Relationship between reasons for intermittent missing patient-reported outcomes data and missing data mechanisms.

PURPOSE: Non-response (NR) to patient-reported outcome (PRO) questionnaires may cause bias if not handled appropriately. Collecting reasons for NR is recommended, but how reasons for NR are related to missing data mechanisms remains unexplored. We aimed to explore this relationship for intermittent NRs. METHODS: Patients with multiple myeloma completed validated PRO questionnaires at enrolment and 12 follow-up time-points. NR was defined as non-completion of a follow-up assessment within seven days, which triggered contact with the patient, recording the reason for missingness and an invitation to complete the questionnaire (denoted "salvage response"). Mean differences between salvage and previous on-time scores were estimated for groups defined by reasons for NR using linear regression with clustered standard errors. Statistically significant mean differences larger than minimal important difference thresholds were interpreted as "missing not at random" (MNAR) mechanism (i.e. assumed to be related to declining health), and the remainder interpreted as aligned with "missing completely at random" (MCAR) mechanism (i.e. assumed unrelated to changes in health). RESULTS: Most (7228/7534 (96%)) follow-up questionnaires were completed; 11% (802/7534) were salvage responses. Mean salvage scores were compared to previous on-time scores by reason: those due to hospital admission, mental or physical reasons were worse in 10/22 PRO domains; those due to technical difficulties/procedural errors were no different in 21/22 PRO domains; and those due to overlooked/forgotten or other/unspecified reasons were no different in any domains. CONCLUSION: Intermittent NRs due to hospital admission, mental or physical reasons were aligned with MNAR mechanism for nearly half of PRO domains, while intermittent NRs due to technical difficulties/procedural errors or other/unspecified reasons generally were aligned with MCAR mechanism.

Nationwide implementation of lenalidomide maintenance in multiple myeloma: A retrospective, real-world study.

Lenalidomide maintenance (LM) has shown benefit in progression-free survival (PFS) and overall survival (OS) in clinical trials. LM is the recommended standard of care in patients with newly diagnosed multiple myeloma (MM) after high-dose melphalan and autologous stem cell transplantation (HDM-ASCT). In Denmark, LM has been approved and publicly funded for all patients treated with HDM-ASCT since June 2019. Patients with newly diagnosed MM treated with their first HDM-ASCT between June 2019 and March 2022 were included and followed until data cut-off in June 2023. To compare outcomes, a historical pre-LM cohort from the Danish MM Registry, consisting of 364 MM patients treated with HDM-ASCT between June 2015 and June 2019, was used. Among 364 patients treated with HDM-ASCT after June 2019, 22.3% received consolidation therapy and 3.7% underwent tandem HDM-ASCT. During follow-up, 297 patients (81.6%) initiated maintenance therapy, with 277 (76.1%) receiving LM. Overall, 145 patients (52.3%) discontinued LM most commonly due to toxicity 75 (51.7%), with fatigue (30.7%), cytopenia (25.3%), and neuropathy (17.3%) being the main reasons. In a 6-month landmark analysis, early discontinuation did not negatively impact PFS or OS. The LM cohort had similar PFS, and OS compared to the pre-LM cohort. The 3-year PFS and OS rates in the LM cohort were 61% and 86%, respectively, while the pre-LM cohort had a 3-year PFS of 55% and a 3-year OS of 89%. In conclusion, the introduction of LM as a nationwide treatment option in Denmark did not lead to improved clinical outcomes.

Diverse roles of the metal binding domains and transport mechanism of copper transporting P-type ATPases.

Copper transporting P-type (P1B-1-) ATPases are essential for cellular homeostasis. Nonetheless, the E1-E1P-E2P-E2 states mechanism of P1B-1-ATPases remains poorly understood. In particular, the role of the intrinsic metal binding domains (MBDs) is enigmatic. Here, four cryo-EM structures and molecular dynamics simulations of a P1B-1-ATPase are combined to reveal that in many eukaryotes the MBD immediately prior to the ATPase core, MBD-1, serves a structural role, remodeling the ion-uptake region. In contrast, the MBD prior to MBD-1, MBD-2, likely assists in copper delivery to the ATPase core. Invariant Tyr, Asn and Ser residues in the transmembrane domain assist in positioning sulfur-providing copper-binding amino acids, allowing for copper uptake, binding and release. As such, our findings unify previously conflicting data on the transport and regulation of P1B-1-ATPases. The results are critical for a fundamental understanding of cellular copper homeostasis and for comprehension of the molecular bases of P1B-1-disorders and ongoing clinical trials.

