Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-10-21
2004-11-09
Eyler, Yvonne (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S069100, C435S253100, C435S320100, C435S325000, C530S350000, C530S300000, C536S023100, C536S024310, C424S130100
Reexamination Certificate
active
06815539
ABSTRACT:
FIELD OF THE INVENTION
The invention provides isolated nucleotide and amino acid sequences of Slo3, a pH sensitive potassium channel expressed in sperm; antibodies to Slo3; methods if of screening for Slo3 inhibitors and activators; and methods of identifying Slo3 homologs.
BACKGROUND OF THE INVENTION
Potassium channels are found in a wide variety of animal cells such as nervous, muscular, glandular, immune or epithelial tissue. The channels regulating these currents open and allow the escape of potassium under certain conditions. The outward flow of potassium ions upon opening of these channels makes the interior of the cell more negative, counteracting depolarizing voltages applied to the cell. These channels are regulated, e.g., by calcium sensitivity, voltage-gating, and ATP-sensitivity.
The Drosophila Slo1 gene encodes a calcium-activated potassium channel present in both neurons and muscle (Elkins et al.,
Proc. Natl. Acad. Sci. U.S.A
. 83:8415 (1986); Atkinson et al.,
Science
253:551 (1991); and Adelman et al.,
Neuron
9:209 (1992)). Mammalian homologs of dSlo1 were cloned and found to be “Maxi” or BK (large conductance) channel types, as the single channel conductance was 272 pS with symmetrical potassium concentrations. Slo1 channels cloned from mouse and human show strong conservation of sequence and functional properties (Butler et al.,
Science
261:221-224 (1993); Dworetzky et al.,
Brain Res. Mol. Brain Res
. 27:189-193 (1994); Tseng-Crank et al.,
Neuron
13:1315-1330 (1994); McCobb et al.,
Am. J. Physiol
. 269:H767-H777 (1995); and Wallner et al.,
Rec. Chan
. 3:185-199 (1995)). One proposed role of the Slo1 channel is to provide negative feedback for the entry of calcium into cells via voltage-dependent calcium channels. Perhaps because of the versatility of this mechanism, Slo1 channels are expressed in many tissues, including brain, skeletal and smooth muscle, auditory hair cells, pancreas, and adrenal gland (Marty,
Nature
291:497-500 (1981); Pallotta et al.,
Nature
293:471-474 (1981); Petersen & Mauryama,
Nature
307:693-696 (1984); Tabcharani & Misler,
Biochim. Biophys. Acta
. 982:62-72 (1990); Neely & Lingle,
J. Physiol
. 453:97-131 (1992)). In these tissues, Slo1 channels are involved in diverse functions such as regulating arteriolar smooth muscle tone (Brayden & Nelson,
Science
256:532-535 (1992)), tuning of hair cell frequency (Fuchs,
Curr. Op. Neurobiol
. 2:457-461 (1992); Wu et al.,
Prog. Biophys. Mol. Bio
. 63:131-158 (1996)), and modulation of transmitter release at nerve terminals (Robitaille & Charlton,
J. Neurosci
. 12:297-305 (1995); Knaus et al.,
J. Neurosci
. 16:955-963 (1996)), all situations in which both membrane potential and intracellular calcium are critical factors. While numerous family members of every type of voltage-gated K
+
channel have been found, to date the Slo1 channel has remained the sole functionally characterized representative of the Slo family (Wei et al.,
Neuropharmacology
35:805-829 (1996)).
