Role of chloride channels in nitric oxide-induced rat hippocampal neuronal apoptosis in vitro

BACKGROUND：Chloride channels participate in non-neuronal apoptosis.However,it remains unclear whether chloride channels are involved in ischemic neuronal apoptosis.OBJECTIVE：To explore the effects of 4-acetamido-4＇-isothiocyanatostilbene-2,2＇-disulfonic acid （SITS） and 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS）,two chloride channel blockers,on the hippocampal neuronal apoptosis induced by 3-morpholinosydnonimine （SIN-1） based on the nitric oxide toxicity theory of neuronal apoptosis following ischemic brain injury.DESIGN,TIME AND SETTING：Comparative observation and in vitro experiments were performed at the laboratory of Zhuhai Campus of Zunyi Medical College from January to May 2009.MATERIALS：SIN-1,SITS,and DIDS were purchased from Sigma,USA.METHODS：Hippocampal neurons from Sprague-Dawley rats,aged 1 day,were cultured In vitro for 12 days and randomly assigned to control,SIN-1,or chloride channel blocker groups.SIN-1 group neurons were induced by SIN-1 for 18 hours to establish a model of ischemic neuronal apoptosis.Neurons in chloride channel blocker groups were treated with SITS or DIDS plus SIN-1 for 18 hours.The controls were cultured in DMEM/Ham＇s F12 complete medium alone.MAIN OUTCOME MEASURES：The apoptotic neurons and nuclear appearance were detected by Hoechst 33258 fluorescence staining; neuronal viability was quantitatively determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide analysis.Caspase-3 activity was analyzed by Western blot.RESULTS：SIN-1 （1 mmol/L） dramatically induced apoptosis （50%-60%）.SITS and DIDS inhibited nitric oxide-induced neuronal injury in a dose-dependent manner,suppressed caspase-3 activation,reduced neuronal apoptosis,and improved neuronal survival.CONCLUSION：Chloride channel blockers can protect against neuronal injury induced by NO.Chloride channels might be involved in neuronal apoptosis following cerebral ischemia.

ClC-3 chloride channel in hippocampal neuronal apoptosis

Over-production of nitric oxide is pathogenic for neuronal apoptosis around the ischemic area fol- lowing ischemic brain injury. In this study, an apoptotic model in rat hippocampal neurons was es- tablished by 0.5 mmol/L 3-morpholinosyndnomine （SIN-l）, a nitric oxide donor. The models were then cultured with 0.1 mmol/L of 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS; the chloride channel blocker）for 18 hours. Neuronal survival was detected using the 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, and apoptosis was assayed by Hoechst 33342-labeled neuronal DNA fluorescence staining. Western blot analysis and immunochemilumi- nescence staining were applied to determine the changes of activated caspase-3 and CIC-3 channel proteins. Real-time PCR was used to detect the mRNA expression of CIC-3. The results showed that SIN-1 reduced the neuronal survival rate, induced neuronal apoptosis, and promoted CIC-3 chloride channel protein and mRNA expression in the apoptotic neurons. DIDS reversed the effect of SIN-I. Our findings indicate that the increased activities of the CIC-3 chloride channel may be involved in hippocampal neuronal apoptosis induced by nitric oxide.

Chloride channel blocker 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid inhibits nitric oxide-induced apoptosis in cultured rat hippocampal neurons

Apoptosis in cultured rat hippocampal neurons was induced using the nitric oxide donor 3-morpholinosydnonimine, and cells were treated with the chloride channel blocker, 4,4- diisothiocyanatostilbene-2,2＇-disulfonic acid. Results showed that the survival rate of neurons was significantly increased after treatment with 4,4-diisothiocyanatostilbene-2,2＇-disulfonic acid, and the rate of apoptosis decreased. In addition, the expression of the apoptosis-related proteins poly（adenosine diphosphate-ribose）polymerase-1 and apoptosis-inducing factor were significantly reduced. Our experimental findings indicate that the chloride channel blocker 4,4- diisothiocyanatostilbene-2,2＇-disulfonic acid can antagonize apoptotic cell death of hippocampal neurons by inhibiting the expression of the apoptosis-related proteins poly（adenosine diphosphate-ribose）polymerase-1 and apoptosis-inducing factor.

