A potassium channel gene is expressed at neural induction

Neuron, Vol. 5, 691-701, November 1990, Copyright 0 1990 by Cell Press

A Potassium Channel Gene Is Expressed at Neural Induction Angeles 6. Ribera* Department of Biology University of California, La Jolla San Diego, California 92093

Summary Voltage-dependent potassium currents exhibit specific time tables of functional differentiation and regulate the development of action potentials in amphibian spinal neurons. A Xenopus nucleotide sequence (XSha2) encoding a potassium current has been isolated by homology screening with the Drosophila Shaker gene. Functional expression in oocytes identifies it as a delayed rectifier. Southern analysis suggests that XSha2 is a member of a family of highly related genes. XSha2 is expressed in the nervous system but is not detectable in skeletal muscle. Transcripts are apparent at the neural fold stage, and subsequent levels parallel those of the neural marker N-CAM. Thus molecular events required for the establishment of electrical excitability in the vertebrate embryo occur early during neurogenesis. tntroduction The acquisition of electrical excitability is an early event during neuronal differentiation in amphibians. Xenopus laevis spinal neurons have been examined physiologically in situ as early as 20-22 hr after fertilization (Baccaglini and Spitzer, 1977), -7 hr after the last round of DNA synthesis of early birth date neurons (Lamborghini, 1980). Action potentials are first detected at the neural tube stage (22 hr), at which point they are calcium dependent and of long duration. During the following day in vivo and in vitro, the impulse matures to a brief sodium-dependent spike (Baccaglini and Spitzer, 1977; Spitzer and Lamborghini, 1976). This program is expressed in a cellautonomous manner, since a neuron developing in the absence of other cells also exhibits a transient period of long duration impulses (Henderson and Spitzer, 1986). Analysis of the voltage-dependent currents that underlie the action potential indicates that the conversion of the neuronal impulse from calcium to sodium dependence is primarily due to the maturation of a delayed rectifier potassium current (garish, 1986; O’Dowd et al., 1988). Delayed rectifier current is present at the time of primary neurite extension early in culture (6 hr in culture, equivalent to 24 hr after fertilization; O’Dowd et al., 1988). During the first day in vitro, it triples in density and its kinetics are accelerated (O’Dowd et al., 1988). However, it shows no fur* Present address: Department of Physiology C-240, University of Colorado, Denver, Colorado 80262.

ther increase in its density or rate of activation during the second day in culture (Ribera and Spitzer, 1989). The later maturation of the delayed rectifier with respect to calcium current permits a period during which impulses are calcium dependent and of long duration (Barish, 1986; O’Dowd et al., 1988); transient expression of calcium-dependent impulses appears to be required for the subsequent elaboration of other phenotypes (Holliday and Spitzer, 1990). RNA synthesis inhibition prevents expression of the mature delayed rectifier and arrests the normal development of the action potential; a critical period of transcription is required for the differentiation of this current (Ribera and Spitzer, 1989). Probes for voltage-dependent channels that underlie the action potential are necessary for molecular analyses of the development of electrical excitability. The Xenopus embryo has permitted examination of the early molecular events and gene activation occurring during the formation of the nervous system. Transcription of N-CAM is an early response to neural induction (Kintner and Melton, 1987). N-cadherin is also expressed at these early times (Detrick et al., 1990). A homeobox-containing gene, XlHbox6 (Sharpe et al., 1987), and a neurofilament gene (Sharpe, 1988) are activated in the developing neural plate, Six neural-specific clones associated with neural plate formation have been identified using subtractive hybridization techniques (Richter et al., 1988). This molecular analysis of electrical excitability in the early nervous system is focused on potassium channel genes, since the delayed rectifier determines the phenotype of the action potential. The Drosophila Shaker gene encodes a voltage-dependent potassium A-current (Baumann et al., 1987; Kamb et al., 1987, 1988; Papazian et al., 1987; Tempel et al., 1987; lverson et al., 1988; Pangs et al., 1988; Schwarz et al., 1988; Timpe et al., 1988a, 1988b). Homologs of Shaker have been isolated in mouse (Tempel et al., 1988; Chandy et al., 1990) and rat (Baumann eta al., 1988; Christie et al., 1989; McKinnon, 1989; Stuhmer et al., 1989b; Douglass et al., 1990; Swanson et al., 1990). Significantly, functional expression of these cloned genes indicates that delayed rectifier type currents are encoded by most of the mammalian sequences (Christie et al., 1989; Stuhmer et al., 1989a, 1989b; Christie et al., 1990; Douglass et al., 1990; Swanson et al., 1990). A Xenopus genomic library was screened at reduced stringency for sequences with identity to a Shaker cDNA (ShAl). A transcript, XSha2, encoding a voltage-dependent delayed rectifier potassium current is expressed in tissues of the nervous system, but not the excitable tissues of skeletal muscle. It is also present in spinal neurons developing in culture that express the same developmental program for the action potential as those developing in situ. XSha2 is de-

