Ubiquitous potassium-channel proteins and their genes

Details Ubiquitous potassium-channel proteins and their genes Ubiquitous potassium-channel proteins and their genes

1998-10-06

2002-12-10

Kemmerer, Elizabeth (Department: 1646)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S252300, C435S254110, C435S320100, C536S023100, C536S023500, C536S024300, C536S024310

Reexamination Certificate

active

06492167

ABSTRACT:

The present invention relates to proteins for novel ATP-sensitive potassium channels, huK
ATP
-1 and ruK
ATP
-1, that are expressed in various tissues of human and rat origins, and to genes encoding the same. The said proteins and genes can be used as diagnostic and therapeutic agents for potassium-channel related diseases such as diabetes, hypertension and endocrine insufficiencies.
BACKGROUND OF THE INVENTION
The etiology for diabetes is known to be mostly owing to disturbances of insulin secretion in the pancreatic &bgr;-cells. Consequently, elucidation of the molecular mechanism of insulin secretion is expected to play an important role in the clarification of causes for diabetes and the development of therapeutic agents against diabetes, but no detail has yet been made known on such molecular mechanism.
It has already been made clear that the ATP-sensitive potassium channel (K
ATP
) being present on the cellular membrane plays a leading role in the cellular functions such as secretions and muscular contraction by coupling the state of metabolism in the cells with the membrane potential.
The K
ATP
channel was first discovered in the cardiac muscle in 1983 [Noma, A., Nature 305:147 (1983)] and was thereafter confirmed to be present in tissues such as the pancreatic &bgr;-cell [Cook, D. L. et al., Nature 311: 271 (1984), Misler, S. et al., Proc. Natl. Acad. Sci. U.S.A. 83: 7119 (1986)], pituitary [Bernardi, H. et al., Proc. Natl. Acad. Sci. U.S.A., 90:1340 (1993)]. skeletal muscle [Spruce, A. E., et al., Nature, 316: 736 (1985)] and brain.
In addition, it has been suggested that there exists the molecular heterogeneity of such K
ATP
channels [Ashcroft, F. M., Annu. Rev. Neurosci. 11: 97 (1988)].
Particularly in the pancreatic &bgr;-cells, ATP produced by the metabolism of glucose brings about calcium ion inflow from the calcium channel by closing the K
ATP
channel to cause depolarization, resulting in secretion of insulin. As is evident from this, the K
ATP
channel plays a leading role in regulating the secretion of insulin.
The K
ATP
channel belongs to a potassium channel family exhibiting electrophysiologically inward rectification, whereby the potassium channel family exhibiting inward rectification is classified into the four subfamilies, ROMK1, IRK1, GIRK1 and cK
ATP
-1, on the basis of the degree of amino acid sequence identity.
Nevertheless, there has not been clarified the molecular architecture for the K
ATP
channel in the pancreatic &bgr;-cells. In addition, no information has been disclosed on the novel ATP-sensitive potassium channels (huK
ATP
-1 and ruK
ATP
-1) of the present invention for the detailed protein structure and the formation of complexes with other proteins, for example, the sulfonylurea binding protein.
SUMMARY OF THE INVENTION
In order to achieve the isolation, identification and functional analyses of a novel membrane channel, there are required the sophisticated techniques, such as molecular biological technique, cellular biological technique and electro-physiological technique.
Such being the case, the present inventors made ample and full use of such techniques to isolate human and rat genomes and cDNAs encoding the novel K
ATP
channel (uK
ATP
-1) expressed in different tissues of mammalians and to identify their amino acid sequences (see
FIGS. 1
,
2
,
3
and
4
). The identified uK
ATP
-1 channel was expressed in the Xenopus oocyte system and mammalian cell lines.
Electrophysiological analysis demonstrated that uK
ATP
-1 is an ATP-sensitive potassium channel exhibiting inward rectification. The uK
ATP
-1 channel being expressed ubiquitously in tissues of mammalians inclusive of man and rats is involved in the maintenance of the membrane potential through the basal energy metabolism.
