Sperm-specific cation channel, catsper-4, and uses therefor

Abstract

Nucleic acid and protein sequences relating to a cation channel which is sperm-specific (CatSper4) are disclosed. The CatSper4 protein is shown to be specifically expressed in sperm. Nucleic acids, vectors, transformed cells, transgenic animals, polypeptides, and antibodies relating to the CatSper4 gene and protein are disclosed. Also provided are methods of in vitro fertilization and contraception, methods of identifying modulators of CatSper4 activity, methods of genotyping subjects with respect to CatSper4, and methods of diagnosing and treating CatSper4-mediated disorders, including infertility. Related business methods are also disclosed.

1 . An isolated nucleic acid having a nucleotide sequence selected from the group consisting of: 
 (a) at least 10 consecutive nucleotides of SEQ ID NO: 1;    (b) at least 12 consecutive nucleotides of SEQ ID NO: 1;    (c) at least 14 consecutive nucleotides of SEQ ID NO: 1;    (d) at least 16 consecutive nucleotides of SEQ ID NO: 1;    (e) at least 18 consecutive nucleotides of SEQ ID NO: 1; and    (f) a sequence complementary to any one of the sequences of (a)-(e).    
     
     
         2 . An isolated nucleic acid having a nucleotide sequence selected from the group consisting of: 
 (a) at least 10 consecutive nucleotides of SEQ ID NO: 3;    (b) at least 12 consecutive nucleotides of SEQ ID NO: 3;    (c) at least 14 consecutive nucleotides of SEQ ID NO: 3;    (d) at least 16 consecutive nucleotides of SEQ ID NO: 3;    (e) at least 18 consecutive nucleotides of SEQ ID NO: 3; and    (f) a sequence complementary to any one of the sequences of (a)-(e).    
     
     
         3 . An isolated nucleic acid having a nucleotide sequence selected from the group consisting of: 
 (a) a sequence encoding a CatSper4 protein;    (b) a sequence encoding at least a transmembrane domain of a CatSper4 protein;    (c) a sequence encoding at least an extracellular loop of a CatSper4 protein;    (d) a sequence encoding at least a pore region of a CatSper4 protein;    (e) a sequence encoding at least an epitope of a CatSper4 protein having high predicted antigenicity; and    (f) a sequence complementary to any one of the sequences of (a)-(e).    
     
     
         4 . An isolated nucleic acid as in  claim 3  selected from the group consisting of: 
 (a) SEQ ID NO: 1;    (b) SEQ ID NO: 3;    (c) a sequence encoding a polypeptide comprising residues 46-75, 86-98, 108-137, 155-202, 209-233 and 242-272 of SEQ ID NO: 2;    (d) a sequence encoding a polypeptide comprising residues 48-73, 81-111, 113-134, 156-204, 208-233 and 245-273 of SEQ ID NO: 4;    (e) a sequence encoding a polypeptide comprising residues 76-85, 138-154 and 234-242 of SEQ ID NO: 2;    (f) a sequence encoding a polypeptide comprising residues 74-80, 135-155 and 234-244 of SEQ ID NO 4;    (g) a sequence encoding a polypeptide comprising residues 222-231 of SEQ ID NO: 2;    (h) a sequence encoding a polypeptide comprising residues approximately residues 222-231 of SEQ ID NO: 4;    (i) a sequence encoding a polypeptide comprising a high predicted antigenicity epitope of SEQ ID NO: 2;    (j) a sequence encoding a polypeptide comprising a high predicted antigenicity epitope of SEQ ID NO: 4; and    (k) a sequence complementary to any one of the sequences of (a)-(j).    
     
     
         5 . An isolated nucleic acid encoding a polypeptide having at least 80% amino acid sequence identity with a polypeptide selected from the group consisting of: 
 (a) a CatSper4 protein;    (b) at least a transmembrane domain of a CatSper4 protein;    (c) at least an extracellular loop of a CatSper4 protein; and    (d) at least a pore region of a CatSper4 protein.    
     
     
         6 . An isolated nucleic acid encoding a polypeptide having at least 80% amino acid sequence identity with a CatSper4 protein and having CatSper4 activity in a cell capable of expressing CatSper4 activity.  
     
     
         7 . An isolated nucleic acid comprising 
 a regulatory element having at least 80% nucleotide sequence identity to at least 100 consecutive nucleotides selected from SEQ ID NO: 5;    wherein said regulatory element is capable of promoting transcription of a coding sequence operably joined thereto in a mammalian cell in which a CatSper4 gene can be expressed.    
     
