Dejian Ren

CatSper1 required for evoked Ca2+ entry and control of flagellar function in sperm

CatSper family proteins are putative ion channels expressed exclusively in membranes of the sperm flagellum and required for male fertility. Here, we show that mouse CatSper1 is essential for depolarization-evoked Ca2+ entry and for hyperactivated movement, a key flagellar function. CatSper1 is not needed for other developmental landmarks, including regional distributions of CaV1.2, CaV2.2, and CaV2.3 ion channel proteins, the cAMP-mediated activation of motility by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}, and the protein phosphorylation cascade of sperm capacitation. We propose that CatSper1 functions as a voltage-gated Ca2+ channel that controls Ca2+ entry to mediate the hyperactivated motility needed late in the preparation of sperm for fertilization.

A voltage-gated ion channel expressed specifically in spermatozoa

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 Cav 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 α-like subunits form either a homo- or heterotetramer. It is possible, therefore, that two independent α subunits, different from other known voltage-gated channels, regulate sperm motility.

TPC Proteins Are Phosphoinositide- Activated Sodium-Selective Ion Channels in Endosomes and Lysosomes

Mammalian two-pore channel proteins (TPC1, TPC2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double-knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P(2) and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na(+), not K(+), as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes and may explain the specificity of PI(3,5)P(2) in regulating the fusogenic potential of intracellular organelles.

The Control of Male Fertility by Spermatozoan Ion Channels

Ion channels control the sperm ability to fertilize the egg by regulating sperm maturation in the female reproductive tract and by triggering key sperm physiological responses required for successful fertilization such as hyperactivated motility, chemotaxis, and the acrosome reaction. CatSper, a pH-regulated, calcium-selective ion channel, and KSper (Slo3) are core regulators of sperm tail calcium entry and sperm hyperactivated motility. Many other channels had been proposed as regulating sperm activity without direct measurements. With the development of the sperm patch-clamp technique, CatSper and KSper have been confirmed as the primary spermatozoan ion channels. In addition, the voltage-gated proton channel Hv1 has been identified in human sperm tail, and the P2X2 ion channel has been identified in the midpiece of mouse sperm. Mutations and deletions in sperm-specific ion channels affect male fertility in both mice and humans without affecting other physiological functions. The uniqueness of sperm ion channels makes them ideal pharmaceutical targets for contraception. In this review we discuss how ion channels regulate sperm physiology.

mTOR Regulates Lysosomal ATP-Sensitive Two-Pore Na+ Channels to Adapt to Metabolic State

Survival in the wild requires organismal adaptations to the availability of nutrients. Endosomes and lysosomes are key intracellular organelles that couple nutrition and metabolic status to cellular responses, but how they detect cytosolic ATP levels is not well understood. Here, we identify an endolysosomal ATP-sensitive Na(+) channel (lysoNa(ATP)). The channel is a complex formed by two-pore channels (TPC1 and TPC2), ion channels previously thought to be gated by nicotinic acid adenine dinucleotide phosphate (NAADP), and the mammalian target of rapamycin (mTOR). The channel complex detects nutrient status, becomes constitutively open upon nutrient removal and mTOR translocation off the lysosomal membrane, and controls the lysosomes membrane potential, pH stability, and amino acid homeostasis. Mutant mice lacking lysoNa(ATP) have much reduced exercise endurance after fasting. Thus, TPCs make up an ion channel family that couples the cells metabolic state to endolysosomal function and are crucial for physical endurance during food restriction.

A Superfamily of Voltage-gated Sodium Channels in Bacteria

NaChBac, a six-α-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na+-selective channel (Ren, D., Navarro, B., Xu, H., Yue, L., Shi, Q., and Clapham, D. E. (2001) Science 294, 2372-2375). As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na+ channels (NaVPZ from Paracoccus zeaxanthinifaciens and NaVSP from Silicibacter pomeroyi). NaVPZ and NaVSP share ∼40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both NaVPZ and NaVSP were Na+-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-α-helical transmembrane-spanning subunits constitute a widely distributed superfamily (NaVBac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of NaVBac structure and function of similarity and dissimilarity among NaVBac proteins. Thus, the NaVBac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.