Effects of antigliomatin from the scorpion venom of Buthus martensii Karsch on chloride channels on C6 glioma cells

Using whole-cell patch-clamp recordings, the effects of antigliomatin were observed on chloride channels on C6 glioma cells cultured in vitro. Antigliomatin was extracted from the venom of the scorpion Buthus martensii Karsch. Chloride channels are closed under normal osmotic pressure. When osmotic pressure was reduced to 120, 110 and 100 mV, the cell volume enlarged, chloride channels opened, and the chloride channel current increased. Three minutes after antigliomatin treatment, the chloride channel current decreased in a dose-dependent manner. These results show that antigliomatin extracted from the venom of the scorpion Buthus martensii Karsch diminishes chloride channel currents on C6 glioma cells.

Synthesis and Characterization of A Small Molecule CFTR Chloride Channel Inhibitor

A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of 1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluorescent and electrophysiological methods. A large amount(100 g) of high-quality small molecule thiazolidinone CFTR chloride channel inhibitor,CFTR_ inh-172 ,can be produced with this simple three-step synthetic procedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl)-5-[4-carboxyphenyl- methylene]-4-thiazolidinone was confirmed by 1H NMR. The overall yield was 58% with a purity over 99% as analyzed by HPLC. The synthesized CFTR_ inh-172 specifically inhibited CFTR chloride channel function in a cell-based fluorescence assay( K _d≈1.5 μmol/L) and in a Ussing chamber-based short-circuit current assay( K _d≈0.2 μmol/L),indicating better quality than that of the commercial combinatorial compound. The synthesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The synthetic procedure developed here can be used to produce a large amount of the high-quality CFTR_ inh-172 suitable for antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be used to synthesize radiolabled CFTR_ inh-172 for in vivo pharmacokinetics studies.

Chloride channel involved in the regulation of curcumin-induced apoptosis of human breast cancer cells

Objective: To investigate the role of ClC-3 chloride channel in the proliferation of breast cancer cell line Mcf-7 treated with curcumin and its specific mechanism. Methods: MTT assay was used to detect the effect of chloride channel blocker(DIDS) and curcumin on Mcf-7 and human normal cell viability. Patch-clamp technique was used to determine the current density before and after drug treatment. Apoptosis assay by flow cytometry was performed for further examination of cell apoptosis. Results: Curcumin had toxicity on Mcf-7 and HUVEC cells and DIDS reduced the survival rate of Mcf-7 cells by inhibiting proliferation. Curcumin could activate the chloride ion current on MCF-7 cell membrane, which would be inhibited by DIDS.Finally, curcumin in low concentration combined with DIDS could significantly promote the MCF-7 cells apoptosis. Conclusions: Our results suggest that ClC-3 protein is involved in the regulation of curcumin induced proliferation inhibiting in breast cancer cells through inducing cell apoptosis. ClC-3 may be a potential target of tumor therapy.

Identification of Herbal Compound Imperatorin with Adverse Effects on ANO1 and CFTR Chloride Channels

Calcium-activated chloride channels（CaCCs） are the crucial regulators of transepithelial fluid secretion, smooth muscle contraction and sensory transduction. Recently, compelling evidence has indicated that TMEM16A（ANO1 or anoctamin-1） is a bona fide calcium-acvtivated chloride channel. A few small molecule CaCCs regulators are available for functional and therapeutic studies. We screened 126 natural compounds from Chinese herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells to coexpress ANO1 and an iodide-sensitive fluorescent indicator（EYFP-H148Q/I152L）. Imperatorin, a coumarin compound, was identified to inhibit ANO1-mediated chloride transport activated by multiple calcium-elevating agonists. The inhibitory effect is dose-dependent with IC50～14.63 μmol/L. Interestingly, imperatorin activated CFTR chloride channel with EC50～35.52 μmol/L. The adverse effects of imperatorin on CaCC and CFTR chloride channels will make it useful in pharmacological dissection of chloride transport in airway and intestinal epithelium. Further studies are required to evaluate the therapeutic effects of imperatorin on hypertension, asthma and certain tumors.