     
         8 . An isolated nucleic acid comprising a nucleotide sequence that hybridizes to at least a portion of a nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 3 under conditions including a wash step of 1.0×SSC at 65° C.  
     
     
         9 . An isolated nucleic acid as in  claim 8  wherein said nucleic acid encodes a polypeptide having CatSper4 activity.  
     
     
         10 . A nucleic acid comprising: 
 (i) a nucleotide sequence encoding a polypeptide having CatSper4 activity, wherein said nucleic acid hybridizes to at least a portion of a nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 3 under conditions including a wash step of 1.0×SSC at 65° C.; and    (ii) a heterologous regulatory region operably joined to said sequence such that said sequence is expressed.    
     
     
         11 . A nucleic acid comprising: 
 (i) a nucleotide sequence encoding a polypeptide having at least 80 percent amino acid sequence identity with an amino acid sequence of SEQ ID NO: 2 or 4; and    (ii) a heterologous regulatory region operably joined to said sequence such that said sequence is expressed.    
     
     
         12 . A kit for detecting at least a portion of a CatSper4 nucleic acid comprising an isolated nucleic acid of  claim 1  and a means for detecting said isolated nucleic acid.  
     
     
         13 . A kit as in  claim 12  wherein said means for detecting said isolated nucleic acid comprises a detectable label bound thereto.  
     
     
         14 . A kit as in  claim 12  wherein said means for detecting said isolated nucleic acid comprises a labeled secondary nucleic acid which specifically hybridizes to said isolated nucleic acid.  
     
     
         15 . A vector comprising an isolated nucleic acid of  claim 1 .  
     
     
         16 . A vector comprising a genetic construct capable of expressing a nucleic acid of  claim 3 .  
     
     
         17 . A vector as in  claim 16  wherein said nucleic acid is operably joined to an exogenous regulatory region.  
     
     
         18 . A vector as in  claim 16  wherein said nucleic is operably joined to heterologous coding sequences to form a fusion vector.  
     
     
         19 . A vector comprising an isolated nucleic acid of  claim 3 .  
     
     
         20 . A vector comprising an isolated nucleic acid of  claim 3  operably joined to a reporter gene.  
     
     
         21 . A cell transformed with a nucleic acid of  claim 3 .  
     
     
         22 . A cell transformed with a genetic construct capable of expressing a nucleic acid of  claim 3 .  
     
     
         23 . A cell as in  claim 22  wherein said nucleic acid is operably joined to heterologous coding sequences to encode a fusion protein.  
     
     
         24 . A cell as in  claim 22  wherein said cell is selected from the group consisting of bacterial cells, yeast cells, insect cells, nematode cells, amphibian cells, rodent cells, and human cells.  
     
     
         25 . A cell as in  claim 22  wherein said cell is selected from the group consisting of mammalian somatic cells, fetal cells, embryonic stem cells, zygotes, gametes, germ line cells and transgenic animal cells.  
     
     
         26 . A non-human transgenic animal, wherein a genetic construct has introduced a modification into a genome of said animal, or an ancestor thereof, and wherein said modification is selected from the group consisting of insertion of a nucleic acid encoding at least a fragment of a CatSper4 protein, inactivation of an endogenous CatSper4 gene, and insertion by homologous recombination of a reporter gene operably joined to CatSper4 regulatory elements.  
     
     
         27 . An animal as in  claim 26  wherein said modification is insertion of a nucleic acid encoding a polypeptide selected from the group consisting of a CatSper4 protein, at least a transmembrane domain of a CatSper4 protein, at least an extracellular loop of a CatSper4 protein, at least a pore region of a CatSper4 protein, and at least an epitope of a CatSper4 protein having high predicted antigenicity.  
     
     
         28 . An animal as in  claim 26  wherein said animal is selected from the group consisting of rats, mice, hamsters, guinea pigs, rabbit, dogs, cats, goats, sheep, pigs, and non-human primates.  
     
     
         29 . A substantially pure protein preparation comprising a polypeptide selected from the group consisting of: 
 (a) a CatSper4 protein;    (b) at least a transmembrane domain of a CatSper4 protein;    (c) at least an extracellular loop of a CatSper4 protein;    (d) at least a pore region of a CatSper4 protein; and    (e) at least an epitope of a CatSper4 protein having high predicted antigenicity.    
     
