Structure and complex transcription pattern of the mouse SK1 K(Ca) channel gene, KCNN1.

Small conductance calcium-gated K(+) channels (SK channels) are encoded by the three SK genes, SK1, SK2, and SK3. These channels likely contribute to slow synaptic afterhyperpolarizations of apamin-sensitive and apamin-insensitive types. SK channels are also widely expressed outside the nervous system. The mouse SK1 gene comprises at least 12 exons extending across 19.8 kb of genomic DNA. This gene encodes a complex pattern of alternatively spliced SK1 transcripts widely expressed among mouse tissues. These transcripts exhibit at least four distinct 5'-nucleotide sequence variants encoding at least two N-terminal amino acid sequences. Optional inclusion of exons 7 and 9, together with two alternate splice donor sites in exon 8, yields transcripts encoding eight variant C-terminal amino acid sequences for SK1. These include an altered putative S6 transmembrane span, modification of the C-terminal cytoplasmic domain binding site for calmodulin, and generation of two alternate predicted binding sites for PDZ domain-containing proteins. 20 of the 32 predicted mouse SK1 transcripts are expressed in brain at levels sufficient to allow consistent detection, and encode 16 SK1 polypeptide variants. Only four of these 16 polypeptides preserve the ability to bind calmodulin in a Ca(2+)-independent manner. Mouse SK1 also exhibits novel, strain-specific, length polymorphism of a polyglutamate repeat in the N-terminal cytoplasmic domain. The evolutionary conservation of this complex transcription pattern suggests a possible role in the tuning of SK1 channel function.

cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation.

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.

Episodic ataxia type-1 mutations in the hKv1.1 cytoplasmic pore region alter the gating properties of the channel.

Episodic ataxia type-1 is a rare human neurological syndrome which occurs during childhood and persists through the whole life of affected patients. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene hKv1.1 of different affected families. V408A and E325D mutations are located in the cytoplasmic putative pore region of hKv1.1 channels and profoundly alter their gating properties. V408A channels showed increased kinetic rates of activation, deactivation and C-type inactivation. Expression of E325D channels in Xenopus oocytes led to an approximately 13-fold current amplitude reduction and to a 52.4 mV positive shift in the voltage dependence of activation. Moreover, the E325D mutation altered the kinetics of activation, deactivation, C-type inactivation and channel open probability. Heteromeric channels composed of two wild-type and two mutated subunits, linked as dimers, showed gating properties intermediate between channels formed from four normal or four mutated subunits. The results demonstrate that the highly conserved residues Val408 and Glu325 play a pivotal role in several gating processes of a human potassium channel, and suggest a pathogenetic mechanism by which the impairment of the delayed-rectifier function of affected neurons is related to the type and number of mutated subunits which make up the hKv1.1 channels.

Inward rectifier potassium channels. Cloning, expression and structure-function studies.

A PCR-based cloning strategy was used to identify novel subunits of the two-transmembrane domain inward rectifier potassium channel family from rat brain, heart, and skeletal muscle. When expressed in Xenopus oocytes, two of these clones (Kir4.1 and Kir2.3) gave rise to inwardly rectifying potassium currents. Two-electrode voltage clamp commands to potentials negative to EK evoked inward potassium-selective currents which rapidly reached a peak amplitude and then relaxed to a steady-state level. Differences in the extent of current relaxation, the degree of rectification, and the voltage-dependent block by external cesium were detected. Two other members of this family (Kir5.1 and Kir3.4) did not produce macroscopic currents, when expressed by themselves, yet both subunits modified the currents when coexpressed with other specific members of the Kir family. Expression of chimeric subunits between Kir4.1 and either Kir5.1 or Kir3.4 suggested that the transmembrane domains determine the specificity of subunit heteropolymerization, while the C-terminal domains contribute to alterations in activation kinetics and rectification. Expression of covalently linked subunits demonstrated that the relative subunit positions, as well as stoichiometry, affect heteromeric channel activity.

A neuron-specific enhancer targets expression of the gonadotropin-releasing hormone gene to hypothalamic neurosecretory neurons.

