Association of prior treatment with nitrogen-containing bisphosphonates on outcomes of COVID-19 positive patients.

COVID-19 infection has resulted in significant morbidity and mortality globally, especially among older adults. Repurposed drugs have demonstrated activity in respiratory illnesses, including nitrogen-containing bisphosphonates. In this retrospective longitudinal study at 4 academic medical centers, we show no benefit of nitrogen-containing bisphosphonates regarding ICU admission, ventilator use, and mortality among older adults with COVID-19 infection. We specifically evaluated the intravenous bisphosphonate zoledronic acid and found no difference compared to oral bisphosphonates. BACKGROUND: Widely used in osteoporosis treatment, nitrogen-containing bisphosphonates (N-BP) have been associated with reduced mortality and morbidity among older adults. Based on prior studies, we hypothesized that prior treatment with N-BP might reduce intensive care unit (ICU) admission, ventilator use, and death among older adults diagnosed with COVID-19. METHODS: This retrospective analysis of the PCORnet Common Data Model across 4 academic medical centers through 1 September 2021 identified individuals age >50 years with a diagnosis of COVID-19. The composite outcome included ICU admission, ventilator use, or death within 15, 30, and 180 days of COVID-19 diagnosis. Use of N-BP was defined as a prescription within 3 years prior. ICU admission and ventilator use were determined using administrative codes. Death included both in-hospital and out-of-hospital events. Patients treated with N-BP were matched 1:1 by propensity score to patients without prior N-BP use. Secondary analysis compared outcomes among those prescribed zoledronic acid (ZOL) to those prescribed oral N-BPs. RESULTS: Of 76,223 COVID-19 patients identified, 1,853 were previously prescribed N-BP, among whom 559 were prescribed ZOL. After propensity score matching, there were no significant differences in the composite outcome at 15 days (HR 1.22, 95% CI: 0.89-1.67), 30 days (HR 1.24, 95% CI: 0.93-1.66), or 180 days (HR 1.17, 95% CI: 0.93-1.48), comparing those prescribed and not prescribed N-BP. Compared to those prescribed oral N-BP, there were no significant differences in outcomes among those prescribed ZOL. CONCLUSION: Among older COVID-19 patients, prior exposure to N-BP including ZOL was not associated with a reduction in ICU admission, ventilator use, or death.

Quantifying biomarkers of axonal degeneration in early glaucoma to find the disc at risk.

To evaluate regional axonal-related parameters as a function of disease stage in primary open angle glaucoma (POAG) and visual field (VF) sensitivity. Spectral domain optical coherence tomography was used to acquire 20° scans of POAG (n = 117) or healthy control (n = 52) human optic nerve heads (ONHs). Region specific and mean nerve fibre layer (NFL) thicknesses, border NFL and peripapillary NFL, minimum rim width (MRW)/ area (MRA) and prelamina thickness; and volume were compared across POAG disease stages and with visual field sensitivity. Differences identified between early glaucoma (EG), preperimetric glaucoma (PG) and control (C) ONHs included thinner PG prelamina regions than in controls (p < 0.05). Mean border NFL was thinner in EG (p < 0.001) and PG (p = 0.049) compared to control eyes; and EG mean, and inferior and ST, border NFL was thinner than in PG (p < 0.01). Mean, superior and inferior PG peripapillary NFL were thinner than in controls (p < 0.05), and EG ST peripapillary NFL was thinner than in PG (p = 0.023). MRW differences included: PG SN and inferior less than in controls (p < 0.05); thinner EG mean regional, inferior, nasal, and ST MRW versus PG MRW (p < 0.05). Regional border NFL, peripapillary NFL, MRW, MRA, prelamina thickness (except centre, p = 0.127) and prelamina volume (p < 0.05) were significantly associated with VF mean deviation (MD). Novel axon-derived indices hold potential as biomarkers to detect early glaucoma and identify ONHs at risk.

Differences in ebullitive methane release from small, shallow ponds present challenges for scaling.