Neuron 692

tectable in the developing Xenopus embryo at the neural fold stage (13.5 hr after fertilization) and is thus present during early development of the nervous system. Molecular probes for voltage-dependent potassium channels will permit developmental manipulations producing altered levels of transcripts and further definition of their role in the establishment of electrical excitability. Results Identification of a Xenopus Shaker Homolog A Xenopus genomic library was screened at reduced stringency for sequences with identity to a cDNA from the fly (ShAl; kindly provided by Dr. Bruce L. Tempel). Several hybridizing clones were identified. A hybridizing restriction fragment of one isolated clone (MCI) was subcloned into M13, sequenced, and found to have identity with Shaker, as shown below. Sequencing of the 1.5 kb EcoRI-Pstl fragment of this clone indicated that the Xenopus gene is probably intronless (Ribera et al., 1988, Sot. Neurosci., abstract); however, a 5’ start ATG codon was not contained within this clone, and a second overlapping clone (DCI) was identified from another genomic library. The restriction maps of hybridizing fragments from the two clones suggested that the clones are derived from the same region of the genome. This conclusion was confirmed directly by the nucleotide sequences of the two clones across the overlapping regions. DC1 contains the entire coding region within 1497 nucleotides (Figure 1). This region is uninterrupted by introns, as found for several mouse and rat sequences (Chandy et al., 1990; Douglass et al., 1990; Swanson et al., 1990). The predicted peptide has hydrophobic domains comprising at least six putative membrane-spanning domains (Sl-S6; Figure I), as proposed in models of voltage-dependent sodium, calcium, and potassium channels (see Catterall, 1988, and Jan and Jan, 1989, for reviews). Similar to these sequences, the fourth putative transmembrane domain, S4, contains a basic residue (arginine or lysine) at every third position. A leucine zipper motif begins within S4 and extends to S5, as reported by McCormack et al. (1989). The Sl-S6 segments show high amino acid conservation with other potassium channel sequences. Amino acids 74-80 are identical to the sequence identified by Frech et al. (1989) as a hallmark of the extended voltage-dependent potassium channel family. A putative site for N-glycosylation, between Sl and S2 (Asn&, is conserved on a proposed extracellular domain. A region rich in basic and serine residues that may be a phosphorylation site (amino acids 445-455) is found near the carboxyl-terminus in a putative intracellular domain; this position is analogous to the location of a consensus sequence for CAMP-dependent phosphorylation in Shaker (e.g., Tempel et al., 1988). The predicted amino acid sequence (Figure 1) indi-

Nucleotide and predicted amino acid sequence of XSha2. The sequence of the Hindlll-Pstl fragment is shown. The Xbal (X) and Pvull (P) sites referred to in the text are indicated with a dashed line and the appropriate letter above the nucleotide sequence. The putative transmembrane domains, Sl-S6, are designated by horizontal bars under the amino acid sequence. The region in the 3’ noncoding sequence that is conserved with MEW and RBK2 is indicated by a dotted line.

cates that this amphibian clone is most conserved with the mammalian sequences MBK2 and RBK2/ RCK5 (RBK2 and RCK5 are 96% equivalent; McKinnon, 1989; Stuhmer et al., 1989b; Chandy et al., 1990;

Embryonic Potassium Current Transcript Expression 693

Christie et al., 1990). The first 7and last 14 amino acids are identical, as are several other regions of the predicted peptide; these terminal sequences distinguish MBK2 and RBK2 from other mammalian family members. Overall, the frog and mammalian peptides are 90% identical and 95% similar. Moreover, in the 3’ noncoding regions of all three sequences, there is a stretch of 33 nucleotides that is 82% identical (Figure 1); this region is found in analogous positions at 76, 99, and 94 nucleotides downstream of the coding regions in XSha2, MBK2 and RBK2, respectively. This sequence is probably transcribed but untranslated, since it is proximate to the stop codon found in the RBK2 cDNA (McKinnon, 1989; Christie et al., 1990). It is possible that this region serves a regulatory function that is conserved between these genes. In view of these similarities, the Xenopus Shaker-like gene has been named XSha2. XSha2 Encodes a Delayed Rectifier Type Current Functional expression of Drosophila Shaker sequences indicates that an A-type potassium current is encoded by the fly gene (Iverson et al., 1988; Timpe et al., 1988a, 1988b). In contrast, mammalian Shaker homologs have been found mostly to induce the expression of a functionally different type of potassium current, a delayed rectifier (Christie et al., 1989; Stuhmer et al., 1989a, 1989b; Christie et al., 1990; Douglass et al., 1990). Both currents are activated by stepping the membrane voltage to depolarized levels. However, there is an important functional difference between these two types of voltage-dependent potassium currents: the A-current inactivates during a depolarizing voltage step, whereas the delayed rectifier shows little inactivation. Functional expression of XSha2 in Xenopus oocytes (Gurdon et al., 1971; Dascal, 1987) was carried out to determine whether XSha2 encodes a sustained or inactivating voltage-dependent potassium current. A 1.6 kb Hindlll-Pstl fragment of DC1 (Figure 1) that spanned the entire coding region was subcloned into a transcription vector, and capped sense transcripts were generated (Melton et al., 1984) and injected into oocytes. The oocyte membrane was clamped at -70 to -80 mV and then stepped for 60-300 ms intervals to depolarized potentials expected to activate delayed rectifier and potassium A-currents. This protocol revealed a voltage-activated current not found in uninjetted oocytes or in oocytes injected with RNA made using the MGI “truncated” sequence as template. The current was sustained during a 60 ms voltage pulse (Figure 2), indicating that it is of the delayed rectifier type. Activation of the current is observed at potentials positive to -20 mV, similar to the delayed rectifier elicited from Xenopus spinal neurons (O’Dowd et al., 1988; Ribera and Spitzer, 1989, 1990). The ionic dependence of the induced current is strongly potassium selective. Elevating the external potassium concentration from 3 mM to IO or 40 mM leads to shifts of 23 f 2 mV or 43 f 4 mV in the reversal