The molecular mechanisms specifying gene expression in individual neurons of the mammalian central nervous system have been difficult to study due to the cellular complexity of the brain and the absence of cultured model systems representing differentiated central nervous system neurons. We have developed clonal, differentiated, neuronal tumor cell lines of the hypothalamic GnRH-producing neurons by targeting tumorigenesis in transgenic mice. These cells (GT1 cells) provide a model system for molecular studies of GnRH gene regulation. Here we present the identification and characterization of a neuron-specific enhancer responsible for directing expression of the rat GnRH gene in GT1 hypothalamic neurons. This approximately 300 base pair (bp) upstream region (-1571 to -1863) confers enhancer activity to a short -173-bp GnRH promoter or to a heterologous promoter only in GT1 cells. The enhancer is bound by multiple GT1 nuclear proteins over its entire length. Deletion of more than 30 bp from either end dramatically reduces activity, and even large internal fragments carrying seven of the eight DNAse I-protected elements show decreased activity. Scanning replacement mutations demonstrate that several of the internal elements are required for activity of the enhancer. Thus, the GnRH gene is targeted to hypothalamic neurons by a complex multicomponent enhancer that relies on the interaction of multiple nuclear-protein binding enhancer elements.

Potassium channel assembly from concatenated subunits: effects of proline substitutions in S4 segments.

A concatenated cDNA was made that comprised four contiguous copies of the rat brain potassium channel subunit Kv1.1. Currents were measured in oocytes that had been injected with in vitro transcribed RNA. A Pro residue was introduced by site-directed mutagenesis into the S4 transmembrane region of one of the four domains, at a position corresponding to that in which a Pro is found in voltage-dependent sodium and calcium channels. This substitution (L305P in any one domain) led to currents having a shallow activation curve, and an additional fast component of deactivation from strongly positive potentials. cDNAs with L305P substitution in two domains formed functional channels only if the domains were non-adjacent; the properties of the currents were similar to the wild-type concatemer. In both cases, 100-1000 x more RNA was injected to obtain maximal currents similar to those seen with the wild-type concantemer. The point mutation Y379V is known to reduce the blocking potency of extracellular tetraethylammonium; this residue from each of the four domains is exposed at the outer mouth of the pore because the progressive introduction of 1, 2, 3 and 4 such mutations causes progressive reductions in tetraethylammonium sensitivity. The Y379V mutation was introduced into concatenated cDNAs with two non-adjacent Pro-containing domains; the sensitivity to TEA of the resulting currents showed that the Pro-containing subunits did not contribute to the pore-forming part of the channel. The results suggest that channels can form with a Pro substitution in a single S4 domain, but they require strong depolarization to open and deactivate rapidly upon repolarization. With Pro substitutions in two domains, channels appear to be formed as a multimerized concatemer, in which the Pro-containing domains are excluded from pore formation.

Cloning and functional expression of a rat heart KATP channel.

Potassium channels that are ATP-sensitive (KATP) couple membrane potential to the metabolic status of the cell. KATP channels are inhibited by intracellular ATP and are stimulated by intracellular nucleotide diphosphates. KATP channels are important regulators of secretory processes and muscle contraction, and are targets for therapeutic treatment of type II diabetes by the inhibitory sulphonylureas and for hypertension by activators such as pinacidil. In cardiac tissue, KATP channels are central regulators of post-ischaemic cardioprotection. Electrophysiological and pharmacological characteristics vary among KATP channels recorded from diverse tissues suggesting extensive molecular heterogeneity. A complementary DNA encoding a KATP channel was isolated from rat heart using the polymerase chain reaction. We report here that the expressed channels possess all of the essential features of native cardiac KATP channels, including sensitivity to intracellular nucleotides. In addition the cloned channels are activated by the potassium channel opener, pinacidil, but are not inhibited by the sulphonylurea, glibenclamide.

Cloned Ca(2+)-dependent K+ channel modulated by a functionally associated protein kinase.