Small, shallow waterbodies are potentially important sites of greenhouse gas release to the atmosphere. The role of ebullition may be enhanced here relative to larger and deeper systems, due to their shallow water, but these features remain relatively infrequently studied in comparison to larger systems. Herein, we quantify ebullitive release of methane (CH4) in small shallow ponds in three regions of North America and investigate the role of potential drivers. Shallow ponds exhibited open-water season ebullitive CH4 release rates as high as 40 mmol m-2 d-1, higher than previously reported for similar systems. Ebullitive release of CH4 varied by four orders of magnitude across our 15 study sites, with differences in flux rates both within and between regions. What is less clear are the drivers responsible for these differences. There were few relationships between open water-season ebullitive flux and physicochemical characteristics, including organic matter, temperature, and sulphate. Temperature was only weakly related to ebullitive CH4 release across the study when considering all observation intervals. Only four individual sites exhibited significant relationships between temperature and ebullitive CH4 release. Other sites were unresponsive to temperature, and region-specific factors may play a role. There is some evidence that where surface water sulphate concentrations are high, CH4 production and release may be suppressed. Missouri sites (n = 5) had characteristically low ebullitive CH4 release; here bioturbation could be important. While this work greatly expands the number of open-water season ebullition rates for small and shallow ponds, more research is needed to disentangle the role of different drivers. Further investigation of the potential thresholding behaviour of sulphate as a control on ebullitive CH4 release in lentic systems is one such opportunity. What is clear, however, is that efforts to scale emissions (e.g., as a function of temperature) must be undertaken with caution.

Predicting delivery of a small-for-gestational-age infant and adverse perinatal outcome in women with suspected pre-eclampsia.

OBJECTIVE: To evaluate the test performance of 47 biomarkers and ultrasound parameters for the prediction of delivery of a small-for-gestational-age (SGA) infant and adverse perinatal outcome in women presenting with suspected pre-eclampsia. METHODS: This was a prospective, multicenter observational study in which 47 biomarkers and ultrasound parameters were measured in 397 women with a singleton pregnancy presenting with suspected preterm pre-eclampsia between 20 + 0 and 36 + 6 weeks' gestation, with the objective of evaluating them as predictors of subsequent delivery of a SGA infant and adverse perinatal outcome. Women with confirmed pre-eclampsia at enrollment were excluded. Factor analysis and stepwise logistic regression were performed in two prespecified groups stratified according to gestational age at enrollment. The primary outcome was delivery of a SGA infant with a birth weight < 3rd customized centile (SGA-3), and secondary outcomes were a SGA infant with a birth weight < 10th customized centile and adverse perinatal outcome. RESULTS: In 274 women presenting at 20 + 0 to 34 + 6 weeks' gestation, 96 (35%) delivered a SGA-3 infant. For prediction of SGA-3, low maternal placental growth factor (PlGF) concentration had a sensitivity of 93% (95% CI, 84-98%) and negative predictive value (NPV) of 90% (95% CI, 76-97%) compared with a sensitivity of 71% (95% CI, 58-82%) and a NPV of 79% (95% CI, 68-87%) for ultrasound parameters (estimated fetal weight or abdominal circumference < 10th centile). No individual biomarker evaluated had a better performance than did PlGF, and marker combinations made only small improvements to the test performance. Similar results were found in 123 women presenting between 35 + 0 and 36 + 6 weeks' gestation. CONCLUSION: In women presenting with suspected preterm pre-eclampsia, measurement of PlGF offers a useful adjunct for identifying those at high risk of delivering a SGA infant, allowing appropriate surveillance and timely intervention. © 2017 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

P2X receptors.

