Nerve conduction features may serve as a diagnostic clue for neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease is a neurodegenerative disorder with a wide phenotypic spectrum, including peripheral neuropathy. This study aims to characterize the nerve conduction features and proposes an electrophysiological criterion to assist the diagnosis of neuronal intranuclear inclusion disease. In this study, nerve conduction studies were performed in 50 genetically confirmed neuronal intranuclear inclusion disease patients, 200 age- and sex-matched healthy controls and 40 patients with genetically unsolved leukoencephalopathy. Abnormal electrophysiological parameters were defined as mean values plus or minus two standardized deviations of the healthy controls or failure to evoke a response on the examined nerves. Compared to controls, neuronal intranuclear inclusion disease patients had significantly slower motor and sensory nerve conduction velocities, as well as lower amplitudes of compound motor action potentials and sensory nerve action potentials in all tested nerves (P < 0.05). Forty-eight of the 50 neuronal intranuclear inclusion disease patients (96%) had at least one abnormal electrophysiological parameter, with slowing of motor nerve conduction velocities being the most prevalent characteristic. The motor nerve conduction velocities of median, ulnar, peroneal and tibial nerves were 44.2 ± 5.5, 45.3 ± 6.1, 37.3 ± 5.3 and 35.6 ± 5.1 m/s, respectively, which were 12.4-13.6 m/s slower than those of the controls. The electrophysiological features were similar between neuronal intranuclear inclusion disease patients manifesting with CNS symptoms and those with PNS-predominant presentations. Thirteen of the 14 patients (93%) who underwent nerve conduction study within the first year of symptom onset exhibited abnormal findings, indicating that clinical or subclinical peripheral neuropathy is an early disease marker of neuronal intranuclear inclusion disease. We then assessed the feasibility of using motor nerve conduction velocity as a diagnostic tool of neuronal intranuclear inclusion disease and evaluated the diagnostic performance of various combinations of nerve conduction parameters using receiver operating characteristic curve analysis. The criterion of having at least two nerves with motor nerve conduction velocity ranging from 35 to 50 m/s in median/ulnar nerves and 30-40 m/s in tibial/peroneal nerves demonstrated high sensitivity (90%) and specificity (99%), with an area under the curve of 0.95, to distinguish neuronal intranuclear inclusion disease patients from healthy controls. The criterion's diagnostic performance was validated on an independent cohort of 56 literature reported neuronal intranuclear inclusion disease cases (area under the curve = 0.93, sensitivity = 87.5%, specificity = 99.0%), and in distinguishing neuronal intranuclear inclusion disease from genetically unresolved leukoencephalopathy cases (sensitivity = 90.0%, specificity = 80.0%). In conclusion, mildly to moderately decreased motor nerve conduction velocity in multiple nerves is a significant electrophysiological hallmark assisting the diagnosis of neuronal intranuclear inclusion disease, regardless of CNS- or PNS-predominant manifestations.

Reduced-penetrance Huntington's disease-causing alleles with 39 CAG trinucleotide repeats could be a genetic factor of amyotrophic lateral sclerosis.

BACKGROUND: Expanded HTT alleles with 40 or more CAG repeats were recently found to be a rare cause of frontotemporal dementia and amyotrophic lateral sclerosis (ALS) spectrum diseases. The aim of this study was to investigate the role of HTT repeat expansions in a Taiwanese cohort with ALS. METHODS: We analyzed the numbers of CAG repeats in exon 1 of HTT in a cohort of 410 Taiwanese patients with ALS and 1514 control individuals by utilizing polymerase chain reaction and amplicon fragment length analysis. RESULTS: Only one of the 410 ALS patients carried a reduced-penetrance HD-causing allele with 39 CAG repeats, and none had an expanded HTT CAG repeats ≥40. The patient presented with rapidly progressive bulbar-onset ALS with disease onset at the age of 64 years. He had neither chorea nor cognitive impairment. He had a family history of chorea, but no other family member manifested with ALS. None of the 1514 control individuals carried an HTT expanded allele with CAG repeats larger than 37 repeats. CONCLUSION: The HTT allele with 39 CAG repeats could be a genetic factor linked to ALS susceptibility.

Assessing the NOTCH2NLC GGC repeat expansion in Taiwanese patients with hereditary spastic paraplegia.