Imaging of extracellular and intracellular ATP in pancreatic beta cells reveals correlation between glucose metabolism and purinergic signalling.

Adenosine triphosphate (ATP) is a universal energy molecule and yet cells release it and extracellular ATP is an important signalling molecule between cells. Monitoring of ATP levels outside of cells is important for our understanding of physiological and pathophysiological processes in cells/tissues. Here, we focus on pancreatic beta cells (INS-1E) and test the hypothesis that there is an association between intra- and extracellular ATP levels which depends on glucose provision. We imaged real-time changes in extracellular ATP in pancreatic beta cells using two sensors tethered to extracellular aspects of the plasma membrane (eATeam3.10, iATPSnFR1.0). Increase in glucose induced fast micromolar ATP release to the cell surface, depending on glucose concentrations. Chronic pre-treatment with glucose increased the basal ATP signal. In addition, we co-expressed intracellular ATP sensors (ATeam1.30, PercevalHR) in the same cultures and showed that glucose induced fast increases in extracellular and intracellular ATP. Glucose and extracellular ATP stimulated glucose transport monitored by the glucose sensor (FLII12Pglu-700uDelta6). In conclusion, we propose that in beta cells there is a dynamic relation between intra- and extracellular ATP that depends on glucose transport and metabolism and these processes may be tuned by purinergic signalling. Future development of ATP sensors for imaging may aid development of novel approaches to target extracellular ATP in, for example, type 2 diabetes mellitus therapy.

Real-world outcomes following third or subsequent lines of therapy: A Danish population-based study on 189 patients with relapsed/refractory large B-cell lymphomas.

Outcome data of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) beyond the second line are scarce outside of clinical trials. Novel therapies in the R/R setting have been approved based on single-arm trials, but results need to be contextualized by real-world outcomes. Medical records from 3753 Danish adults diagnosed with DLBCL were reviewed. Patients previously treated with rituximab and anthracycline-based chemotherapy who received the third or later line (3 L+) of treatment after 1 January 2015, were included. Only 189 patients with a median age of 71 years were eligible. The median time since the last line of therapy was 6 months. Patients were treated with either best supportive care (22%), platinum-based salvage therapy (13%), low-intensity chemotherapy (22%), in clinical trial (14%) or various combination treatments (32%). The 2-year OS-/PFS estimates were 25% and 12% for all patients and 49% and 17% for those treated with platinum-based salvage therapy. Age ≥70, CNS involvement, elevated LDH and ECOG ≥2 predicted poor outcomes, and patients with 0-1 of these risk factors had a 2-year OS estimate of 65%. Only a very small fraction of DLBCL patients received third-line treatment and were eligible for inclusion. Outcomes were generally poor, but better in intensively treated, fit young patients with limited disease.

Progressive multifocal leukoencephalopathy in a patient with multiple myeloma: a case report and analysis of the FDA adverse event reporting system.

This paper demonstrates a case of progressive multifocal leukoencephalopathy (PML) in a patient with multiple myeloma (MM) treated with nine different MM therapies. This case report contributes to the already published 16 cases of PML in patients with MM. Additionally, this paper presents an analysis of cases from the United States Food and Drug Administration Adverse Event Report System database (n = 117) with a description of demographics and MM-specific therapies. Patients with MM, that developed PML, were treated with immunomodulatory drugs (97%), alkylating agents (52%), and/or proteasome inhibitors (49%). Prior to PML diagnosis, 72% of patients received two or more MM therapies. These results indicate that PML in MM is underreported and could be related to treatment with multiple immunosuppressive therapies rather than MM as a disease itself. Physicians should be aware of potential PML in the late stage of heavily treated MM patients.

Geographical and ecological analyses of multiple myeloma in Denmark: Identification of potential hotspot areas and impact of urbanisation.