Extracellular adenosine 5'-triphosphate (ATP) activates cell surface P2X and P2Y receptors. P2X receptors are membrane ion channels preferably permeable to sodium, potassium and calcium that open within milliseconds of the binding of ATP. In molecular architecture, they form a unique structural family. The receptor is a trimer, the binding of ATP between subunits causes them to flex together within the ectodomain and separate in the membrane-spanning region so as to open a central channel. P2X receptors have a widespread tissue distribution. On some smooth muscle cells, P2X receptors mediate the fast excitatory junction potential that leads to depolarization and contraction. In the central nervous system, activation of P2X receptors allows calcium to enter neurons and this can evoke slower neuromodulatory responses such as the trafficking of receptors for the neurotransmitter glutamate. In primary afferent nerves, P2X receptors are critical for the initiation of action potentials when they respond to ATP released from sensory cells such as taste buds, chemoreceptors or urothelium. In immune cells, activation of P2X receptors triggers the release of pro-inflammatory cytokines such as interleukin 1ß. The development of selective blockers of different P2X receptors has led to clinical trials of their effectiveness in the management of cough, pain, inflammation and certain neurodegenerative diseases.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.

Socio-economic status influences the relationship between obesity and antenatal depression: Data from a prospective cohort study.

BACKGROUND: Obesity has been associated with increased risk of antenatal depression, but little is known about this relationship. This study tested whether socio-economic status (SES) influences the relationship between obesity and antenatal depression. METHODS: Data were taken from the Screening for Pregnancy Endpoints (SCOPE) cohort. BMI was calculated from measured height and weight at 15±1 weeks' gestation. Underweight women were excluded. SES was indicated by self-reported household income (dichotomised around the median: low SES ≤£45,000; high SES >£45,000). Antenatal depression was defined as scoring ≥13 on the Edinburgh Postnatal Depression Scale at both 15±1 and 20±1 weeks' gestation, to identify persistently elevated symptoms of depression. RESULTS: Five thousand five hundred and twenty two women were included in these analyses and 5.5% had persistently elevated antenatal depression symptoms. There was a significant interaction between SES and BMI on the risk of antenatal depression (p=0.042). Among high SES women, obese women had approximately double the odds of antenatal depression than normal weight controls (AOR 2.11, 95%CI 1.16-3.83, p=0.014, adjusted for confounders). Among low SES women there was no association between obesity and antenatal depression. The interaction effect was robust to alternative indicators of SES in sensitivity analyses. LIMITATIONS: 1) Antenatal depression was assessed with a self-reported screening measure; and 2) potential mediators such as stigma and poor body-image could not be examined. CONCLUSIONS: Obesity was only associated with increased risk of antenatal depression among high SES women in this sample. Healthcare professionals should be aware that antenatal depression is more common among low SES women, regardless of BMI category.

Previous pregnancy loss has an adverse impact on distress and behaviour in subsequent pregnancy.

OBJECTIVE: To investigate whether women with previous miscarriages or terminations have higher levels of anxiety, depression, stress, and altered behaviours in a subsequent pregnancy. DESIGN: A retrospective analysis of 5575 women recruited into the Screening for Pregnancy Endpoints (SCOPE) study, a prospective cohort study. SETTING: Auckland, New Zealand, Adelaide, Australia, Cork, Ireland, and Manchester, Leeds, and London, UK. POPULATION: Healthy nulliparous women with singleton pregnancies. METHODS: Outcomes were recorded at 15 and 20 weeks of gestation. MAIN OUTCOME MEASURES: Short-form State-Trait Anxiety Inventory (STAI) score, Perceived Stress Scale score, Edinburgh Postnatal Depression Scale score, and pregnancy-related behaviour measured using behavioural responses to pregnancy score. RESULTS: Of the 5465 women included in the final analysis, 559 (10%) had one and 94 (2%) had two previous miscarriages, and 415 (8%) had one and 66 (1%) had two previous terminations of pregnancy. Women with one previous miscarriage had increased anxiety (adjusted mean difference 1.85; 95% confidence interval, 95% CI 0.61-3.09), perceived stress (adjusted mean difference 0.76; 95% CI 0.48-1.03), depression (adjusted odds ratio, aOR 1.26; 95% CI 1.08-1.45), and limiting/resting behaviour in pregnancy (adjusted mean difference 0.80; 95% CI 0.62-0.97). In women with two miscarriages, depression was more common (aOR 1.65; 95% CI 1.01-2.70) and they had higher scores for limiting/resting behaviour in pregnancy (adjusted mean difference 1.70; 95% CI 0.90-2.53) at 15 weeks of gestation. Women with one previous termination displayed elevated perceived stress (adjusted mean difference 0.65; 95% CI 0.08-1.23) and depression (aOR 1.25; 95% 1.08-1.45) at 15 weeks of gestation. Women with two previous terminations displayed increased perceived stress (adjusted mean difference 1.43; 95% CI 0.00-2.87) and depression (aOR 1.67; 95% 1.28-2.18). CONCLUSIONS: This study highlights the psychological implications of miscarriage and termination of pregnancy.