Activation Effect of Cathartic Natural Compound Rhein to CFTR Chloride Channel

The cystic fibrosis transmembrane conductance regulator （CFFR） is a cAMP-activated chloride channel expressed in intestinal exoerine glands, which plays a key role in intestinal fluid secretion. A natural anthraquinone ac tivator of CFTR Cl^- channel, rhein, was identified by screening 217 single compounds from Chinese herbs via a cellbased halide-sensitive fluorescent assay. Rhein activates CFTR Cl^- transportation in a dose-dependent manner in the presence of cAMP with a physiological concentration. This study provides a novel molecular pharmacological mechanism for the laxative drugs in Treditional Chinese Medicine such as aloe, cascara and senna.

Impact of Chloride Channel on Spiking Patterns of Morris-Lecar Model

In this paper,we study the complicated dynamics of general Morris-Lecar model with the impact of Cl- fluctuations on firing patterns of this neuron model. After adding Cl- channel in the original Morris-Lecar model, the dynamics of the original model such as its bifurcations of equilibrium points would be changed and they occurred at different values compared to the primary model. We discover these qualitative changes in the point of dynamical systems and neuroscience. We will conduct the co-dimension two bifurcations analysis with respect to different control parameters to explore the complicated behaviors for this new neuron model.

Chloride channels in vascular function and disease

Vascular smooth muscle cells（VSMCs） are the major component of vascular wall which are often stretched and compressed by pounding intravascular pressure.These mechanical signals are usually transformed to electrical signals by the opening or closing of ion channels in VSNCs and endothelial cells.Intravascular pressure causes a graded membrane potential depolarization of the VSMCs and leads to vasoconstriction（i.e.,myogenic response）,independent of the vascular endothelium. Although the important role of cation channels including L-type Ca2+ channels,K+ channels,and TRP channels in the regulation of vascular tone has been well established the functional roles played by Cl- channels in the regulation of the membrane potential and vascular tone remain essentially obscure. Recent emerging evidence implicates very important roles of Cl- channels in vascular function ranging from the control of membrane potential equilibrium, vascular contraction and relaxation to the regulation of intracellular pH,cell volume homeostasis,cell proliferation,migration,and apoptosis.

Increased expression of human calcium-activated chloride channel 1 is correlated with mucus overproduction in the airways of Chinese patients with chronic obstructive pulmonary disease

Background Chronic obstructive pulmonary disease （COPD） is usually complicated with mucus overproduction in airway. Recently the increased expression of the human calcium-activated chloride channel 1 （CaCC1） was found to play an important role in mucus overproduction in the asthmatic airways. To investigate the relationship of CaCC1 and mucus overproduction in the airway of Chinese patients with COPD, the expressions of CaCC1, MUC5AC and mucus in bronchial tissues were examined. Methods Bronchial tissues were obtained from fiberoptic bronchoscopy and bronchial biopsy in West China Hospital from April to July in 2004. Twenty-five patients were diagnosed as the patients with COPD overproduction, and other 20 were the control subjects. The expressions of CaCC1, MUC5AC and mucin in bronchial tissues were detected by reverse transcriptase-polymerase chain reaction （RT-PCR）, in situ hybridization with digoxigenin （DIG）-Iabeled RNA probe, immunohistochemical and alcian blue-periodic acid Schiff （AB-PAS） staining, respectively. Results Compared with the control group, the stronger expressions of CaCC1 were further detected throughout the bronchial tissues from patients with COPD （P〈0.01）. Furthermore, the stronger expressions of the CaCC1 mRNA were related to the severity of airflow obstruction. Samples from COPD showed a stronger staining for MUC5AC than those in control subjects （P〈0.01） and AB-PAS staining revealed more mucins in COPD patients＇ submucosal gland comparing with that in control subjects （P〈0.01）. Expression levels of the CaCC1 mRNA were respectively negatively correlated with the patients＇ forced expiratory volume in one second （FEV~） / forced vital capacity （FVC） data, FEV1% predicted data, V50% predicted data, V25% predicted data （r=-0.43, r=-0.43, r=-0.35, r=-0.36, P〈0.01, P〈0.01, P〈0.05, P〈0.05）. While the expression levels of the CaCC1 mRNA were well correlated with the expression levels of the MUC5AC mRNA of airway epithelium and the PAS-AB stained area of submucosal glands （r=0.39, r=0.46, P〈0.05, P〈0.01）. Expression levels of the MUC5AC mRNA were negatively correlated with the patients＇ FEV1/FVC data （P=0.01）, FEV1% pred data （P=-0.01）, V50% predicted data, V25% predicted data（r=-0.53, r=-0.53, r=-0.48, r=-0.43, P〈0.01, P〈0.01, P〈0.01, P〈0.01）. While the expression levels of the MUC5AC mRNA were well correlated with the positively PAS-AB stained area of submucosal gland （P〈0.05）, and the correlation coefficients were 0.43. Conclusion These results suggest that the stronger gene expression of CaCC1 exists, complicated with mucus overproduction in the airwav of Chinese patients with COPD.