The Cation Selectivity Filter of the Bacterial Sodium Channel, NaChBac

The Bacillus halodurans voltage-gated sodium-selective channel (NaChBac) (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001b. Science. 294:2372–2375), is an ideal candidate for high resolution structural studies because it can be expressed in mammalian cells and its functional properties studied in detail. It has the added advantage of being a single six transmembrane (6TM) orthologue of a single repeat of mammalian voltage-gated Ca2+ (CaV) and Na+ (NaV) channels. Here we report that six amino acids in the pore domain (LESWAS) participate in the selectivity filter. Replacing the amino acid residues adjacent to glutamatic acid (E) by a negatively charged aspartate (D; LEDWAS) converted the Na+-selective NaChBac to a Ca2+- and Na+-permeant channel. When additional aspartates were incorporated (LDDWAD), the mutant channel resulted in a highly expressing voltage-gated Ca2+-selective conductance.

CATSPER Channel-Mediated Ca2+ Entry into Mouse Sperm Triggers a Tail-to-Head Propagation

Abstract Many Ca2+ channel proteins have been detected in mammalian sperm, but only the four CATSPER channels have been clearly shown to be required for male fertility. Ca2+ entry through the principal piece-localized CATSPER channels has been implicated in the activation of hyperactivated motility. In the present study, we show that the Ca2+ entry also triggers a tail-to-head Ca2+ propagation in the mouse sperm. When activated with 8-Br-cAMP, 8-Br-cGMP, or alkaline depolarization, a CATSPER-dependent increase in intracellular Ca2+ concentration starts in the principal piece, propagates through the midpiece, and reaches the head in a few seconds. The Ca2+ propagation through the midpiece leads to a Ca2+-dependent increase in NADH fluorescence. In addition, CatSper1-mutant sperm have lower intracellular ATP levels than wild-type sperm. Thus, a Ca2+ influx in the principal piece through CATSPER channels can not only initiate hyperactivated motility, but can also trigger a tail-to-head Ca2+ propagation that leads to an increase in [NADH] and may regulate ATP homeostasis.

CATSPERβ : A NOVEL TRANSMEMBRANE PROTEIN IN THE CATSPER CHANNEL COMPLEX *

Four CatSper ion channel subunit genes (CatSpers 1-4) are required for sperm cell hyperactivation and male fertility. The four proteins assemble (presumably as a tetramer) to form a sperm-specific, alkalinization-activated Ca2+-selective channel. We set out to identify proteins associating with CatSper that might help explain its unique role in spermatozoa. Using a transgenic approach, a CatSper1 complex was purified from mouse testis that contained heat shock protein 70-2, a testis-specific chaperone, and CatSperβ, a novel protein with two putative transmembrane-spanning domains. Like the CatSper ion channel subunits, CatSperβ was restricted to testis and localized to the principal piece of the sperm tail. CatSperβ protein is absent in CatSper1-/- sperm, suggesting that it is required for trafficking or formation of a stable channel complex. CatSperβ is the first identified auxiliary protein to the CatSper channel.

Extracellular Calcium Controls Background Current and Neuronal Excitability via an UNC79-UNC80-NALCN Cation Channel Complex

In contrast to its extensively studied intracellular roles, the molecular mechanisms by which extracellular Ca(2+) regulates the basal excitability of neurons are unclear. One mechanism is believed to be through Ca(2+)s interaction with the negative charges on the cell membrane (the charge screening effect). Here we show that, in cultured hippocampal neurons, lowering [Ca(2+)](e) activates a NALCN channel-dependent Na(+)-leak current (I(L-Na)). The coupling between [Ca(2+)](e) and NALCN requires a Ca(2+)-sensing G protein-coupled receptor, an activation of G-proteins, an UNC80 protein that bridges NALCN to a large novel protein UNC79 in the same complex, and the last amino acid of NALCNs intracellular tail. In neurons from nalcn and unc79 knockout mice, I(L-Na) is insensitive to changes in [Ca(2+)](e), and reducing [Ca(2+)](e) fails to elicit the excitatory effects seen in the wild-type. Therefore, extracellular Ca(2+) influences neuronal excitability through the UNC79-UNC80-NALCN complex in a G protein-dependent fashion.