotide sequences that differ from Kv2.1 sites are shown below the corresponding Kv2.1 nucleotides (nucleotides 762 and 1056). B, Nucleotide and amino acid sequence of XShab12 (Xenopus Kv2.2). Dashes appear under the region spanned by the pXb3 PCR product. pXb3 nucleotide sequences that differ from Kv2.2 are shown underneath the corresponding Kv2.2 nucleotides. Except for one nucleotide change at position 854, all nucleotide changes occur in the third codon position. A tyrosine phosphorylation site is marked by a triangle. Although two of three CAMP phosphorylation sites are conserved between Kv2.1 and Kv2.2, the putative tyrosine phosphorylation site is unique to Kv2.2 (amino acid 512)|
|Figure 2. Xenopus Kv2.1 and Kv2.2 mRNAs induce expression of delayed rectifier-type potassium currents in Xenopus oocytes. For A-C, currents were generated in response to 60 msec voltage steps to potentials ranging from -50 to + 100 mV from a holding potential of -80 mV; leak-subtracted currents. are shown (see Materials and Methods). A, Kv2.1 mRNA induces expression of delayed rectifier-type potassium currents in Xenopus oocytes. The average current size at +20 mV is 1.1 -t 0.6 FA (mean t- SD; n = 7) for 1.7 ng of injected cRNA. B, Kv2.2 mRNA-induced current is sustained for the duration of the depolarizing pulse. The average current size at +20 mV is 1.3 -t 0.3 PA (mean t- SD; n = 6) for 1 ng of injected cRNA. C, Comparison of the kinetics of activation of Kv2.1 and Kv2.2 mRNA-induced currents. Current traces from A and B were superimposed for comparison. The Kv2.1 mRNA-induced current (dotted line) rises faster than the Kv2.2 mRNA-induced current (solid line). D, Tail current analysis indicates the Kv2.1 mRNA-induced channels are potassium-selective. Currents were activated with prepulses of 100 msec duration to +25 mV, then stepped to different hyperpolarizing pulses ranging from -100 to 10 mV (inset). Varying the external K+ concentration shifts the reversal potential as predicted by the Nernst equation for a K+-selective channel. Results from a representative experiment are shown, and average results are indicated in the text. E, Sensitivity of Kv2.1 and Kv2.2 currents to TEA. Current amplitude was measured in the presence of variable concentrations of TEA. Current amplitude was plotted against TEA concentration. Current values + SEM were taken from voltage pulses to +20 mV. F, Normalized conductance versus voltage relationships for Xenopus Kv2.1, Kv2.2, Kvl.1, and Kv1.2 mRNA-induced currents. The conductance was calculated for a given voltage command (v) and its corresponding steady-state current response (0 from the formula G = 1/(V - EK), where the equilibrium potential for Kt (EK) is -116 mV. Half-maximal activation (V,,,) is achieved at values of +25.0 2 0.1 mV (mean 5 SD; II = 7) for Kv2.1 currents and at +30.0 + 5.5 mV (mean + SD; n = 3) for Kv2.2 currents. Kvl currents activate and achieve G,,, at less positive membrane potentials than do the Kv2 currents. Data from representative recordings were used to generate the graphs. Single Boltzmann distributions fit Kvl.1 and Kv1.2 data well (Jones and Ribera, 1994) (A. Ribera, unpublished observations), whereas Kv2.1 and Kv2.2 data deviate consistently from the Boltzmann isotherm (data not shown). This type of behavior has been observed previously in human Kv2.1 channels expressed in oocytes (Benndorf et al., 1994).|
|Figure 3. Xenopus Kv2.1 and Kv2.2 mRNA levels are differentially regulated during embryonic development. RNA extracted from embryos of the indicated stages was hybridized simultaneously to Kv2.1, Kv2.2, N-CAM, and EF-la antisense probes. After RNase treatment, the hybridized products were run on an 8% acrylamide gel. Kv2.1 transcripts are first detected by stage 22 (1 d, early tail bud), and the levels gradually increase at subsequent times. Kv2.2 mRNA is present in the fertilized egg, and its levels change during development. N-CAM signal is initially detected in the stage 15 embryo (neural plate stage). EF-la mRNA increases dramatically between stages 1 and 15, reflecting the fact that zygotic transcription initiates during this interval (Krieg et al., 1989). The relatively constant levels of EF-la! after stage 15 indicate that equal amounts of total mRNA were hybridized to the cRNA probes. Yeast RNA (Y, control lane) was used to control for nonspecific and self-hybridization of the cRNA probes. Undigested probes contain -50 nucleotides that correspond to vector sequences and thus run slightly slower than the protected bands; the positions of protected bands are indicated by arrows at right. EF-lcuprotected bands were visible after an overnight exposure to x-ray film. N-CAM-, Kv2.1-, and Kv2.2-protected bands were visible after a 15 d exposure. The probes for Kv2.1 and Kv2.2 had different specific activities than the EF-101 and N-CAM probes did. Thus, it was not possible to estimate the relative abundance of Kv2.1 or Kv2.2 transcripts with respect to N-CAM or EF-la mRNA during development.|