25

-80 (F”

1

200 nA

‘.L-

Figure 2. Characterization of the Voltage-Dependent sium Current Induced by XSha2 Transcripts

10 ms

Potas-

Currents were elicited by depolarizing steps to voltage levels ranging between -30 and +25 mV from a holding potential of about -80 mV. In the example shown, the current is sustained for the duration of the 60 ms pulse, indicating that is a delayed rectifier-like current. Leak subtraction was employed using the P/4 protocol from the Clampex set of pCLAMP programs.

potential of tail currents (n = 7). The Nernst equation predicts 30 and 65 mV shifts, respectively, for a purely potassium-dependent current; the data are in good agreement at 3 and IO mM external potassium, as found for voltage-dependent potassium currents recorded from Xenopus spinal neurons in culture (O’Dowd et al., 1988; Ribera and Spitzer, 1990) and fly or rat potassium clones (Timpe et al., 1988a, 1988b; lverson et al., 1988; Christie et al., 1989; Stuhmer et al., 1989b; Douglass et al., 1990; Swanson et al., 1990). At 40 mM external potassium, the potassium dependence is less strong. The recording conditions used here are similar to those used for study of the endogenous neuronal current, but are substantially different from most expression studies, in which the divalent cation concentration and composition differ. Divalent cations have been found to activate endogenous currents (Miledi et al., 1989) as well as to affect the currents induced in oocytes (Douglass et al., 1990). The reversal potential noted in 40 mM external potassium may be due to the experimental conditions (high external divalent cation concentration) and/or the encoded peptides. Pharmacological analysis indicates that the potassium current inhibitor tetraethylammonium (TEA) is not an efficient blocker of this current at a concentration of 40 mM (19% + 5% reduction at 25 mV, n= 5). However, 4aminopyridine (1 mM) reduces the current by 71% f 6% (25 mV, n = 4). The block by Caminopyridine is voltage dependent, being strongest at positive membrane potentials (at neutral pH, Caminopyridine is negatively charged). Recovery from Caminopyridine block is slow and incomplete (>I5 min). The sensitivities of the delayed rectifier in Xenopus spinal neurons (O’Dowd et al., 1988; Harris et al., 1988; Ribera and Spitzer, 1990) are substantially different from those of the expressed clone. The endogenous neuronal delayed rectifier is more sensitive to TEA and is moder-

Neuron 694

ately sensitive to Qaminopyridine. Altered sensitivities to TEA, Qaminopyridine, and charybdotoxin are observed for RBK21RCK5,rat homologs of XSha2, as well as for other rat and fly sequences (MacKinnon et al., 1988; Christie et al., 1989,199O; Stuhmer et al., 1989b). It is possible that in the oocyte the protein is not processed in the manner required for the neuronal sensitivities to these blockers. In addition, the neuronal channels may consist of hetero-oligomers, rather than the homo-oligomeric form that was studied here (see, for example, Christie et al., 1990; lsacoff et al., 1990; Ruppersberg et al., 1990). XSha2 May Be a Member of a Gene Family Diversity within the Shaker gene of Drosophila is generated by alternative splicing (Kamb et al., 1988; Pongs et al., 1988; Schwarz et al., 1988). Molecular analyses of potassium channel genes in mammals have not provided evidence for alternative splicing. Instead, potassium channel diversity is generated by a Shakerlike gene family with several members (see Stuhmer et al., 1989b; Chandy et al., 1990). In addition, related potassium channel genes in Drosophila (Butler et al., 1989; Wei et al., 1990) and mammals (Frech et al., 1989; Wei et al., 1990; Yokoyama et al., 1989) have also been identified. The possibility of an XSha2 gene family was examined by Southern analysis. After washes of reduced stringency, several hybridizing bands are detected, suggesting a family of closely related genes in Xenopus (Figure 3, right); this is consistent with the gene families reported for mouse and rat voltage-dependent potassium channel genes (Chandy et al., 1990; Stuhmer et al., 1989b; Swanson et al., 1990). After final high stringency washes, a few lanes show only one or two hybridizing bands (Figure 3, left). Given that Xenopus is tetraploid and exhibits a high degree of polymorphism (for review, see Kobel and DuPasquier, 1986), this hybridization pattern is consistent with a single XSha2 gene. XSha2 Is Expressed in the Nervous System The tissue specificity of potassium channel transcripts has been examined to varying extents in other systems. In Drosophila, Shaker transcripts are found throughout the animal (Baumann et al., 1987; Kamb et al., 1987, 1988; Pongs et al., 1988; Schwarz et al., 1988). In mammals expression has been found in brain, heart, skeletal muscle, and the immune system (Tempel et al., 1988; McKinnon, 1989; Douglass et al., 1990; Beckh and Pongs, 1990; Swanson et al., 1990). The tissue-specific expression of XSha2 in Xenopus embryos was addressed by extracting RNA from various tissues of 3- to 4week old tadpoles. The presence of XSha2 transcripts was determined by RNAase protection (Melton et al., 1984). The Xbal-Pvull fragment of the XSha2 sequence was subcloned into the RNA transcription vector pSP73, and a2P-labeled riboprobes were generated. To compare the relative amounts of RNA in different samples, a a2P-labeled riboprobe for