We investigated 98 Taiwanese patients with molecularly unassigned hereditary spastic paraplegia (HSP) and found none of them had the NOTCH2NLC GGC repeat expansion, which is the cause of neuronal intranuclear inclusion disease (NIID). Our findings suggest that the NOTCH2NLC GGC repeat expansion may not contribute to HSP.

GGC Repeat Expansion of NOTCH2NLC in Taiwanese Patients With Inherited Neuropathies.

BACKGROUND AND OBJECTIVES: The GGC repeat expansion in the 5' untranslated region of NOTCH2NLC was recently identified as the cause of neuronal intranuclear inclusion disease (NIID), which may manifest with peripheral neuropathy. The aim of this study is to investigate its contribution to inherited neuropathy. METHODS: This cohort study screened patients with molecularly undiagnosed Charcot-Marie-Tooth disease (CMT) and healthy controls for the GGC repeat expansion in NOTCH2NLC using repeat-primed PCR and fragment analysis. The clinical and electrophysiologic features of the patients harboring the GGC repeat expansion were scrutinized. Skin biopsy with immunohistochemistry staining and electric microscopic imaging were performed. RESULTS: One hundred twenty-seven unrelated patients with CMT, including 66 cases with axonal CMT (CMT2), and 200 healthy controls were included. Among them, 7 patients with CMT carried a variant NOTCH2NLC allele with GGC repeat expansion, but it was absent in controls. The sizes of the expanded GGC repeats ranged from 80 to 104 repeats. All 7 patients developed sensory predominant neuropathy with an average age at disease onset of 37.1 years (range 21-55 years). Electrophysiologic studies revealed mild axonal sensorimotor polyneuropathy. Leukoencephalopathy was absent in the 5 patients who received a brain MRI. Skin biopsy from 2 patients showed eosinophilic, ubiquitin- and p62-positive intranuclear inclusions in the sweat gland cells and dermal fibroblasts. Two of the 7 patients had a family history of NIID. DISCUSSION: The NOTCH2NLC GGC repeat expansions are an underdiagnosed and important cause of inherited neuropathy. The expansion accounts for 10.6% (7 of 66) of molecularly unassigned CMT2 cases in the Taiwanese CMT cohort. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that in Taiwanese patients with genetically undiagnosed CMT, 10.6% of the CMT2 cases have the GGC repeat expansion in NOTCH2NLC.

Genetic and Functional Analysis of Glycosyltransferase 8 Domain-Containing Protein 1 in Taiwanese Patients With Amyotrophic Lateral Sclerosis.

BACKGROUND AND OBJECTIVES: To investigate the frequency, spectrum, and molecular functional effect of glycosyltransferase 8 domain-containing protein 1 (GLT8D1) variations in Taiwanese patients with amyotrophic lateral sclerosis (ALS). METHODS: We performed genetic analyses of GLT8D1 in 410 unrelated patients with ALS by Sanger sequencing. The 410 patients were selected from a cohort of 477 unrelated patients with ALS after excluding variations in common ALS disease genes. Functional effects of the GLT8D1 variation were investigated by in vitro functional analysis. RESULTS: We identified a novel heterozygous missense variation in GLT8D1, p.I290M (c.870C>G), in 1 single patient with familial ALS. The patient with the p.I290M variation had a spinal-onset ALS with disease onset at age 60 years and a survival of 6 years. Functional studies demonstrated that the variant I290M GLT8D1 protein was mislocalized to the endoplasmic reticulum (ER), provoked ER stress and unfolded protein response, compromised the glycosyltransferase activity, and led to an increased cytotoxicity. DISCUSSION: GLT8D1 variations account for 0.2% (1/477) of the patients with ALS in Taiwan. These findings expand the spectrum of GLT8D1 variation and support the pathogenic role of GLT8D1 variations in ALS.

Analysis of NOTCH2NLC GGC repeat expansion in Taiwanese patients with amyotrophic lateral sclerosis.

The GGC repeat expansion in the 5'-untranslated region of NOTCH2NLC was recently identified as the cause of neuronal intranuclear inclusion disease (NIID). To determine if the NIID repeat expansion contributes to amyotrophic lateral sclerosis (ALS), we screened 304 unrelated ALS patients and 637 healthy controls for the GGC repeat expansion in NOTCH2NLC using repeat-primed PCR and fragment analysis. None of the ALS patients carried the GGC repeat expansion. The sizes of the GGC repeats ranged from 7 to 36 in the ALS patients and 4 to 46 in the controls. The distribution of the GGC repeat sizes did not differ between the two groups. Our findings indicate that the NOTCH2NLC GGC repeat expansion is absent or extremely rare in Taiwanese patients with ALS.