As is described in the above, the present invention relates to an ATP-sensitive potassium channel (uK
ATP
-1) which is ubiquitously present in mammalians, and encompasses the ATP-sensitive potassium channel proteins, identified DNA sequences encoding the same, plasmid having such sequences incorporated therein and furthermore recombinant cells (tranformants) having such plasmid transfected therein. In addition, this invention comprises the isolated UK
ATP
-1 proteins and recombinant proteins, their related materials such as agonists and antagonists, and drug designs inclusive of diagnostics and drugs for gene therapy.
DETAILED DESCRIPTION
huK
ATP
-1 of a human origin is composed of 424 amino acid residue (See
FIG. 1
(SEQ ID NO: 1)) with a molecular weight of 47,965, while the one of a rat origin is likewise composed of 424 amino acid residue (see
FIG. 4
(SEQ ID NO: 4)) with a molecular weight of 47,960. These two potassium channels exhibit 98% amino acid sequence identity, and such a marked homology leads us to the assumption that uK
ATP
-1 performs common, structurally and functionally basic actions in all mammalian cells. Among others, uK
ATP
-1 participates in the membrane potential and energy metabolism, suggesting that it could find application as a drug substance acting to prevent disturbances under unusual, extreme metabolic conditions inclusive of endocrine diseases, e.g. diabetes, starvation and ischemia.
For example, the inflow and outflow of calcium ions caused by the opening and closing of UK
ATP
-1 during the onset of ischemia is closely connected with ischemic disturbances. In other words, there is a possibility that the agonists and antagonists for the opening and closing of uK
ATP
-1 would constitute a suppressory agent against ischemic disturbances.
From the comparative studies of huK
ATP
-1 and ruK
ATP
-1 with other potassium channels for the amino acid sequence, it was confirmed that uK
ATP
-1 of the present invention belongs to a novel family of the inward rectifier potassium channels; the central region of the uK
ATP
-1 protein showed incresed homology with other inward rectifier potassium channels. A hydropathy plot indicated the presence of two hydrophobic regions, which are composed of two transmembrane regions characteristic of the inward rectifier potassium channels and one pore region [Nicholas, C. G., Trends Pharmacol. Sci., 14: 320 (1993), Jan, L. Y. and Jan, Y. N., Nature, 371: 119 (1994)].
With reference to ruK
ATP
-1 (Inagaki, N. et al., J. B. C., 270: 5691 (1995)], it was reported that in the second intracellular region, there are two potential cAMP-dependent protein kinase phosphorylation sites (Thr-234 and Ser-385) and seven potential protein kinase C dependent phosphorylation sites (Ser-224, Thr-345, Ser-354, Ser-379, Ser-385, Ser-391 and Ser-397), while there are one (Thr-63) and four potential casein kinase II dependent phosphorylation sites (Thr-234, Ser-281, Thr-329 and Ser-354) in the first and second intracellular regions, respectively, with no N-linked glycosylation site being present in the intracellular regions. The same findings were obtained with huK
ATP
-1 [Inagaki, N., et al., in press (1995)].
Then, the present inventors identified the nucleotide sequences and entire amino acid sequences of huK
ATP
-
1
and ruK
ATP
-1, thus enabling not only proteins themselves of huK
ATP
-1 and ruK
ATP
-1 but also their mutants to be synthesized in large quantities by expressing the DNAs encoding huK
ATP
-1 and ruK
ATP
-1 and their mutants in bacteria or animal cells with use of the known genetic engineering techniques. It is furthermore added that huK
ATP
-
1
and its fragments are useful for the hybridization diagnosis of depleted huK
ATP
-1 DNA, with the mutants of huK
ATP
-1 being of use in the studies on the sugar metabolism in cells, particularly insulin-dependent and independent diabetes.
The DNAs of novel huK
ATP
-1 and ruK
ATP
-1 according to the present invention were identified based on a cDNA library and genome library. The DNA encoding huK
ATP
-1 shows a length of about 9.7 kb, being composed of three exons and is present on the chromosome at 12p11.23. The chromosomal DNA can be obtained by probing a genome DNA library with use o

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