BACKGROUND: The aetiology of multiple myeloma (MM) is unknown but various environmental exposures are suspected as risk factors. We present the first paper analysing the geographical distribution of MM in Denmark at the municipal level to investigate variations that could be explained by environmental exposures. METHODS: Patients diagnosed with MM in Denmark during 2005-2020 were identified from nationwide registries and grouped into the 98 Danish municipalities based on residence. The age- and sex-standardised incidence rate (SIR) of each municipality was compared to the national incidence in a funnel plot with 95% control limits. Differences in SIRs of rural, suburban, and urban areas were evaluated with incidence rate ratios. RESULTS: In total, 5243 MM patients were included. Overall, we found a heterogeneous geographical distribution of MM and a potential hotspot in southern Denmark. This hotspot contains three municipalities with SIRs above the 95% control limit assuming considerably higher rate of MM compared to the national incidence rate. A significant higher SIR was found in rural areas compared to urban areas. CONCLUSION: The geographical distribution of MM in Denmark indicates that the risk of developing MM depends on place of residence probably due to environmental factors.

Patients in complete remission after R-CHOP(-like) therapy for diffuse large B-cell lymphoma have limited excess use of health care services in Denmark.

For most patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL), R-CHOP immunochemotherapy leads to complete remission and 60-70% of patients remain progression-free after 5 years. Given a median age of 65, it is relevant to disentangle how DLBCL and DLBCL therapy influence health care use among the survivors. In this nationwide study, the health care use among Danish DLBCL patients diagnosed in 2007-2015, who achieved complete remission after R-CHOP(-like) therapy, was explored and compared to matched comparators from the Danish general population. The post-remission 5-year risk of hospitalization was significantly higher among DLBCL survivors (55%) compared to matched comparators (49%, P < 0.001). DLBCL survivors had on average 10.3 (9.3-11.3) inpatient bed days within 5 years of response evaluation, whereas matched comparators had 8.4 (7.9-8.8). The rate of outpatient visits was also significantly higher(excluding routine follow-up visits, incidence rate ratio, 1.3, P < 0.001), but translated into only a very small absolute difference of <1 outpatient visits within 5 years between DLBCL survivors (4.2 visits, 95% CI, 4.0-4.4) and matched comparators (3.8 visits, 95% CI, 3.7-3.9). In conclusion, DLBCL survivors have an increased incidence of hospital visits due to a wide range of conditions, but in absolute terms the excess use of health care services in DLBCL survivors was small.

Mechanisms of irreversible aquaporin-10 inhibition by organogold compounds studied by combined biophysical methods and atomistic simulations.

The inhibition of glycerol permeation via human aquaporin-10 (hAQP10) by organometallic gold complexes has been studied by stopped-flow fluorescence spectroscopy, and its mechanism has been described using molecular modelling and atomistic simulations. The most effective hAQP10 inhibitors are cyclometalated Au(III) C^N compounds known to efficiently react with cysteine residues leading to the formation of irreversible C-S bonds. Functional assays also demonstrate the irreversibility of the binding to hAQP10 by the organometallic complexes. The obtained computational results by metadynamics show that the local arylation of Cys209 in hAQP10 by one of the gold inhibitors is mapped into a global change of the overall free energy of glycerol translocation across the channel. Our study further pinpoints the need to understand the mechanism of glycerol and small molecule permeation as a combination of local structural motifs and global pore conformational changes, which are taking place on the scale of the translocation process and whose study, therefore, require sophisticated molecular dynamics strategies.

Overproduction of Human Zip (SLC39) Zinc Transporters in Saccharomyces cerevisiae for Biophysical Characterization.

Zinc constitutes the second most abundant transition metal in the human body, and it is implicated in numerous cellular processes, including cell division, DNA and protein synthesis as well as for the catalytic activity of many enzymes. Two major membrane protein families facilitate zinc homeostasis in the animal kingdom, i.e., Zrt/Irt-like proteins (ZIPs aka solute carrier 39, SLC39, family) and Zn transporters (ZnTs), essentially conducting zinc flux in the opposite directions. Human ZIPs (hZIPs) regulate import of extracellular zinc to the cytosol, being critical in preventing overaccumulation of this potentially toxic metal, and crucial for diverse physiological and pathological processes, including development of neurodegenerative disorders and several cancers. To date, our understanding of structure-function relationships governing hZIP-mediated zinc transport mechanism is scarce, mainly due to the notorious difficulty in overproduction of these proteins for biophysical characterization. Here we describe employment of a Saccharomyces cerevisiae-based platform for heterologous expression of hZIPs. We demonstrate that yeast is able to produce four full-length hZIP members belonging to three different subfamilies. One target (hZIP1) is purified in the high quantity and homogeneity required for the downstream biochemical analysis. Our work demonstrates the potential of the described production system for future structural and functional studies of hZIP transporters.