Investigating TBP CAG/CAA trinucleotide repeat expansions in a Taiwanese cohort with ALS.

Intermediate-length CAG repeats in ATXN2 have been well recognized as a genetic risk factor for amyotrophic lateral sclerosis (ALS). However, the role of similar trinucleotide repeat expansions in the TATA-box binding protein gene (TBP), another disease-associated gene for inherited ataxia, in ALS remains elusive. To assess the association between TBP trinucleotide repeat expansions and ALS, we investigated the repeat lengths in 325 unrelated ALS patients and 1500 controls in the Taiwanese population. The most common size of repeats in the patients and controls were both 36. The repeat lengths ranged from 29 to 46 repeats in the ALS patients and 27 to 43 repeats in the controls. Two ALS patients carried a TBP allele with a repeat number equal or greater than 44 (44 and 46). The patient with the 46 trinucleotide repeats also had a C9ORF72 GGGGCC hexanucleotide repeat expansion. The odds ratio of an individual carrying the CAG/CAA repeats ≥ 44 to have ALS is 23.2 (95% confidence interval: 1.11-484.24; p = 0.04). Our findings suggest that intermediate-length CAG/CAA repeat expansions in TBP may associate with ALS risk.

Hand-onset weakness is a common feature of ALS patients with a NEK1 loss-of-function variant.

OBJECTIVE: The NEK1 gene has been recently implicated in amyotrophic lateral sclerosis (ALS). This study aims to assess the influence of NEK1 variants on the occurrence of ALS and investigate the spectrum and clinical features of NEK1 loss-of-function (LOF) variants in a Taiwanese ALS cohort. METHODS: We screened 325 unrelated ALS patients for coding variants in NEK1 by targeted resequencing and queried the Taiwan Biobank database for NEK1 coding variants in 1000 Taiwanese healthy individuals. The clinical features of the patients with a NEK1 LOF variant were analyzed. RESULTS: Six patients and two healthy individuals carried NEK1 LOF variants. The rare missense variants with minor allele frequencies <0.1% in Taiwanese population were present in 2.8% of the ALS patients and 1.6% of the healthy subjects. NEK1 LOF variants, but not rare missense variants, are significantly enriched in the ALS patients (P = 0.0037 and 0.24, Fisher's exact test). The odds ratio of an individual carrying a NEK1 LOF variant to develop ALS is 9.39 (95% confidence interval: 1.88-46.7). All the six patients carrying a NEK1 LOF variant had a hand-onset ALS with an onset age from 52 to 64 years. Comparing with ALS patients without a NEK1 LOF variant, patients with a NEK1 LOF variant tend to have a hand-onset disease (P = 0.0008, Fisher's exact test). INTERPRETATION: Our study supports the pathogenic role of NEK1 LOF variants and demonstrates their spectrum and clinical features in a Taiwanese cohort with ALS.

Clinical characteristics of Taiwanese patients with Hereditary spastic paraplegia type 5.

OBJECTIVES: To investigate the clinical, electrophysiological, neuroimaging characteristics and genetic features of SPG5 in Taiwan. METHODS: Mutational analysis of the coding regions of CYP7B1 was performed by utilizing targeted resequencing analysis of the 187 unrelated Taiwanese HSP patients. The diagnosis of SPG5 was ascertained by the presence of biallelic CYP7B1 mutations. The SPG5 patients received clinical, electrophysiological, and neuroimaging evaluations. Disease severity was assessed by using the Spastic Paraplegia Rating Scale (SPRS) and the disability score. Two microsatellite markers as well as 18 single-nucleotide polymorphism (SNP) markers flanking CYP7B1 were genotyped to assess the founder effect of the CYP7B1 p.R112* mutation. RESULTS: Nineteen SPG5 patients from 17 families were identified. They typically presented an insidious onset progressive spastic paraparesis with proprioception involvement beginning at age 8 to 40 years. Their MRIs often showed white matter abnormalities in bilateral occipito-parietal regions, spinal cord atrophy, and mild cerebellar atrophy. Six different mutations in CYP7B1 were recognized, including three novel ones (p.N131Ifs*4, p.A295V, and p.L439R). CYP7B1 p.R112* was the most common mutation and present in 88.2% of the 17 SPG5 pedigrees. The patients with homozygous CYP7B1 p.R112* mutations had a milder clinical severity. Detailed haplotype analyses demonstrated a shared haplotype in the 25 individuals carrying at least one single allele of CYP7B1 p.R112*, suggesting a founder effect. INTERPRETATION: This study delineates the distinct clinical and genetic features of SPG5 in Taiwan and provides useful information for the diagnosis and management of SPG5, especially in patients of Chinese descent.