Chloride channels are involved in sperm motility and are downregulated in spermatozoa from patients with asthenozoospermia

Human spermatozoa encounter an osmotic decrease from 330 to 290 mOsm I-z when passing through the female reproductive tract. We aimed to evaluate the role of chloride channels in volume regulation and sperm motility from patients with asthenozoospermia. Spermatozoa were purified using Percoll density gradients. Sperm volume was measured as the forward scatter signal using flow cytometry. Sperm motility was analyzed using computer-aided sperm analysis （CASA）. When transferred from an isotonic solution （330 mOsm I-z） to a hypotonic solution （290 mOsm I-Z）, cell volume was not changed in spermatozoa from normozoospermic men; but increased in those from asthenozoospermic samples. The addition of the chloride channel blockers, 4,4＇-diisothiocyanatostilbene-2,2＇- isulfonic acid （DIDS） or 5-nitro-2-（3-phenylpropylamino） benzoic acid （NPPB） to the hypotonic solution caused the normal spermatozoa to swell but did not increase the volume of those from the asthenozoospermic semen. DIDS and NPPB decreased sperm motility in both sets of semen samples. The inhibitory effect of NPPB on normal sperm motility was much stronger than on spermatozoa from the asthenozoospermic samples. Both sperm types expressed CIC-3 chloride channels, but the expression levels in the asthenozoospermic samples were much lower, especially in the neck and mid-piece areas. Spermatozoa from men with asthenozoospermia demonstrated lower volume regulating capacity, mobility, and CIC-3 expression levels （especially in the neck） than did normal spermatozoa. Thus, chloride channels play important roles in the regulation of sperm volume and motility and are downregulated in cases of asthenozoospermia.

Effects of glycoprotein Ⅱb/Ⅲa antagonists and chloride channel blockers on platelet cytoplasmic free calcium

Platelet activation plays an important role in thrombosis. Platelet glycoprotein Ⅱ b/Ⅲ a （ GPⅡ b/Ⅲ a ） is the receptor of fibrinogen. Platelet cross-linking with fibrinogen through GP Ⅱ b/Ⅲ a is the process of thrombosis. Ca^2＋ is an important intracellular second messenger in platelet activation. It has been reported that GP Ⅱ b/Ⅲ a receptors were involved in the calcium influx of activated platelet, and GP Ⅱ b/Ⅲ a receptor had characteristics of calcium channel or an adjacent calcium channel.

Murine calcium-activated chloride channel family member 3 induces asthmatic airway inflammation independently of allergen exposure

Background Expression of murine calcium-activated chloride channel family member 3 （mCLCA3） has been reported to be increased in the airway epithelium of asthmatic mice challenged with ovalbumin （OVA）. However, its role in asthmatic airway inflammation under no OVA exposure has not yet been clarified. Methods mCLCA3 plasmids were transfected into the airways of normal BALB/c mice. mCLCA3 expression and airway inflammation in mouse lung tissue were evaluated. Cell differentials and cytokines in bronchoalveolar lavage fluid （BALF） were analyzed. The expression of mCLCA3 protein and mucus protein mucin-5 subtype AC （MUC5AC） were analyzed by Western blotting. The mRNA levels of mCLCA3, MUC5AC and interleukin-13 （IL-13） were determined quantitatively. Results mCLCA3 expression was not detected in the control group while strong immunoreactivity was detected in the OVA and mCLCA3 plasmid groups, and was strictly localized to the airway epithelium. The numbers of inflammatory cells in lung tissue and BALF were increased in both mCLCA3 plasmid and OVA groups. The protein and mRNA levels of mCLCA3 and MUC5AC in the lung tissue were significantly increased in the mCLCA3 plasmid and OVA groups compared to the control group. The level of IL-13, but not IL-4, IL-5, IFN-y, CCL2, CCL5 or CCL11, was significantly increased compared with control group in BALF in the mCLCA3 plasmid and OVA groups. The level of IL-13 in the BALF in the mCLCA3 plasmid group was much higher than that in the OVA group （P 〈0.05）. The level of mCLCA3 mRNA in lung tissue was positively correlated with the levels of MUC5AC mRNA in lung tissue, IL-13 mRNA in lung tissue, the number of eosinophils in BALF, and the content of IL-13 protein in BALE The level of IL-13 mRNA in lung tissue was positively correlated with the number of eosinophils in BALF and the level of MUC5AC mRNA in lung tissue. Conclusion These findings suggest that increased expression of a single-gene, mCLCA3, could simulate an asthma attack, and its mechanism may involve mCLCA3 overexpression up-regulating IL-13 expression.