Figure3. SouthernAnalysisof Genomic DNA XenopusgenomicDNA wasdigestedin different reactions with the following restriction enzymes: BarnHI,Hindlll, Pstl, Pvull, EcoRI, and Xbal. The restricted DNA was fractionated on a 1% agarose gel by electrophoresis. The DNA was transferred to Nytran and hybridized to a radiolabeled probe derived from the XSha2 sequence. Washes were carried out at increasing degrees of stringency. (Left) Under conditions of high stringency (0.1 x SSPE, 1% SDS, W’C), a few lanes contain only one hybridizing band; at lower stringency (0.5x SSPE, 1% SDS, 62OC), more hybridizing bands are detected fright). These findings are consistent with the existence of a family of highly related potassium channel genes and a single XSha2 gene.

EF-la was also made (Krieg and Melton, 1989). EF-la codes for an enzyme involved in protein synthesis (a “housekeeping” enzyme). Spinal cords, brains, eyes, skeletal muscle, heart, and livers were dissected from 12 stage 52 embryos. RNA was extracted from these tissues and hybridized to the XSha2 and EF-la probes. XSha2 protection was observed for spinal cord, brain, and eye RNA, but not skeletal muscle, heart, or liver RNA (Figure 4). However, in one instance (n = l/3), the RNA extracted from heart tissue did provide a weak XSha2 signal, indicating that there may be low levels present. In the example shown, the signals generated by the EF-la probe suggest that the lack of protection of XSha2 by skeletal muscle, heart, and liver RNA was not due simply to lack of RNA in the sample. These results indicate that XSha2 is expressed in excitable tissues of the nervous system. XSha2 Expression In Vivo Is Developmentally Regulated Developmental regulation of expression of the Drosophila and related mammalian transcripts has been examined in a few cases. The expression of transcripts for the Shab and Shaw genes is modified during development of the fly (Butler et al., 1989). In rat, the K,l transcript becomes enriched in cardiac muscle as a function of development; a more modest developmentally regulated increase in expression is observed in brain for this as well as the K,2 transcript (Swanson et al., 1990). RCKl and RCK5 transcripts are abundant in the nervous system of the adult rat, whereas RCK3 and RCK4 are found at all stages of em-

Embryonic Potassium Current Transcript 695

Expression

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Figure 4. Tissue Specificity Xenopus Tadpoles

of XShaZ Expression

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RNAase protection assay indicates that XSha2 (Xba-Pvu probe) is expressed in spinal cord, brain, and eye, but not in skeletal muscle, heart, or liver. EF-la protection is presented to indicate the relative amounts of RNA that were hybridized to the probes. (The tissue specificity of the Pvu-Pst probe is demonstrated in Figure 7.) The tRNA lane demonstrates, in this as in subsequent

assays, that incubation with nonhybridizing tRNA does not protect the probes from degradationand that protection signals are not due to nonspecific hybridization.

Figure 5. RNAase Protection Assay on RNA from Difterent Xenopus Embryos

Protection of XSha2 (Pvu-Pst probe) is seen with RNA extracted from stage 21, 27, and 30 embryos, but not from stage 12 or 15 embryos, indicating that the transcript is detectable under these conditions at stage 21 in viva (corresponding to -5 hr in culture). N-CAM signals are detectable at stages 15,21, 27, and 30. EF-la signals indicate the relative amounts of RNA that were present in the samples. For stages 12, 15, and 21, 5 embryo equivalents of RNA were hybridized to the probes; 2.5 embryo equivalents of stage 27 and 30 RNA were used.