Aberrant Sensory Gating of the Primary Somatosensory Cortex Contributes to the Motor Circuit Dysfunction in Paroxysmal Kinesigenic Dyskinesia.

Paroxysmal kinesigenic dyskinesia (PKD) is conventionally regarded as a movement disorder (MD) and characterized by episodic hyperkinesia by sudden movements. However, patients of PKD often have sensory aura and respond excellently to antiepileptic agents. PRRT2 mutations, the most common genetic etiology of PKD, could cause epilepsy syndromes as well. Standing in the twilight zone between MDs and epilepsy, the pathogenesis of PKD is unclear. Gamma oscillations arise from the inhibitory interneurons which are crucial in the thalamocortical circuits. The role of synchronized gamma oscillations in sensory gating is an important mechanism of automatic cortical inhibition. The patterns of gamma oscillations have been used to characterize neurophysiological features of many neurological diseases, including epilepsy and MDs. This study was aimed to investigate the features of gamma synchronizations in PKD. In the paired-pulse electrical-stimulation task, we recorded the magnetoencephalographic data with distributed source modeling and time-frequency analysis in 19 patients of newly-diagnosed PKD without receiving pharmacotherapy and 18 healthy controls. In combination with the magnetic resonance imaging, the source of gamma oscillations was localized in the primary somatosensory cortex. Somatosensory evoked fields of PKD patients had a reduced peak frequency (p < 0.001 for the first and the second response) and a prolonged peak latency (the first response p = 0.02, the second response p = 0.002), indicating the synchronization of gamma oscillation is significantly attenuated. The power ratio between two responses was much higher in the PKD group (p = 0.013), indicating the incompetence of activity suppression. Aberrant gamma synchronizations revealed the defective sensory gating of the somatosensory area contributes the pathogenesis of PKD. Our findings documented disinhibited cortical function is a pathomechanism common to PKD and epilepsy, thus rationalized the clinical overlaps of these two diseases and the therapeutic effect of antiepileptic agents for PKD. There is a greater reduction of the peak gamma frequency in PRRT2-related PKD than the non-PRRT PKD group (p = 0.028 for the first response, p = 0.004 for the second response). Loss-of-function PRRT2 mutations could lead to synaptic dysfunction. The disinhibiton change on neurophysiology reflected the impacts of PRRT2 mutations on human neurophysiology.

Genetic analysis of ANXA11 variants in a Han Chinese cohort with amyotrophic lateral sclerosis in Taiwan.

Mutations in the annexin A11 gene (ANXA11) have been recently identified in British patients and Italian patients with amyotrophic lateral sclerosis (ALS), and their role in other ALS populations remains unclear. The aim of this study was to investigate the ANXA11 mutations in a Taiwanese ALS cohort. Mutational analysis of ANXA11 was performed in 286 unrelated Taiwanese patients with ALS by Sanger sequencing. Eight ANXA11 missense variants were identified initially, and only one of them was absent from population databases. This missense variant, p.Q362L, was identified in 1 single patient with apparently sporadic ALS, and no further strong evidence was available to support its pathogenicity. Therefore, it is classified as a variant of uncertain significance. Our data indicate that pathogenic ANXA11 mutations are absent or rare in ALS patients in Taiwan.

Investigating CCNF mutations in a Taiwanese cohort with amyotrophic lateral sclerosis.

Mutations in the cyclin F gene (CCNF) have been recently identified in a small number of patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia, and their role in patients with ALS in Taiwan remains elusive. The aim of this study was to elucidate the frequency and spectrum of CCNF mutations in a Taiwanese ALS cohort of Han Chinese origin. Mutational analyses of the CCNF gene were performed using Sanger sequencing in a cohort of 255 unrelated patients with ALS. Among these patients, the genetic diagnoses of 204 patients remained unclear after mutations in SOD1, C9ORF72, TARDBP, FUS, ATXN2, OPTN, VCP, UBQLN2, SQSTM1, PFN1, HNRNPA1, HNRNPA2B1, MATR3, CHCHD10, TUBA4A, and TKB1 had been investigated. Two novel heterozygous missense mutations in CCNF, p.S222P (c.664T>C) and p.S532R (c.1596C>T), were identified; 1 in each patient with apparently sporadic ALS. In vitro functional study demonstrated that both mutations result in a general and cyclin F-mediated ubiquitin-proteasome pathway dysfunction. The frequency of CCNF mutations in ALS patients in Taiwan is, therefore, approximately 0.8% (2/255). These findings expand the mutational spectrum of CCNF and also emphasize the pathogenic role of CCNF mutations in ALS.