Failed back surgery syndrome: who has failed?

INTRODUCTION: Failed back surgery syndrome (FBSS) results from a cascade of medical and surgical events that lead to or leave the patient with chronic back and radicular pain. This concept is extremely difficult to understand, both for the patient and for the therapist. The difficulty is related to the connotations of failure and blame directly associated with this term. The perception of the medical situation varies enormously according to the background and medical education of the clinician who manages this type of patient. Eight health system experts (2 pain physicians, 1 orthopaedic spine surgeon, 1 neuro spine surgeon, 1 functional neurosurgeon, 1 physiatrist, 1 psychologist and one health-economic expert) were asked to define and share their specialist point of view concerning the management of postoperative back and radicular pain. Ideally, it could be proposed that the patient would derive optimal benefit from systematic confrontation of these various points of view in order to propose the best treatment option at a given point in time to achieve the best possible care pathway. CONCLUSION: The initial pejorative connotation of FBSS suggesting failure or blame must now be replaced to direct the patient and therapists towards a temporal concept focusing on the future rather than the past. In addition to the redefinition of an optimised care pathway, a consensus based on consultation would allow redefinition and renaming of this syndrome in order to ensure a more positive approach centered on the patient.

Relationship between water quality parameters and bacterial indicators in a large prairie reservoir: Lake Diefenbaker, Saskatchewan, Canada.

Lake Diefenbaker (LD) is a large reservoir on the South Saskatchewan River used for agricultural irrigation, drinking water, and recreation. Our objectives were to determine the distribution and abundance of bacterial indicators in embayments and the main channel of LD and to relate these to environmental factors. Total coliforms (TCs), fecal coliforms (FCs), and fecal indicator bacteria (i.e., Escherichia coli) were measured concurrently with water quality parameters. Although TCs, FCs, and E. coli were present in LD, they rarely exceeded the TC and FC Canadian Council of Ministers of the Environment (CCME) water quality standards for agricultural use (1000 colony-forming units (CFU) per 100 mL and 100 CFU per 100 mL, respectively). The correlation between the bacterial indicators in the sediments and the water column indicates that higher embayment abundances may be related to sediment loading and (or) resuspension events in these frequently mixed embayments. With higher water temperatures and water levels, as well as higher microbial activity, CCME bacterial limits may be exceeded. The greatest contributor to bacterial indicator abundance was water temperature. We predict that water quality standards will be exceeded more frequently with climate warming.

Ectodomain movements of an ATP-gated ion channel (P2X2 receptor) probed by disulfide locking.

The ectodomain of the P2X receptor is formed mainly from two- or three-stranded ß-sheets provided symmetrically by each of the three subunits. These enclose a central cavity that is closed off furthest from the plasma membrane (the turret) and that joins with the transmembrane helices to form the ion permeation pathway. Comparison of closed and open crystal structures indicates that ATP binds in a pocket positioned between strands provided by different subunits and that this flexes the ß-sheets of the lower body and enlarges the central cavity: this pulls apart the outer ends of the transmembrane helices and thereby opens an aperture, or gate, where they intersect within the membrane bilayer. In the present work, we examined this opening model by introducing pairs of cysteines into the rat P2X2 receptor that might form disulfide bonds within or between subunits. Receptors were expressed in human embryonic kidney cells, and disulfide formation was assessed by observing the effect of dithiothreitol on currents evoked by ATP. Substitutions in the turret (P90C, P89C/S97C), body wall (S65C/S190C, S65C/D315C) and the transmembrane domains (V48C/I328C, V51C/I328C, S54C/I328C) strongly inhibited ATP-evoked currents prior to reduction with dithiothreitol. Western blotting showed that these channels also formed predominately as dimers and/or trimers rather than monomers. The results strongly support the channel opening mechanism proposed on the basis of available crystal structures.