     
         30 . A substantially pure protein preparation as in  claim 29  wherein said polypeptide is selected from the group consisting of: 
 (a) SEQ ID NO: 2;    (b) SEQ ID NO: 4;    (c) residues 46-75, 86-98, 108-137, 155-202, 209-233 and 242-272 of SEQ ID NO: 2;    (d) residues 48-73, 81-111, 113-134, 156-204, 208-233 and 245-273 of SEQ ID NO: 4;    (e) residues 76-85, 138-154 and 234-242 of SEQ ID NO: 2;    (f) residues 74-80, 135-155 and 234-244 of SEQ ID NO 4;    (g) residues 222-231 of SEQ ID NO: 2;    (h) residues 222-231 of SEQ ID NO: 4;    (i) a high predicted antigenicity epitope of SEQ ID NO: 2; and    (j) a high predicted antigenicity epitope of SEQ ID NO: 4.    
     
     
         31 . A substantially pure protein preparation comprising a polypeptide having at least 80% amino acid sequence identity with a polypeptide selected from the group consisting of: 
 (a) a CatSper4 protein;    (b) at least a transmembrane domain of a CatSper4 protein;    (c) at least an extracellular loop of a CatSper4 protein; and    (d) at least a pore region of a CatSper4 protein.    
     
     
         32 . A substantially pure protein preparation comprising a polypeptide having at least 80% amino acid sequence identity with a CatSper4 protein and having CatSper4 activity in a cell capable of expressing CatSper4 activity.  
     
     
         33 . A substantially pure antibody preparation comprising an antibody raised against a CatSper4 epitope.  
     
     
         34 . A substantially pure antibody preparation as in  claim 33  wherein said epitope has high predicted antigenicity.  
     
     
         35 . A substantially pure antibody preparation as in  claim 33  wherein said epitope comprises an amino acid sequence within the an amino acid sequence selected from the group consisting of high predicted antigenicity epitopes of SEQ ID NO: 2, and high predicted antigenicity epitopes of SEQ ID NO: 4.  
     
     
         36 . A substantially pure antibody preparation as in  claim 33  wherein said antibody is a monoclonal antibody.  
     
     
         37 . A substantially pure antibody preparation as in  claim 33  wherein said antibody is an antibody fragment selected from the group consisting of an Fab fragment, an F(ab′) 2  fragment, an Fv fragment, and a single-chain Fv fragment (scFv).  
     
     
         38 . A kit for detecting at least an epitope of a CatSper4 protein comprising an anti-CatSper4 antibody of  claim 33  and a means for detecting said antibody.  
     
     
         39 . A kit as in  claim 38  wherein said means for detecting said anti-CatSper4 antibody comprises a detectable label bound thereto.  
     
     
         40 . A kit as in  claim 38  wherein said means for detecting said anti-CatSper4 antibody comprises a labeled secondary antibody which specifically binds to said anti-CatSper4 antibody.  
     
     
         41 . A method of identifying a potential modulator of CatSper4 activity comprising: 
 contacting a candidate compound with a cell expressing a CatSper4 protein;    measuring an indicator of CatSper4 activity in said cell;    determining whether said candidate compound caused an increase or decrease in said indicator relative to a reference level; and    identifying said candidate compound as a potential modulator of CatSper4 activity if said increase or decrease is significant.    
     
     
         42 . A method as in  claim 41  wherein said indicator is an indicator of the level of mRNA encoding said CatSper4 protein.  
     
     
         43 . A method as in  claim 41  wherein said indicator is an indicator of the level of CatSper4 protein.  
     
     
         44 . A method as in  claim 41  wherein said indicator is an indicator of cation flux across a membrane of said cell.  
     
     
         45 . A method as in  claim 41  wherein said indicator is an indicator of whole cell or channel currents of said cell.  
     
     
         46 . A method as in  claim 41  wherein said cell has been transformed with a genetic construct capable of expressing a CatSper4 protein.  
     
     
         47 . A method as in  claim 41  wherein said cell is a mature sperm cell and said indicator is a measure of sperm motility.  
     
     
         48 . A method of identifying a potential modulator of CatSper4 activity comprising: 
 contacting under physiological conditions a candidate compound with CatSper4 moiety comprising at least a structural domain of a CatSper4 protein;    measuring binding, if any, between said candidate compound and said CatSper4 moiety;    identifying said candidate compound as a potential modulator of CatSper4 activity if said binding is significant.    
     