Spermatocytes require proteins tailored to fulfill roles unique to the process of germ cell development and fertilization. Cellular signaling in spermatic cells is tightly regulated to prevent inappropriate activation of the irreversible steps that prepare the sperm to fertilize the oocyte. Many of these steps are triggered and coordinated by changes in membrane potential and intracellular Ca
2+
concentration and pH. Between mating and fertilization, sperm undergo capacitation, a process which later enables them to fertilize the oocyte. Capacitation involves an increase in cytosolic pH (pHi), which promotes metabolic and swimming activity (Babcock et al.,
Proc. Natl. Acad. Sci. USA
80:1327-1331 (1983); Babcock & Pfeiffer,
J. Biol. Chem
. 262:15041-15047 (1987); Vredenburgh-Wilberg & Parrish,
Mol. Reprod. Dev
. 40:490-502 (1995)). This increase in pHi is accompanied by changes in membrane potential and a rise in cytoplasmic Ca
2+
, which trigger the acrosome reaction upon contact with the oocyte (Amoult et al.,
J. Cell Biol
. 134:637-645 (1996); Florman,
Dev. Biol
. 165:152-164 (1994)). Because of the central importance of these events in development, many efforts have been made to identify the specific proteins, including ion channels, which regulate spermatic function. In particular, there have been reports of channels present in spermatocytes and spermatids that have been proposed to play central roles in these reactions (Cook & Babcock,
J. Biol. Chem
. 268:22402-22407 (1993), including voltage dependent calcium channels (Florman et al.,
Dev. Biol
. 152:304-214 (1992); Arnoult et al.,
Proc. Natl. Acad. Sci. USA
93:13004-13009 (1996); Lievano et al.,
FEBS Lett
. 388:150-154(1996); Santi et al.,
Am. J. Physiol
. 271:C1583-C1593 (1996)). Apart from a cyclic nucleotide gated channel, however, few of these channels have been directly cloned from testis (Weyand et al.,
Nature
368:859-863 (1994)).
SUMMARY OF THE INVENTION
Potassium channels have evolved to play specialized roles in many inexcitable tissues. The present invention provides for the first time isolated nucleotide and amino acid sequences of Slo3, a potassium channel with novel functional properties, abundantly expressed in spermatocytes. The physiological reactions that sperm undergo to achieve fertilization include changes in both pHi and membrane potential. Although Slo3 is a member of the Slo family, to which the large-conductance, calcium-activated Slo1 potassium channel belongs, Slo3 channels are not gated by calcium. Slo3 channels, however, are activated by changes in intracellular pH and membrane potential. Slo3 channels also exhibit markedly lower selectivity for K
+
over Na
+
than most voltage-gated K
+
channels.
In one aspect, the invention provides an isolated nucleic acid encoding a polypeptide monomer of a pH sensitive potassium channel. The monomer (i) has a calculated molecular weight of between 120-156 kDa; (ii) has a unit conductance of approximately 80-120 pS when the monomer is in a functional tetrameric form of a potassium channel and is expressed in a Xenopus oocyte; (iii) has increased activity above approximately intracellular pH of 7.1; and (iv) specifically binds to polyclonal antibodies generated against SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:16 or SEQ ID NO:18.
In one embodiment, the nucleic acid encodes mSlo3 or hSlo3. In another embodiment, the nucleic acid encodes SEQ ID NO:1, SEQ ID NO:16, or SEQ ID NO:18. In one embodiment, the nucleic acid selectively hybridizes under moderate stringency hybridization conditions to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:17, or SEQ ID NO:19. In one embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:17, or SEQ ID NO:19.
In one embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as the primer sets selected from the group consisting of:
CTCGAACTCCCTAAAATCTTACAGAT (SEQ ID NO:8) and
TTCCGTTGAGCCAGGGGTCACCAGAATT (SEQ ID NO:9);
TCTGCTTTGTGAAGCTAAATCT (SEQ ID NO:10) and
TTTCAAAGCCTCTTTAGCGGTAA (SEQ ID NO:11); and
TTATGCCTGGATCTGCACTCTACATG (SEQ ID NO:12) and
ATAGTTTCCGTCTACTACCGAAA (SEQ ID NO:13).
In another embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as the primer sets selected from the group consisting of:
GGCAGCGCTCATTCTTTCCTCCTT (SEQ ID NO:14) and
TGCCCAAAACCTCAACCCAAAATA (SEQ ID NO:15).
In another aspect, the invention provides an isolated nuclcic acid encoding at least 15 contiguous amino acids from a pH sensitive potassium channel polypeptide monomer, said monomer having an amino acid sequence of SEQ ID NO:1, SEQ ID NO:16, or SEQ ID NO:18 and conservatively modified variants thereof.
In one embodiment, the nucleic acid encodes a pH sensitive potassium channel polypeptide monomer having: (i) a unit conductance of 80-120 pS when the monomer is in a functional tetrameric form of a potassium channel and is expressed in a Xe
Salkoff Lawrence
Schreiber Matthew
Silvia Chris
Basi Nirmal S.
Eyler Yvonne
Icagen, Incorporated
Townsend and Townsend / and Crew LLP
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