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Figure 6. RNAase Protection Assayon RNAfrom Dissected Early Stage Xenopus Embryos (Stages 13, 15, and 18) The embryo was divided into dorsal anterior, dorsal posterior, and ventral parts. Protection of XSha2 (Pvu-Pst probe) and N-CAM is seen with RNA extracted from the dorsal posterior embryo at stage 13, consistent with the direction of neural induction (Spemann, 1938); at later times, its expression is relatively higher in the RNA from the anterior portion, consistent with a relative abundance of neurons in the brain (dorsal anterior) as compared with the spinal cord (dorsal posterior). Twenty dissected parts were included in each sample. Similar patterns of expression are observed for N-CAM, an early marker of neural induction (Kintner and Melton, 1987). Ventral RNA provided weak signals that were not developmentally regulated. EF-la signals indicate the relative amounts of RNA that were recovered in the samples. The inset shows a standard curve for quantifying XSha2 concentrations; the indicated amounts of synthetic XSha2 RNA were included in the four samples.

since they are prepared from the posterior two-thirds of the neural plate (Eagleson and Harris, 1990). RNA was extracted from cultures at 6 hr, 1 day, and 2 days after plating. Six hours corresponds to stage 22, the time of initial neurite extension in vitro (Spitzer and Lamborghini, 1976) and in vivo (Taylor and Roberts, 1983). The RNA from 6 hr, 1, and 2 day cultures protected the XSha2 probe from degradation by RNAases (Figure 7), indicating that the transcript is present as soon as neurons are morphologically distinguishable in culture. In parallel experiments, XSha2 expression was not detected in cultures lacking neurons (data not shown), consistent with its neural specificity. The XSha2 signal decreases as a function of time in culture, as does the neural N-CAM signal. Counts of neurons in the culture indicate that ~50% of the neurons die between 1 and 2 days in culture (Ribera and Spitzer, submitted). The reduction in signals from the neural-specific probes XSha2 and neural N-CAM is consistent with the observed decline in neuron number. In vivo, both XSha2 and neural N-CAM signals increase as a function of development (Figure 5), reflecting the increased number of postmitotic neurons during the period examined (Lamborghini, 1980; Hartenstein, 1989).

PROBES

Culture RNA

Tissue RNA

XSha

Expression of XSha2 in Cultured Neural Plate Cells Parallels That In Vivo The time table for the functional differentiation of voltage-dependent potassium currents in Xenopus spinal neurons developing in culture has been identified (O’Dowd et al., 1988; Ribera and Spitzer, 1990). At the time of primary neurite extension (6 hr in vitro), neurons have a delayed rectifier current but no A-current. At 1 day, the delayed rectifier current has undergone a dramatic increase in its density and the A-current is present in 50% of the neurons. By 2 days, all the neurons possess A-current and the delayed rectifier shows no further changes. The correlation between the expression of XSha2 and the functional differentiation of voltage-dependent potassium currents was examined by determining the developmental regulation of XSha2 transcripts in culture. Xenopus neural plate stage cultures were prepared from stage 15 embryos as previously described (Spitzer and Lamborghini, 1976; Blair, 1983; Ribera and Spitzer, 1989). The cultures are enriched in spinal neurons and low in anterior brain neurons,

NCAM

EF-la

Figure 7. RNAase Protection Assay with RNA from Xenopus Cells and Dissected Tissues

Cultured

RNA extracted from cultures at 6 hr, 1 day, and 2 day cultures protect the XSha2 probe (Pvu-Pst probe). N-CAM signals are present at all stages and decrease in parallel with development, as does the XSha2 signal. In each case the RNA extracted from 30 cultured neural plates was hybridized to the probes. The tissue specificity of the Pvu-Pst probe was also examined; as for the Xba-Pvu probe (Figure 4), neural specificity is demonstrated. EF-la protection is presented to demonstrate that the muscle sample contained comparable levels of cellular RNA.

Embryonic 697

Potassium

Current

Transcript

Expression

These results indicate that at the time of earliest physiological and molecular examination in vitro, the neurons already possess XSha2 transcripts. In the developing embryo, analyses were carried out at earlier times, and transcripts were detectable m3 hr before neural plates were dissected in preparation for culture (see above and Figures 5 and 6). This pattern of expression is most simply correlated with the appearance of the delayed rectifier current, as indicated by functional expression of XSha2 in oocytes. Discussion The goal of the present study was to examine the molecular program establishing electrical excitability in the developing vertebrate nervous system. This type of analysis is recently possible because of the advances in the cloning of voltage-dependent channel genes (see Catterall, 1988, and Jan and Jan, 1989, for reviews). This study examined transcripts encoding voltage-dependent potassium channels in Xenopus spinal neurons, for which the functional differentiation of electrical excitability has been well characterized (Spitzer and Lamborghini, 1976; Baccaglini and Spitzer, 1977). By homology screening with the Drosophila Shaker gene, an amphibian gene, XSha2, has been cloned and sequenced. This gene is most homologous to the mammalian MBK2 and RBK2/RCK5 genes (McKinnon, 1989; Stuhmer et al., 1989b; Chandy et al., 1990; Christie et al., 1990). The expression of XSha2 transcripts is tissue specific; it is detected in tissues of the nervous system, but not in other excitable tissues, such as skeletal muscle. Functional expression of XSha2 in oocytes induces a delayed rectifier-type potassium current that has activation properties similar to those of the delayed rectifier current studied in Xenopus spinal neurons developing in culture (O’Dowd et al., 1988; Harris et al., 1988; Ribera and Spitzer, 1990). It will be of interest to study the single-channel behavior of XSha2 homooligomeric channels and compare their properties to those of the 15 and 30 pS channels that underlie the delayed rectifier current (Harris et al., 1988). The cloning of other XSha family members and reconstitution in oocytes of hetero-oligomers will provide further information about the diversity of function encoded by the XSha potassium channel genes (see, for example, Christie et al., 1990; lsacoff et al., 1990; Ruppersburg et al., 1990). The developmental pattern of expression observed for XSha2 is consistent with its role in encoding the delayed rectifier in spinal neurons. The XSha2 transcript is detected in vivo at early stages of formation of the nervous system (stage 13). Electrical recordings from early birth date spinal neurons indicate that they exhibit delayed rectification a few hours later (Spitzer and Lamborghini, 1976; Baccaglini and Spitzer, 1977; O’Dowd et al., 1988). In the amphibian Ambystoma, there are indications of a delayed rectifier current in