Effects of calcium channel on 3-morpholinosydnonimine-induced rat hippocampal neuronal apoptosis

Previous studies have demonstrated that increased chloride channel activity plays a role in nitric oxide-induced neuronal apoptosis in the rat hippocampus. The present study investigated the effects of the broad-spectrum calcium channel blocker CdCI2 on survival rate, percentage of apoptosis, and morphological changes in hippocampal neurons cultured in vitro, as well as the effects of calcium channels on neuronal apoptosis. The chloride channel blockers 4-acetamido-4＇-isothiocyanatostilbene-2, 2＇-disulfonic acid （SITS） or 4, 4＇-diisethiocyanostilbene-2, 2＇-disulfonic acid （DIDS） increased the survival rate of 3-morpholinosydnonimine （SIN-1）-treated neurons and suppressed SIN-l-induced neuronal apoptosis. The calcium channel blocker CdCI2 did not increase the survival rate of neurons and did not affect SIN-l-induced apoptosis or SITS- or DIDS-suppressed neuronal apoptosis. Results demonstrated that calcium channels did not significantly affect neuronal apoptosis.

Identification and Functional Analysis on Abiotic Stress Response of Soybean Cl^- Channel Gene GmCLCnt

The Cl^- homeostasis was known as the major mechanism of soybean to achieve NaCl tolerance, but studies on the role of chloride channel under abiotic stress were relatively few. We cloned a putative CLC-type chloride channel gene GmCLCnt from soybean via RACE and it was predicted to encode a protein of 783 amino acids with 9 possible transmembrane domains and 2 tandem CBS domains. Real-time RT-PCR analysis showed that the GmCLCnt gene was expressed in all tissues of soybean but enriched in leaves and its expression was induced by NaCl, polyethylene glycol （PEG）, coldness and ABA treatments. The Arabidopsis seedlings overexpressing GmCLCnt were more tolerant to higher concentration of NaCl than those of wild type. The results suggested that the GmCLCnt may be a CLC-type chloride channel and play an important role in salt tolerance.

Functional Characteristics and Molecular Identification of Swelling-activated Chloride Conductance in Adult Rabbit Heart Ventricles

Outwardly rectifying swelling-activated chloride conductance （ICl, Swell） in rabbit heart plays a critical role in cardioprotection following ischemic preconditioning （IP）. But the functional characterization and molecular basis of this chloride conductance in rabbit heart ventricular myocytes is not clear. Candidate chloride channel clones （e.g. ClC-2, ClC-3, ClC-4 and ClC-5） were determined using RT-PCR and Western blot analysis.Whole cell ICl,Swell was recorded from isolated rabbit ventricular myocytes using patch clamp techniques during hypo-osmotic stress. The inhibitory effects of 4,4＇ isothiocyanato-2,2-disulfonic acid （DIDS）, 5-nitro-2（3-phenylroylamino） benzoic acid （NPPB） and indanyloxyacetic acid 94 （LAA-94） on ICl,Swell were examined. The expected size of PCR products for ClC-2, ClC-3 and ClC-4 but not for ClC-5 was obtained. ClC-2 and ClC-3 expression was confirmed by automated fluorescent DNA sequencing. RT-PCR and Western blot showed that ClC-4 was expressed in abundance and ClC-2 was expressed at somewhat lower levels. The biological and pharmacological properties of ICl,Swell, including outward rectification, activation due to cell volume change, sensitivity to DIDS, LAA-94 and NPPB were identical to those known properties of ICl,Swell in exogenously expressed systems and other mammals hearts. It was concluded that ClC-3 or ClC-4 might be responsible for the outwardly rectifying part of ICl,Wwell and may be the molecular targets of cardioprotection associated with ischemic preconditioning or hypo-osmotic shock.