Validation of the UK myeloma research alliance risk profile, a new clinical prediction model for outcome in patients with newly diagnosed multiple myeloma not eligible for autologous stem cell transplantation; a population-based study from the Danish national multiple myeloma registry.

In 2019 the UK Myeloma Research Alliance introduced the Myeloma Risk Profile (MRP) for prediction of outcome in patients with newly diagnosed multiple myeloma (MM), ineligible for autologous stem cell transplantation. To validate the MRP in a population-based setting we performed a study of the entire cohort of transplant ineligible MM patients above 65 years in the Danish National MM Registry. Our data confirmed the value of the MRP. In a cohort of 1,377 patients, the MRP score separated patients into three distinct risk-groups with an observed hazard ratio of 2.91 for early death in high-risk versus low-risk patients.

Prognostic significance of infectious episodes occurring during first-line therapy for diffuse large B-cell lymphoma - A nationwide cohort study.

Infections during first-line therapy for DLBCL are often associated with chemotherapy dose reductions and increased mortality. Systemic infections have also been suggested as beneficial promotors of immunological responses. However, whether there is an association between the timing of an infectious episode and outcome during treatment has not yet been clarified. We investigated how the occurrence and timing of infectious episodes during the first line of treatment for "de novo" DLBCL influenced patient outcome. We used data on DLBCL patients from the Danish Lymphoma Registry, the Danish National Patient Registry, and the Danish National Pathology Registry. Infections were categorized according to type (ICD-10) and time of occurrence after treatment start. "Early" infections were defined as occurring between days 7 and 42 and "late" infections between days 100 and 150 from treatment start. Patients experiencing both "early and late" infections were categorized separately. We used multivariable Cox regression and Kaplan-Meier estimates to assess the association between infections and survival adjusting for NCCN-IPI, sex, comorbidity, and rituximab treatment. We identified 3546 patients, median age 65 years (IQR 56,73). Infectious episodes occurred in 1171 (33%) patients, of which 666 had "early," 303 "late," and 202 both "early and late" events. Patients without registered infections had a 5-year overall survival (OS) rates of 74%. Those with "early," "late," or "early+late" had 5-year OS of 65%, 62%, and 53%, respectively. Compared with patients without any registered infections, hazard rate ratios (HR) were 1.24 (95% CI 1.05-1.47), 1.32 (95% CI 1.06-1.63), and 1.59 (95% CI 1.27-2.00), respectively, in the multivariable model. We observed that infectious episodes during first-line treatment for "de novo" DLBCL occurred in 44% of the patients. Irrespective of timing, patients with infectious episodes had an inferior outcome compared to those without. Outcome patterns were similar for patients registered with sepsis.

Strategies to improve patient-reported outcome completion rates in longitudinal studies.

PURPOSE: The quality of patient-reported outcome (PRO) data can be compromised by non-response (NR) to scheduled questionnaires, particularly if reasons for NR are related to health problems, which may lead to unintended bias. The aim was to investigate whether electronic reminders and real-time monitoring improve PRO completion rate. METHODS: The population-based study "Quality of life in Danish multiple myeloma patients" is a longitudinal, multicentre study with consecutive inclusion of treatment-demanding newly diagnosed or relapsed patients with multiple myeloma. Education of study nurses in the avoidance of NR, electronic reminders, 7-day response windows and real-time monitoring of NR were integrated in the study. Patients complete PRO assessments at study entry and at 12 follow-up time points using electronic or paper questionnaires. The effect of the electronic reminders and real-time monitoring were investigated by comparison of proportions of completed questionnaires before and after each intervention. RESULTS: The first 271 included patients were analysed; of those, 249 (85%) chose electronic questionnaires. Eighty-four percent of the 1441 scheduled PRO assessments were completed within the 7-day response window and 11% after real-time monitoring, achieving a final PRO completion rate of 95%. A significant higher proportion of uncompleted questionnaires were completed after the patients had received the electronic reminder and after real-time monitoring. CONCLUSIONS: Electronic reminders and real-time monitoring contributed to a very high completion rate in the study. To increase the quality of PRO data, we propose integrating these strategies in PRO studies, however highlighting that an increase in staff resources is required for implementation.