|AFigure 4. Kv2.2 transcripts are present in excitable tissues of developing Xenopus embryos. A-C, In each view, the embryo on the left was hybridized to a digoxigenin sense control cRNA probe, whereas the embryo on the right was hybridized to a Kv2.2 antisense cRNA probe (scale bars: A, C, 0.5 mm; B, 1 mm).A, Early gastrula stage embryo (stage 9; 7 hr after fertilization). Staining is apparent in the dorsal lip of the blastopore (arrow). B, Late gastrula stage (stage 12; 14 hr). Kv2.2 staining localizes to dorsal ectoderm and presumptive neural tissue (arrow). C, Neurula stage (stage 19; 20 hr). Kv2.2 mRNA is localized along the entire neural tube (arrows). Rostra1 is up; dorsal is to the leff. The dark shadow in the gut of the embryo is attributable to incomplete clearing of the embryo. D-F, In these lateral views, rostra1 is to the right, and dorsal is up (scale bars: D-F, 0.5 mm). Sense controls for these older stages are not shown but are similar to those shown in A-C. D, Expression of Kv2.2 mRNA in the early tail bud embryo (stage 23; 1 d). Staining is present in the brain, spinal cord, and anterior midsomite regions. E, A similar pattern of Kv2.2 mRNA expression is observed 6 hr later in the stage 26 embryo, except that the signal in the midsomite regions has extended caudally. F, In the stage 35 embryo (2 d), Kv2.2 staining is faint in the majority of the brain and spinal cord but still visible in the midsomite regions. The dark signal at the tip of the embryo corresponds to the forebrain.|
|F/g~re 5. Kv2.2 mRNA localizes to the ventral spinal cord and perinuclcar somite regions during development of excitability. These views are obtained from 30 pm sections of whole-mount embryos that were hybridized to a Kv2.2 antisense probe. Sections were counterstained with methyl green, which intensely colors nuclei. Dorsal is UP in all panels. In the sagittal sections, rostra1 1s to the r&zt. A, In a cross-section from a stage 23 embryo, Kv2.2 mRNA is restricted to the ventrolateral portion of the neural tube (open u~.rdws) and region around the somite nuclei (filled arrowhead). B, A sense control stage 23 embryo is observed in cross-section; the color is attributable solely to methyl green countcrstaining. Sense controls for other stages and planes of section provide similar results. C, At stage 26, the signal in the ventral portion of the neural tube (open arrowheads) resembles the pattern observed at stage 23 (A). In the somitcs, the signal predominates around nuclei @Ned arrowheads). D, In a parasagittal section of a stage 26 embryo, the Kv2.2 hybridization signal concentrates around the somite nuclei. E, In a sagittal section of a stage 26 embryo, the Kv2.2 hybridization signal concentrates in the ventral region of the neural tube in discrete patches. The Kv2.2 signal delimits the ventral extent of the neural tube, whereas the nf is positioned within the dorsal region. F, G, Sections obtained from stage 35 embryos probed with either anti,cnse Kv2.2 (F) or Kvl.1 (G) probes. The black varicosities surrounding the spinal cord (durk thzn avowheuds) are attributable to pigmented cells found in the embryos used for this experiment, which were caused by the mating of an albino female and a pigmented malt. By stage 35, pigment begins to appear in albino/pigmented embryos. F, By stage 35, Kv2.2 expression in the neural tube (open arrowheud,) appears fainter than at stage 23 (C), which is consistent with what is observed at the whole-mount level (Fig. 4F). G, Kvl.1 mRNA localizes to the dorsal region of the spinal cord indicated by the open arowa. Scale bar (shown in A): A, 50 pm; R, 75 km; C, 100 km; D, 100 Km; E, 200 km; F, 100 pm; G, 100 pm. Abbreviations: nt, neural tube (spinal cord); 5, somites; /zot, notochord.|