Altered expression of renal bumetanide-sensitive sodium-pota-ssium-2 chloride cotransporter and Cl- channel -K_2 gene in angiotensin Ⅱ-infused hypertensive rats

Background Little information is available regarding the effect of angiotensin Ⅱ （Ang Ⅱ ） on the bumetanide-sensitive sodium-potassium-2 chloride cotransporter （NKCC2 ）, the thiazide-sensitive sodiumchloride cotransporter （ NCC）, and the Cl^- channel （ CLC ）-K2 at both mRNA and protein expression level in Ang Ⅱ-induced hypertensive rats. This study was conducted to investigate the influence of Ang Ⅱ with chronic subpressor infusion on nephron-specific gene expression of NKCC2, NCC and CLC-K2.Methods Sprague Dawleys rats were treated subcutaneously with either Ang Ⅱ （100 ng·kg^-1·min^-1） or vehicle for 14 days. Expression of NKCC2, NCC and CLC-K2 mRNA in kidneys was determined by real time polymerase chain reaction （PCR）. Western blotting analysis was used to measure NKCC2 and NCC protein expression.Results Ang Ⅱ significantly increased blood pressure and up-regulated NKCC2 mRNA and protein expression in the kidney. Expression of CLC-K2 mRNA in the kidney increased 1.6 fold （P 〈 0.05 ） . There were no changes in NCC mRNA or protein expression in AngⅡ-treated rats versus control.Conclusions Chronic subpressor Ang Ⅱ infusion can significantly alter NKCC2 and CLC-K2 mRNA expression in the kidney, and protein abundance of NKCC2 in kidney is positively regulated by Ang Ⅱ. These effects may contribute to enhanced renal Na^＋ and Cl^- reabsorption in response to Ang Ⅱ.

Effect of Residue Mutation on the Electrostatic Potential in EcClC

The effect of mutation of strongly conserved porelining residues in the chloride channel EcClC on the electrostatic potential and binding free energy of the chloride ion was studied using explicit protein-membrane structures. Electrostatic potential distribution and binding free energy of the chloride ion at different binding sites in the wild-type and mutated EcClC were calculated with APBS. The potential data reveal that the electrostatic potential around the selectivity filter, especially around the site Sext and Scen becomes more negative as the residue R147 was mutated to C147. The electrostatic binding free energy shows that the binding free energy of the chloride ion at all binding sites becomes more positive as R147 was mutated. It follows that mutation of R147 decreases ion stabilization at binding sites and affects channel＇s gating.

Autosomal dominant osteopetrosis typeⅡresulting from a de novo mutation in the CLCN7 gene:A case report

BACKGROUND Osteopetrosis is a family of extremely rare diseases caused by failure of osteoclasts and impaired bone resorption. Among them, autosomal dominant osteopetrosis type Ⅱ(ADO Ⅱ), related to the chloride channel 7(CLCN7) gene, is the most frequent form of osteopetrosis. In this study, we report a de novo mutation of CLCN7 in a patient without the family history of ADO Ⅱ.CASE SUMMARY A 5-year-old Chinese boy with ADO Ⅱ was found to have a de novo mutation in the CLCN7 gene [c.746 C>T(p.P249 L)]. Typical clinical manifestations, including thickening of the cortex of spinal bones and long bones, non-traumatic fracture of the femoral neck, and femoral head necrosis, were found in this patient. The patient is the first reported case of ADO Ⅱ with the missense mutation c.746 C>T(p.P249 L) of the CLCN7 gene reported in China. We also review the available literature on ADO Ⅱ-related CLCN7 mutations, including baseline patient clinical features, special clinical significance, and common mutations.CONCLUSION Our report will enrich the understanding of mutations in ADO Ⅱ patients. The possibility of a de novo mutation should be considered in individuals who have no family history of osteopetrosis.