Calcium-dependent potassium (KCa) channels carry ionic currents that regulate important cellular functions. Like some other ion channels, KCa channels are modulated by protein phosphorylation. The recent cloning of complementary DNAs encoding Slo KCa channels has enabled KCa channel modulation to be investigated. We report here that protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that KCa channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch.

Calcium-activated potassium channels expressed from cloned complementary DNAs.

Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.

Regulation by second messengers of the slowly activating, voltage-dependent potassium current expressed in Xenopus oocytes.

1. Voltage-clamp recordings of membrane current were made from Xenopus oocytes that had been injected with RNA which had been transcribed in vitro from a cloned complementary DNA. 2. Depolarization from -80 mV evoked outward potassium currents that developed very slowly. At -20 mV the time constant for activation was about 50 s, and at +40 mV about 6 s. 3. The potassium current was increased by the calcium ionophore A23187 or by intracellular injection of inositol 1,4,5-trisphosphate (IP3), each of which should increase the intracellular calcium concentration ([Ca2+]i). The current was decreased by injection of BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). The current was also reduced by phorbol esters; this effect was blocked by staurosporine. 4. In oocytes that had also been injected with RNA encoding the 5-hydroxytryptamine (5-HT2) receptor, 5-HT increased the potassium current. After caffeine pretreatment, to block the release of intracellular calcium, 5-HT decreased the current; this decrease was prevented by staurosporine. 5. It is concluded that the slowly activating, voltage-dependent potassium current expressed in Xenopus oocytes is increased by increases in [Ca2+]i and is decreased by activation of protein kinase C. Stimulation of 5-HT2 receptors can have both these effects, but the former normally predominates.

Partial characterization of the gonadotropin-releasing hormone (GnRH) gene transcript in the rat ovary.

It has been hypothesized that GnRH or a GnRH-like peptide is produced in the rat ovary, but the presence of GnRH in the ovary has not been unequivocally demonstrated. This study was undertaken to determine whether the GnRH gene is expressed in the rat ovary and to compare the GnRH gene transcripts from the ovary and the hypothalamus. Twelve samples of total RNA from ovaries of individual rats were screened by reverse transcription-polymerase chain reaction (RT-PCR) for the presence of GnRH gene transcripts. Fragments of GnRH cDNA were amplified using pairs of specific primers. GnRH transcripts were detected in all the ovaries examined, and differed from hypothalamic GnRH transcripts in two ways: first, in the ovaries a greater proportion of GnRH transcripts contained intronic sequences; second, the major transcription start utilized in the ovary differed from that used in the hypothalamus. Although fully processed GnRH gene transcripts were detected by RT-PCR in both, ovary and hypothalamus, they were not detected in the ovary by Northern blot. The GnRH probe hybridized specifically to the predicted 0.6 kb transcript in the hypothalamus, and to a 3.3 kb transcript in the ovary. We conclude that in the ovary, most GnRH gene transcripts retain intronic sequences.

The rat gonadotropin-releasing hormone: SH locus: structure and hypothalamic expression.

The rat GnRH gene as expressed in the central nervous system is comprised of four exons and three introns and spans 4.5 kilobases of genomic DNA. Recently it has been shown that the DNA strand opposite that which is transcribed to produce GnRH mRNA is transcribed in heart to produce a set of transcripts, SH RNAs, which share significant exonic sequences with the GnRH gene. The nucleotide sequence of this locus and approximately 3 kilobases on either side has been determined. Northern analysis of hypothalamic RNA probed with GnRH and SH strand specific probes demonstrate that both GnRH and SH RNAs are present within the preoptic hypothalamus. The cap sites for GnRH and SH transcripts have been localized using polymerase chain reaction technology. Results from these experiments indicate that in the preoptic hypothalamus GnRH transcription initiates from three sites. The majority of GnRH transcripts is spliced efficiently and gives rise to the major class of GnRH mRNA. A second spliced population is present in lower abundance, while a third population is not spliced. The SH gene contains at least two distinct promoters, from which two populations of transcripts are derived containing unique 5'-sequences spliced to a common 3'-region.