CFTR potentiators: from bench to bedside.

One major breakthrough in cystic fibrosis research in the past decade is the development of drugs that target the root cause of the disease-dysfunctional CFTR protein. One of the compounds, Ivacaftor or Kalydeco, which has been approved for clinical use since 2012, acts by promoting the gating function of CFTR. Our recent studies have led to a gating model that features energetic coupling between nucleotide-binding domain (NBD) dimerization and gate opening/closing in CFTR's transmembrane domains (TMDs). Based on this model, we showed that ATP analogs can enhance CFTR gating by facilitating NBD dimerization, whereas Ivacaftor works by stabilizing the open channel conformation of the TMDs. This latter idea also explains the near omnipotence of Ivacaftor. Furthermore, this model identifies multiple approaches to synergistically boost the open probability of CFTR by influencing distinct molecular events that control gating conformational changes.

A single amino acid substitution in CFTR converts ATP to an inhibitory ligand.

Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. The third most common pathogenic mutation, a glycine-to-aspartate mutation at position 551 or G551D, shows a significantly decreased open probability (Po) caused by failure of the mutant channel to respond to ATP. Recently, a CFTR-targeted drug, VX-770 (Ivacaftor), which potentiates G551D-CFTR function in vitro by boosting its Po, has been approved by the FDA to treat CF patients carrying this mutation. Here, we show that, in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit with an unusual time course. In marked contrast to wild-type channels, which are stimulated by ATP, sudden removal of ATP in excised inside-out patches elicits an initial increase in macroscopic G551D-CFTR current followed by a slow decrease. Furthermore, decreasing [ATP] from 2 mM to 20 µM resulted in a paradoxical increase in G551D-CFTR current. These results suggest that the two ATP-binding sites in the G551D mutant mediate opposite effects on channel gating. We introduced mutations that specifically alter ATP-binding affinity in either nucleotide-binding domain (NBD1 or NBD2) into the G551D background and determined that this disease-associated mutation converts site 2, formed by the head subdomain of NBD2 and the tail subdomain of NBD1, into an inhibitory site, whereas site 1 remains stimulatory. G551E, but not G551K or G551S, exhibits a similar phenotype, indicating that electrostatic repulsion between the negatively charged side chain of aspartate and the γ-phosphate of ATP accounts for the observed mutational effects. Understanding the molecular mechanism of this gating defect lays a foundation for rational drug design for the treatment of CF.

Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle.

Vx-770 (Ivacaftor), a Food and Drug Administration (FDA)-approved drug for clinical application to patients with cystic fibrosis (CF), shifts the paradigm from conventional symptomatic treatments to therapeutics directly tackling the root of the disease: functional defects of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel caused by pathogenic mutations. The underlying mechanism for the action of Vx-770 remains elusive partly because this compound not only increases the activity of wild-type (WT) channels whose gating is primarily controlled by ATP binding/hydrolysis, but also improves the function of G551D-CFTR, a disease-associated mutation that abolishes CFTR's responsiveness to ATP. Here we provide a unified theory to account for this dual effect of Vx-770. We found that Vx-770 enhances spontaneous, ATP-independent activity of WT-CFTR to a similar magnitude as its effects on G551D channels, a result essentially explaining Vx-770's effect on G551D-CFTR. Furthermore, Vx-770 increases the open time of WT-CFTR in an [ATP]-dependent manner. This distinct kinetic effect is accountable with a newly proposed CFTR gating model depicting an [ATP]-dependent "reentry" mechanism that allows CFTR shuffling among different open states by undergoing multiple rounds of ATP hydrolysis. We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Our data corroborate that Vx-770 increases the open time of WT-CFTR by stabilizing a posthydrolytic open state and thereby fosters decoupling between the gating cycle and ATP hydrolysis cycle. The current study also suggests that this unique mechanism of drug action can be further exploited to develop strategies that enhance the function of CFTR.