neural tissue at the mid-neural fold stage (equivalent to stage 14 in Xenopus; Warner, 1973). These findings suggest that very little time is required to consolidate the expression of XSha2 transcripts in the functional expression of voltage-dependent delayed rectifier current. Similarly, transcripts encoding the muscle nicotinic acetylcholine receptor are first detected at stage 14 (Baldwin et al., 1988), whereas physiological measurements demonstrate that chemosensitivity appears at stages 18-19 (Blackshaw and Warner, 1976; Chow and Cohen, 1983), indicating a comparable 3-5 hr delay between the appearance of these transcripts and the encoded function. The precise timing of the program of potassium channel function is essential for the early period of long-duration, calcium-dependent impulses. If potassium channels matured earlier, this period could be abolished. Conversely, late differentiation of potassium current would prolong this period. The regulation of calcium and potassium channel function appears to be coordinated during development to permit not only the transience of the calcium-dependent phenotype, but also the specific developmental stages over which it occurs. The identification and analysis of other voltage-dependent channel transcripts expressed in these developing neurons will further an understanding of the molecular events required for the regulation of electrical excitability. Misexpression experiments have been used in Xenopus to examine the roles of homeoboxes in axis and somite formation (Harvey and Melton, 1988; Ruiz i Altaba and Melton, 1989) and evaluate the function of N-CAM and N-cadherin in the establishment of the nervous system (Kintner, 1988; Detrick et al., 1990). Similarly, early overexpression of XSha2 may be achieved by injection of synthetic XSha2 transcripts into embryos at stages preceding nervous system development. Subsequent examination of the action potential and potassium currents in embryonic spinal neurons would identify the functional consequences of this developmental manipulation. Of particular interest would be the possible persistence or elimination of the early transient period of calcium-dependent impulses, providing further insight into the roles of early spontaneous calcium influx in embryonic neurons (Holliday and Spitzer, 1990). The 3’ noncoding region conserved in XSha2, MBKZ, and RBK2 (Figure 1) may serve a regulatory function; elimination or mutation of this region may alter the effect of misexpression. The maturation of the action potential is due largely to a 3-fold change in the density of functional delayed rectifier current (O’Dowd et al., 1988). This developmental change in potassium current could be achieved at the level of transcription or translation. Misexpression studies may point to the diverse levels of regulation of function that are normally operative. It is known that the differentiation of the delayed rectifier current in Xenopus spinal neurons is critically dependent upon a 9 hr period of RNA synthesis (Ribera and

Neuron 698

Spitzer, 1989). Understanding the basis of the developmental program of electrical excitability will require the identification of the molecules regulated during this critical period. Potassium channel transcripts are likely candidates, and the tools with which to investigate this issue are now available. ExperimentalProcedures Materials Restriction enzymes and SP6 and T7 RNA polymerases were obtained from Bethesda Research laboratories; RNasin and RQI DNAase were obtained from Promega. Radionucleotides were purchased from Amersham.