Expression of a cloned rat brain potassium channel in Xenopus oocytes.

Potassium channels are ubiquitous membrane proteins with essential roles in nervous tissue, but little is known about the relation between their function and their molecular structure. A complementary DNA library was made from rat hippocampus, and a complementary DNA clone (RBK-1) was isolated. The predicted sequence of the 495-amino acid protein is homologous to potassium channel proteins encoded by the Shaker locus of Drosophila and differs by only three amino acids from the expected product of a mouse clone MBK-1. Messenger RNA transcribed from RBK-1 in vitro directed the expression of potassium channels when it was injected into Xenopus oocytes. The potassium current through the expressed channels resembles both the transient (or A) and the delayed rectifier currents reported in mammalian neurons and is sensitive to both 4-aminopyridine and tetraethylammonium.

Effects of ovariectomy on GnRH mRNA, proGnRH and GnRH levels in the preoptic hypothalamus of the female rat.

Experiments were carried out to investigate the effects of ovariectomy on gonadotropin-releasing hormone (GnRH) messenger RNA (mRNA), proGnRH and GnRH peptide levels in the hypothalamus of female rats. Intact proestrous female rats and female rats, which had been ovariectomized for 2 weeks, were sacrificed at 9.00 h and the preoptic area (POA) and basal hypothalamus (BH) were dissected out and frozen on dry ice. One group of tissues from proestrous control and ovariectomized females were extracted in acetic acid, centrifuged at 13,000 g and the supernatant purified on a C18 column. The purified extract was then radioimmunoassayed for proGnRH, using a specific antiserum to rat proGnRH (ARK-2), and for GnRH using the E1-14 antiserum. Total cellular RNA was isolated from another group of tissues and prepared as Northern blots. Hybridization with 32P-labeled GnRH cRNA was used to detect GnRH mRNA. A third group of proestrous and ovariectomized female rats were perfused, and 50 microns vibratome sections were cut. These were immunostained with proGnRH or GnRH antiserum, followed by in situ hybridization with 35S-labeled GnRH cRNA to detect GnRH mRNA. Based on the histochemical staining, mRNA was colocalized to the cell soma of neurons containing proGnRH and GnRH throughout the POA and BH. Based on the radioimmunoassay, proGnRH levels were 2 times higher in the POA versus the BH, but GnRH levels were 6-7 times higher in the BH. Ovariectomy significantly decreased proGnRH levels in both the POA and BH, while GnRH decreased in the BH. In contrast, quantitative Northern blot analysis demonstrated that ovariectomy had no effect on mRNA levels in the POA and BH. These data indicate that the effects of ovariectomy on proGnRH and GnRH levels are a result of altered translation, posttranslational processing and/or secretion of GnRH.

Two mammalian genes transcribed from opposite strands of the same DNA locus.

This report describes the characterization of a genomic locus in the rat that encodes overlapping genes occupying both strands of the same piece of DNA. One gene (strand) encodes gonadotropin-releasing hormone (GnRH). A second gene, SH, is transcribed from the other DNA strand to produce RNA of undefined function. The RNAs transcribed from each DNA strand are spliced and polyadenylated, and share significant exon domains. GnRH is expressed in the central nervous system while SH transcripts are present in the heart. Thus, the genome of a mammalian organism encodes two distinct genes by using both strands of the same DNA.

Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hormone and prolactin release-inhibiting factor in human and rat.

Cloned cDNAs encoding the precursor protein for gonadotropin-releasing hormone (Gn-RH) and prolactin release-inhibiting factor (PIF) were isolated from libraries derived from human and rat hypothalamic mRNA. Nucleotide sequence analyses predict precursor proteins of 92 amino acids for both species and show identity between the human placental and human hypothalamic precursor proteins. Whereas the Gn-RH peptide structure is completely conserved in human and rat, the PIF domain of the precursor displays 70% interspecies homology. Genomic analyses revealed the presence of a single Gn-RH-PIF gene in human and rat containing sequences corresponding to the cDNA distributed across four exons.