     
         49 . A method as in  claim 48  wherein said CatSper4 moiety is a polypeptide selected from the group consisting of: 
 (a) a CatSper4 protein;    (b) at least a transmembrane domain of a CatSper4 protein;    (c) at least an extracellular loop of a CatSper4 protein; and    (d) at least a pore region of a CatSper4 protein.    
     
     
         50 . A method of decreasing the fertility of a male subject comprising: 
 administering to said male a compound which decreases CatSper4 activity.    
     
     
         51 . A method of causing reversible infertility in a male subject comprising: 
 administering to said male a compound which decreases CatSper4 activity.    
     
     
         52 . A method of contraception comprising: 
 administering to a male subject a compound which decreases CatSper4 activity.    
     
     
         53 . A method of contraception comprising: 
 administering to a female subject a compound which decreases CatSper4 activity.    
     
     
         54 . A method as in  claim 50  wherein said compound is in a formulation selected from the group consisting of an injection, a transdermal patch, a bioerodable implant, a lubricant, a moisturizer, a foam, a jelly, and a sponge.  
     
     
         55 . A method of contraception as in  claim 53  wherein: 
 said female subject is a mammal and said compound is administered into at least one of the vagina, uterus and fallopian tubes of said female.    
     
     
         56 . A method as in  claim 50  wherein said compound is selected from the group consisting of a nucleic acid which is antisense to at least a portion of a CatSper4 gene and an antibody to a CatSper4 protein.  
     
     
         57 . A method as in  claim 56  wherein said compound is an antibody fragment selected from the group consisting of an Fab fragment, an F(ab′) 2  fragment, an Fv fragment, and an scFv fragment.  
     
     
         58 . A method as in  claim 50  wherein said subject is a mammal.  
     
     
         59 . A method as in  claim 58  wherein said mammal is selected from the group consisting of humans, dogs, cats, cows, sheep, horses, mice, rats, raccoons, and gophers.  
     
     
         60 . A method as in  claim 58  wherein said subject is selected from the group consisting of a fish, an amphibian and an insect.  
     
     
         61 . Use of a compound which decreases CatSper4 activity in the preparation of a medicament for decreasing the fertility of a male subject.  
     
     
         62 . Use of a compound which decreases CatSper4 activity in the preparation of a medicament for causing reversible infertility in a male subject.  
     
     
         63 . Use of a compound which decreases CatSper4 activity in the preparation of a contraceptive for administration to a male.  
     
     
         64 . Use of a compound which decreases CatSper4 activity in the preparation of a contraceptive for administration to a female.  
     
     
         65 . A use as in  claim 61  wherein said compound is in a formulation selected from the group consisting of an injection, a transdermal patch, a bioerodable implant, a lubricant, a moisturizer, a foam, a jelly, and a sponge.  
     
     
         66 . A use as in  claim 64  wherein: 
 said female subject is a mammal and said compound is administered into at least one of the vagina, uterus and fallopian tubes of said female.    
     
     
         67 . A use as in  claim 61  wherein said compound is selected from the group consisting of a nucleic acid which is antisense to at least a portion of a CatSper4 gene and an antibody to a CatSper4 protein.  
     
     
         68 . A use as in  claim 67  wherein said compound is an antibody fragment selected from the group consisting of an Fab fragment, an F(ab′) 2  fragment, an Fv fragment, and an scFv fragment.  
     
     
         69 . A use as in  claim 61  wherein said subject is a mammal.  
     
     
         70 . A use as in  claim 69  wherein said mammal is selected from the group consisting of humans, dogs, cats, cows, sheep, horses, mice, rats, raccoons, and gophers.  
     
     
         71 . A use as in  claim 69  wherein said subject is selected from the group consisting of a fish, an amphibian and an insect.  
     
     
         72 . A contraceptive preparation comprising a compound which decreases CatSper4 activity.  
     
     
         73 . A preparation as in  claim 72  wherein said compound is selected from the group consisting of a nucleic acid which is antisense to at least a portion of a CatSper4 gene and an antibody to a CatSper4 protein.  
     
     
         74 . A preparation as in  claim 72  wherein said preparation is in a formulation selected from the group consisting of an injection, a transdermal patch, a bioerodable implant, a lubricant, a moisturizer, a foam, a jelly, and a sponge.  
     
     
         75 - 111 . (canceled)