Elemental Composition and Organic Petrology of a Lower Carboniferous-Age Freshwater Oil Shale in Nova Scotia, Canada.

A 59 m-thick section of a freshwater oil shale interbedded with marlstone of Lower Carboniferous (Tournaisian) age from the Big Marsh area in Antigonish Basin, Nova Scotia, Canada, was examined using reflected light microscopy, Rock-Eval pyrolysis, X-ray diffractometry analysis, inductively coupled plasma-mass spectrometry for elemental analysis, and prompt γ for boron concentration. The oil shale was deposited in a lacustrine environment based on geology, sedimentology, variation in organic matter, and boron content (28-54 ppm). Organic petrology classified the oil shale into three broadly distinct types. Type A oil shale is a coastal facies shale containing terrestrially derived macerals, such as vitrinite and inertinite, sporinite, with some lamalginite, and amorphous bituminous matrix. Type B oil shale was deposited in a shallow-water facies and contains mostly lamalginite and some vitrinite and sporinite. Type C oil shale is a relatively deep-water facies, associated with open-water Torbanite-type oil shale and contains mostly Botryococcus colonial telalginite. The oil shale is thermally mature (T max is 441-443 °C). Total organic carbon (TOC) varies from 5.8 to 7.3 wt %, and the hydrogen index is between 507 and 557 mg HC/g TOC. The rate of sedimentation as determined by the Th/U ratio indicates possibility of three sedimentation periods: an irregular but mostly slow rate of sedimentation from the base of the section up to 68 m, followed by a regular and slow rate between 68 and 53 m, and a regular and fast rate between 53 m and the top of the section. The higher Th/U ratio during deposition of the shallow-water facies was due to the input of allochthonous U. The redox conditions, as reflected in the variation of Cr to Mo, U, and Ni + V, indicate that the oil shale was deposited under suboxic-dysoxic conditions. The high organic productivity by phytoplankton and bacteria is characterized by a low Cr and high V/Cr ratio and suboxic conditions. In contrast, the well-oxygenated and uniform, warm-temperature upper water level supports a dysoxic environment. Variation of Sr/Ca vs Mn/Ca ratios indicates that most samples have low values, a characteristic of colder water and high terrigenous influx. The post-Archean Australian shale (PAAS)-normalized rare earth elements (REEs) follow three trends. Type A oil shale has the highest concentration of total REEs (648 ppm) and light REEs (LREEs, 605 ppm) as compared with type C (269 and 233 ppm), which are less than half of type A. Type B oil shale has the lowest total REEs (184 ppm) and LREEs (152 ppm). The concentration of heavy REEs decreased from 43 ppm in type A oil shale to 36 ppm in type C oil shale. Comparison of PAAS-normalized REEs for the three oil shale types indicates a reduction of the negative Eu anomaly with depth, which is possibly related to sedimentary sorting as a result of accumulation of fine sediments in the deeper water zone of the lake. The concentration of most elements of environmental concern is similar to and/or lower than the world shale. However, there are instances of higher concentrations of hazardous elements (e.g., As, Cd, Mo, and Se).

Pretreatment Hemoglobin Adds Prognostic Information To The NCCN-IPI In Patients With Diffuse Large B-Cell Lymphoma Treated With Anthracycline-Containing Chemotherapy.