Animalsand Cell

Culture Embryos were produced by breeding pairs of adult Xenopus primed with human chorionic gonadotropin (United Stages Biochemicals) and staged according to Nieuwkoop and Faber (1956). Neural plate stage (stage 15) cultures were prepared as described previously (Spitzer and Lamborghini, 1976; Blair, 1983; Ribera and Spitzer, 1989) from the posterior two-thirds of the neural plate (the future spinal cord), thus reducing contributions from brain neuronal tissue. Neuron-free cultures were prepared as described by Kidokoro et al. (1980) with minor modifications (Ribera and Spitzer, submitted). Isolation of Xenopus Shaker Homolog Cenomic Clones Xenopus genomic libraries (Krieg and Melton, 1985; Wahli and Dawid, 1980) were screened for sequences homologous to the fly Shaker gene. In the initial screen 5 x IO5 hybridizing recombinant phage (Krieg and Melton, 1985) were identified with a nick-translated probe made from the ShakerShAl cDNA (kindly provided by B. Tempel). Screening was carried out under conditions of reduced stringency. Hybridization was performed in 7% SDS, 200 mM sodium phosphate (pH 7), 15% formamide, IO mM EDTA, 1% bovine serum albumin, Pentax fraction Vat 3PC overnight, and filters were washed 4 times for 45 min in 2x SSPE, 1% SDS at 37”C-65”C, as recommended by B. Tempel. Under these conditions, several hybridizing clones were identified, and one (MCI was plaque purified. Hybridizing restriction fragments (Xbal-Pstl =: 700 bp; EcoRI-Xbal 9 800 bp) were subcloned into Ml3 for sequencing by the Sanger chain-termination DNA sequencing method (Sanger et al., 1977) with Sequenase (United States Biochemicals). The MCI clone was used to screen a second library (Wahli and Dawid, 1980) at high stringency(hybridization: 1 M NaCI, 50 mM Tris [pH 7.51, 1% SDS, 1% Denhardt’s, 100 @ml salmon sperm DNA at 65OC for 14-16 hr; wash: 0.1x SSPE, 0.1% SDS 6S°C, 4 times for 45 min). One hybridizing clone (DCI) was identified. EcoRl digestion of this clone released three insert fragments, and a 5 kb fragment was hybridized to the probe. A Hindlll-Pstl internal fragment was shown to overlap with the EcoRI-Pstl segment of MCI. Restriction mapping and sequencing over the entire length of the sequence indicated that MC1 and DC1 are derived from the same gene. Sequencing was carried out over both strands; sequencing reactions were repeated with dlTP substitution for dGTP to identify possible compression artifacts (Sequenase, United States Biochemicals). Sequence analyses were carried out using the Wisconsin Genetics Computer Groups programs (Devereux et al., 1984). Cenomic Southern Analysis Cenomic DNA (20 bg; generously provided by C. Coffman) was digested with one of the following enzymes: BamHI, Hindlll, Pstl, Pvull, EcoRI, and Xbal. The DNA was fractionated on 1% agarose gels and transferred to Nytran (Schleicher & Schuell). A random-primed probe was synthesized from the Xbal-Pstl fragment of MCI. Hybridization conditions were according to the manufacturer’s recommendations. Washes were carried out first at low stringency (5x SSPE, 0.1% SDS, 37°C) and were gradually

increased to a final stringency of 0.1x SSPE, 0.1% SDS, 65OC. Blots were exposed to film at -70°C with an intensifying screen for varying times.

RNA Extraction and Purification RNA was extracted from whole or partially dissected embryos, dissected tissues, or cell cultures. Samples were collected and incubated in an SDS-proteinase K buffer, followed by phenol-chloroform extraction; nucleic acids were precipitated in ethanol, and RNAwas separated by incubation in 4M LiCl (Krieg and Melton, 1984; Rebagliati et al., 1985). RNAase Protection RNA from 5 (stages 1’2-22) or 2.5 (stages 27-42) embryo equivalents, from 15-20 dissected embryos, or from 30 cultured neural plates was used in protection experiments. For examination of tissue specificity, the RNA extracted from tissues dissected from 15 3-to 4-week-old (stage 52) tadpoles was pooled. Two different probes from XSha2 were used as indicated. The first was derived from the Xbal-Pvull fragment (Xba-Pst probe), and the second was derived from the Pvull-Pstl segment (Pvu-Pst probe; Figure I). The Xba-Pvu probe corresponds to sequence encoding the S5 and S6 transmembrane domains, whereas Pvu-Pst probe corresponds to the carboxyl terminus and 3’untranslated sequence and presumably is more specific. Nonetheless, similar patterns of expression were revealed with both probes. These regions have been shown directly by nucleotide sequencing to be identical in the MCI and DC1 clones. Both fragments were subcloned into pSP73 (Promega). The Xba-Pvu probe was synthesized by T7 transcription of EcoRI-linearized plasmid; the Pvu-Pst probe was synthesized by SP6 transcription of Pstllinearized plasmid. Probe synthesis and purification, hybridization, digestion, and polyacrylamide gel analysis of protected fragments were carried out by standard methods (Melton et al., 1984; Kintner and Melton, 1987). Two other probes, kindly provided by C. Kintner, were used. An N-CAM probe specific for a neural-splice variant (long cytoplasmic domain form; Kintner and Melton, 1987) was used to control for the amount of neural tissue; EF-la was used to control for the amount of cellular RNA in the samples. For syntheses of these probes, the amount of radioactivity in the reaction mix was diluted I:4 and I:9 for N-CAM and EF-la, respectively, as compared with the amount of radioactivity used for XSha probe syntheses. Oocyte Recording The coding region of the putative potassium channel gene (Hindill-Pstl fragment of DGI) was subcloned into the transcription vector pSP73 (Promega). The construct was linearized with Pstl and capped RNA was synthesized in vitro by T7 transcription (Stratagene). Fifty nanoliters of a 1 mg/ml RNA solution was injected into stage VI defolliculated Xenopus oocytes as described by Gurdon et al. (1971). Oocytes were incubated at 18OC in Barth’s solution (Dascal, 1987). After 2-4 days, two-electrode voltage-clamp technique (Axoclamp 2A amplifier) was used to record voltage-activated currents induced in the oocytes by the injection of RNA (Dascal, 1987). Bath changes were achieved by exchanging with >30 volumes of new solution. The compositions of the bath solutions were as follows: -Standard solution: 80 mM NaCl, 3 mM KCI, 5 mM MgCI,, 10 mM CoC12, 5 mM HEPES (pH Z4), IO-‘g/ml lTX. -10 mM K+ solution: 80 mM NaCI, IO mM KCI, 5 mM MgC12, 10 mM CoC12, 5 mM HEPES (pH 714), IO-‘g/ml lTX. - 40 mM K+ solution: 40 mM NaCI, 40 mM KCI, 5 mM MgC&, 10 mM CoC12, 5 mM HEPES (pH 74), 10-7glml mX. -TEA solution: 40 mM NaCI, 3 mM KCI, 40 mM TEA, 5 mM MgCI,, 10 mM CoCl?, 5 mM HEPES (pH Z4), IO-‘g/ml TTX. The Caminopyridine solution was prepared by adding 1 mM 4aminopyridine to the standard solution and readjusting the pH to 7’4. Electrodes were filled with 3 M KCI and had resistances of 0.5-3.0 MQ; the current electrode typically had 0.5-l Ma lower resistance than the voltage electrode. The membrane voltage