Nonequilibrium gating of CFTR on an equilibrium theme.

Malfunction of cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC protein superfamily that functions as an ATP-gated chloride channel, causes the lethal genetic disease, cystic fibrosis. This review focuses on the most recent findings on the gating mechanism of CFTR. Potential clinical relevance and implications to ABC transporter function are also discussed.

Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC proteins that function as active transporters, CFTR is an ATP-gated chloride channel. The opening of CFTR's gate is associated with ATP-induced dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate closure is facilitated by ATP hydrolysis-triggered partial separation of the NBDs. This generally held theme of CFTR gating-a strict coupling between the ATP hydrolysis cycle and the gating cycle-is put to the test by our recent finding of a short-lived, post-hydrolytic state that can bind ATP and reenter the ATP-induced original open state. We accidentally found a mutant CFTR channel that exhibits two distinct open conductance states, the smaller O1 state and the larger O2 state. In the presence of ATP, the transition between the two states follows a preferred O1→O2 order, a telltale sign of a violation of microscopic reversibility, hence demanding an external energy input likely from ATP hydrolysis, as such preferred gating transition was abolished in a hydrolysis-deficient mutant. Interestingly, we also observed a considerable amount of opening events that contain more than one O1→O2 transition, indicating that more than one ATP molecule may be hydrolyzed within an opening burst. We thus conclude a nonintegral stoichiometry between the gating cycle and ATP consumption. Our results lead to a six-state gating model conforming to the classical allosteric mechanism: both NBDs and transmembrane domains hold a certain degree of autonomy, whereas the conformational change in one domain will facilitate the conformational change in the other domain.

Identification of a novel post-hydrolytic state in CFTR gating.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC protein family may share an evolutionary origin. Although ABC exporters harness the free energy of ATP hydrolysis to fuel a transport cycle, for CFTR, ATP-induced dimerization of its nucleotide-binding domains (NBDs) and subsequent hydrolysis-triggered dimer separation are proposed to be coupled, respectively, to the opening and closing of the gate in its transmembrane domains. In this study, by using nonhydrolyzable ATP analogues, such as pyrophosphate or adenylyl-imidodiphosphate as baits, we captured a short-lived state (state X), which distinguishes itself from the previously identified long-lived C2 closed state by its fast response to these nonhydrolyzable ligands. As state X is caught during the decay phase of channel closing upon washout of the ligand ATP but before the channel sojourns to the C2 closed state, it likely emerges after the bound ATP in the catalysis-competent site has been hydrolyzed and the hydrolytic products have been released. Thus, this newly identified post-hydrolytic state may share a similar conformation of NBDs as the C2 closed state (i.e., a partially separated NBD and a vacated ATP-binding pocket). The significance of this novel state in understanding the structural basis of CFTR gating is discussed.

The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP binding cassette (ABC) superfamily. The deletion of the phenylalanine 508 (ΔF508-CFTR) is the most common mutation among cystic fibrosis (CF) patients. The mutant channels present a severe trafficking defect, and the few channels that reach the plasma membrane are functionally impaired. Interestingly, an ATP analogue, N6-(2-phenylethyl)-2'-deoxy-ATP (P-dATP), can increase the open probability (Po) to ∼0.7, implying that the gating defect of ΔF508 may involve the ligand binding domains, such as interfering with the formation or separation of the dimeric states of the nucleotide-binding domains (NBDs). To test this hypothesis, we employed two approaches developed for gauging the stability of the NBD dimeric states using the patch-clamp technique. We measured the locked-open time induced by pyrophosphate (PPi), which reflects the stability of the full NBD dimer state, and the ligand exchange time for ATP/N6-(2-phenylethyl)-ATP (P-ATP), which measures the stability of the partial NBD dimer state wherein the head of NBD1 and the tail of NBD2 remain associated. We found that both the PPi-induced locked-open time and the ATP/P-ATP ligand exchange time of ΔF508-CFTR channels are dramatically shortened, suggesting that the ΔF508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of ΔF508-CFTR, namely the solubilizing mutation F494N/Q637R and ΔRI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with ΔF508-CFTR. Our results indicate that although these mutations increase the membrane expression and function of ΔF508-CFTR, they have limited impact on the stability of both full and partial NBD dimeric states for ΔF508 channels. The structure-function insights gained from this mechanism may provide clues for future drug design.