BACKGROUND: Hemoglobin (Hgb) concentration at diagnosis is associated with outcome in cancer. In a recently reported simplified 3-factor prognostic score in Hodgkin lymphoma, Hgb, along with age and clinical stage, outperformed the classical International Prognostic Score with seven parameters. METHODS: In the present study, we investigated if pretherapeutic Hgb concentration added prognostic information to the NCCN-IPI in diffuse large B-cell lymphoma. We included patients from the Danish Lymphoma Registry (LYFO; N = 3499) and from the Molecular Epidemiology Resource (MER; N = 1225), Mayo Clinic and University of Iowa. Four sex-specific Hgb groups were defined: below transfusion threshold, from transfusion threshold to below lower limit of normal, from lower limit of normal to the population mean, and above the mean. We used multivariable Cox regression to estimate the hazard rate ratios (HR) and 95% CIs for overall survival (OS) and event-free survival (EFS), adjusting for sex, NCCN-IPI, comorbidity, and rituximab treatment. RESULTS: Approximately half of the patients had Hgb levels below the lower limit of normal. Compared to patients with Hgb levels above the mean, an inferior OS was directly correlated with lower pretreatment Hgb within the predefined groups (HR=1.23, HR=1.51, and HR=2.05, respectively). These findings were validated in the MER. CONCLUSION: Based on multivariable analysis, lower pretreatment Hgb, even within the normal range but below the mean, added prognostic information to established indices such as the NCCN-IPI and the Charlson comorbidity index.

Characterization of Hailey-Hailey Disease-mutants in presence and absence of wild type SPCA1 using Saccharomyces cerevisiae as model organism.

Hailey-Hailey disease is an autosomal genetic disease caused by mutations in one of the two ATP2C1 alleles encoding the secretory pathway Ca2+/Mn2+-ATPase, hSPCA1. The disease almost exclusively affects epidermis, where it mainly results in acantholysis of the suprabasal layers. The etiology of the disease is complex and not well understood. We applied a yeast based complementation system to characterize fourteen disease-causing ATP2C1 missense mutations in presence or absence of wild type ATP2C1 or ATP2A2, encoding SERCA2. In our yeast model system, mutations in ATP2C1 affected Mn2+ transport more than Ca2+ transport as twelve out of fourteen mutations were unable to complement Mn2+ sensitivity while thirteen out of fourteen to some extent complemented the high Ca2+requirement. Nine out of fourteen mutations conferred a cold sensitive complementation capacity. In absence of a wild type ATP2C1 allele, twelve out of fourteen mutations induced an unfolded protein response indicating that in vivo folding of hSPCA1 is sensitive to disease causing amino acid substitutions and four of the fourteen mutations caused the hSPCA1 protein to accumulate in the vacuolar membrane. Co-expression of either wild type ATP2C1 or ATP2A2 prevented induction of the unfolded protein response and hSPCA1 mis-localization.

Human Aquaporin-5 Facilitates Hydrogen Peroxide Permeation Affecting Adaption to Oxidative Stress and Cancer Cell Migration.

Reactive oxygen species (ROS), including H2O2, contribute to oxidative stress and may cause cancer initiation and progression. However, at low concentrations, H2O2 can regulate signaling pathways modulating cell growth, differentiation, and migration. A few mammalian aquaporins (AQPs) facilitate H2O2 diffusion across membranes and participate in tumorigenesis. AQP3 and AQP5 are strongly expressed in cancer tissues and AQP3-mediated H2O2 transport has been related to breast cancer cell migration, but studies with human AQP5 are lacking. Here, we report that, in addition to its established water permeation capacity, human AQP5 facilitates transmembrane H2O2 diffusion and modulates cell growth of AQP5-transformed yeast cells in response to oxidative stress. Mutagenesis studies revealed that residue His173 located in the selective filter is crucial for AQP5 permeability, and interactions with phosphorylated Ser183 may regulate permeation through pore blockage. Moreover, in human pancreatic cancer cells, the measured AQP5-mediated H2O2 influx rate indicates the presence of a highly efficient peroxiporin activity. Cell migration was similarly suppressed by AQP3 or AQP5 gene silencing and could be recovered by external oxidative stimuli. Altogether, these results unveiled a major role for AQP5 in dynamic fine-tuning of the intracellular H2O2 concentration, and consequently in activating signaling networks related to cell survival and cancer progression, highlighting AQP5 as a promising drug target for cancer therapies.

Structure of the human ClC-1 chloride channel.

ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue ("fast gate") known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-ß-synthase (CBS) domains and the intracellular vestibule ("slow gating"). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1-related diseases.