A voltage-gated ion channel expressed specifically in spermatozoa

Abstract

Calcium ions play a primary role in the regulation of sperm cell behavior. We report finding a voltage-gated ion channel (CatSper2) that is expressed in male germ cells but not in other cells. The putative channel contains 6 transmembrane segments, making it more similar to the voltage-gated potassium channels, but the ion selectivity pore domain sequence resembles that of a Ca(v) channel. The mRNA is expressed during the meiotic or postmeiotic stages of spermatogenesis, and the protein is localized to the sperm flagellum, suggesting a role in the regulation of sperm motility. The mRNA for the channel is present in mouse, rat, and human sperm cells, and the gene is found on chromosome 2 E5-F1 in the mouse and 15q13 in the human. Recently, another voltage-gated channel (CatSper) that has features similar to the one reported here was discovered. It also is expressed within the flagellum and is required for normal fertility of mice. However, expression of CatSper2 alone or coexpression with CatSper in cultured cells, or attempts to coimmunoprecipitate the two proteins from germ cells failed to demonstrate that these two unique but similar alpha-like subunits form either a homo- or heterotetramer. It is possible, therefore, that two independent alpha subunits, different from other known voltage-gated channels, regulate sperm motility.

Figure 1

(A) Amino acid sequence of CatSper2. The transmembrane segments (S1–S6) and ion selectivity P region are underlined. The basic residues of S4 are highlighted in bold. (B) Pairwise alignment of CatSper2 and CatSper transmembrane regions. The P regions are boxed with residues highly conserved among the Ca_V families highlighted in blue. The red Asp residue is critical for calcium ion selectivity.

Figure 2

(A) Northern blot of mouse tissue total RNA (15 μg). The blot was probed with a mouse CatSper2 cDNA probe (nucleotides 113–531). Br, brain; He, heart; In, intestine; K_i, kidney; Li, liver; Ov, ovary; Sk, skeletal muscle; Sp, spleen; St, stomach; Te, testis. (B) Northern blot of testis poly(A)⁺ RNA, 2 μg each, from mouse (M), rat (R), and human (H) probed with a mouse CatSper2 cDNA probe (nucleotides 919-1299).

Figure 3

In situ hybridization of testicular tissue. Paraformaldehyde-fixed, paraffin-embedded mouse testis sections were probed with ³⁵S-labeled CatSper2 antisense (1 and 2) or sense (3 and 4) probes (nucleotides 113–531). Bright-field (1 and 3) and dark-field (2 and 4) microscopy. (×200.)

Figure 4

Immunoblot and immunolocalization of CatSper2 protein. (A) SDS polyacrylamide gel electrophoresis/immunoblots of mouse testis (T) and mouse sperm cell membrane (S) probed with NII antibody (1:1000) or C antibody (1:5000). (B) Methanol-fixed epididymal spermatozoa were labeled with C-peptide antibody preincubated without (Upper) or with (Lower) competing peptide. Bound antibody was detected with AlexaFluor-488-conjugated secondary antibody. Phase contrast (Left) and epifluorescence microscopy (Right). (×600.)

Figure 5

Immunoprecipitation of CatSper2 from mouse testis extracts. CatSper2 C-terminal antibody immunoprecipitate fractions obtained from a mouse testis extract (1% Triton X-100) were separated on SDS/PAGE and immunoblotted with both CatSper2 (1:5,000) and CatSper (1:2,000) antibodies. Lanes: T, testis homogenate; I, insoluble fraction; S, soluble fraction; P, preimmune IgG Protein A agarose; C, C peptide IgG protein A agarose without (−) and with (+) competing peptide.