Embryonic Potassium Current Transcript Expression 699

value used in calculations was that measured by the voltage electrode. Voltage protocols and data analysis were accomplished with the pCLAMP suite of programs. Comparison of the current induced by XSha2 with the current recorded from Xenopus spinal neurons was of particular interest, and thus the bath solutions were as described previously for the study of the endogenous neuronal current (O’Dowd et al., 1988; Ribera and Spitzer, 1989, 1990). In a few instances, recordings were carried out in 115 mM NaCI, 2.5 mM KCI, 1.8 mM CaC&, 10 mM HEPES (pH %4); similar but slightly larger currents were elicited. Douglass et al. (1990) found that removal of calcium reduced inactivation of the current induced by expression of RGK5 in oocytes; 0.3-1.0 mM cobalt caused a similar reduction and decreased the current amplitude and shifted the activation curve. Similarly, O’Dowd et al. (1988) noted that in the presence of the divalent cations used for the present recordings, the delayed rectifier was reduced ~20% compared with that recorded in a lower external divalent cation concentration. Acknowledgments The nucleotide sequence reported in this paper has been submitted to CenBank and assigned the accession number M35664. I thank Nicholas C. Spitzer for enthusiastic support and encouragement of this project, as well as for facilities and insights; Chris R. Kintner for generously instructing, suggesting many of the analyses used, and providing facilities during the initial stages of the work; Leslie Blair, Clark Coffman, Vince Dionne, Jane Dixon, Robert Duvoisin, Chris Kintner, Bih-Hwa Shieh, and Bruce Tempel for advising on procedures; Rosario C. de Baca and Doug Tisdale for technical support; Leslie Blair, Chris Kintner, Bih-Hwa Shieh, and Nick Spitzer for comments on the manuscript; Bruce Tempel and Clark Coffman for kindly providing the ShAl cDNA and Xenopus genomic DNA, respectively; and Chris Kintner for N-CAM and EF-la DNAs and Xenopus genomic libraries. This work was supported by National Institutes of Health grants NS25217 to A. B. R. and NS25916 to N. Spitzer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “iadverfisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received July 11, 1990; revised August 16, 1990, References Baccaglini, P. I., and Spitzer, N. C. (1977). Developmental changes in the inward current of the action potential of RohonBeard neurones. J. Physiol. 277, 93-117. Baldwin, T J., Yoshikara, C. M., Blackner, K., Kintner, C. R., and Burden, S. J. (1988). Regulation of acetylcholine receptor transcript expression during development in Xenopus laevis. J. Cell Biol. 106, 469-478. Barish, M. E. (1986). Differentiation of voltage-gated potassium current and modulation of excitability in cultured amphibian spinal neurones. 1. Physiol. 375, 229-250. Baumann, A., Krah-Jentgens, I., Miller, R., Miiller, Holtkamp, F., Seidel, R., Kecskemethy, N., Casal, J., Ferrus, A., and Pongs, 0. (1987). Molecular organization of the maternal effect region of the Shaker complex of Drosophila: characterization of an IA channel transcript with homology to vertebrate Na+ channel. EMBO J. 6, 3419-3429. Baumann, A., Grupe, A., Ackermann, A., and Pongs, 0. (1988). Structure of the voltage-dependent potassium channel is highly conserved from Drosophila to vertebrate central nervous systems. EMBO J. 7, 2457-2463. Beckh, S., and Pongs, 0. (1990). Members of the RCK family are differentially expressed in the rat nervous system. EMBO J. 9, 777-782. Blackshaw, S. E., and Warner, A. (1976). Onset of acetylcholine

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