Direct gating of ATP-activated ion channels (P2X2 receptors) by lipophilic attachment at the outer end of the second transmembrane domain.

The ionic pore of the P2X receptor passes through the central axis of six transmembrane (TM) helices, two from each of three subunits. Val(48) and Ile(328) are at the outer end of TM1 and TM2, respectively. Homology models of the open and closed states of P2X2 indicate that pore opening is associated with a large lateral displacement of Ile(328). In addition, molecular dynamics simulations suggest that lipids enter the interstices between the outer ends of the TM domains. The P2X2(I328C) receptor was activated by propyl-methanethiosulfonate (MTS) as effectively as by ATP, but cysteine substitutions elsewhere in TM2 had no such effect. Other lipophilic MTS compounds (methyl, ethyl, and tert-butylethyl) had a similar effect but not polar MTS. The properties of the conducting pathway opened by covalent attachment of propyl-MTS were the same as those opened by ATP, with respect to unitary conductance, rectification, and permeability of N-methyl-d-glucamine. The ATP-binding residue Lys(69) was not required for the action of propyl-MTS, although propyl-MTS did not open P2X2(K308A/I328C) receptors. The propyl-MTS did not open P2X2 receptors in which the Val(48) side chain was removed (P2X2(V48G/I328C)). The results suggest that an interaction between Val(48) and Ile(328) stabilizes the closed channel and that this is broken by covalent attachment of a larger lipophilic moiety at the I328C receptors. Lipid intercalation between the separating TM domains during channel opening would be facilitated in P2X2(I328C) receptors with attached propyl-MTS. The results are consistent with the channel opening mechanism proposed on the basis of closed and open crystal structures and permit the refinement of the position of the TMs within the bilayer.

Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel.

Rab GTPases play key roles in the delivery, docking and fusion of intracellular vesicles. However, the mechanism by which spatial and temporal regulation of Rab GTPase activity is controlled is poorly understood. Here we describe a mechanism by which localized calcium release through a vesicular ion channel controls Rab GTPase activity. We show that activation of P2XA, an intracellular ion channel localized to the Dictyostelium discoideum contractile vacuole system, results in calcium efflux required for downregulation of Rab11a activity and efficient vacuole fusion. Vacuole fusion and Rab11a downregulation require the activity of CnrF, an EF-hand-containing Rab GAP found in a complex with Rab11a and P2XA. CnrF Rab GAP activity for Rab11a is enhanced by the presence of calcium and the EF-hand domain. These findings suggest that P2XA activation results in vacuolar calcium release, which triggers activation of CnrF Rab GAP activity and subsequent downregulation of Rab11a to allow vacuole fusion.

Optical control of trimeric P2X receptors and acid-sensing ion channels.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

P2X receptor intermediate activation states have altered nucleotide selectivity.

Purinergic P2X receptors are widely distributed in the nervous system and are known to play roles in primary afferent transmission and central respiratory regulation. They are trimeric membrane proteins, with the extracellular domain that provides three intersubunit ATP binding sites. We expressed the rat P2X7 receptor in human embryonic kidney cells and measured membrane currents before and after photo-affinity labeling with the agonist 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP). After tethering BzATP with ultraviolet light, a persistent current remained after washing out the agonist. Additional current could now be elicited by other nucleotides (CTP and ADP) that are not normally effective as P2X receptor agonists. Similar results were obtained at P2X2 receptors even without previous agonist tethering: exposure to low concentrations of ATP caused the receptor to become sensitive to activation by CTP and ADP. The results show that ATP binding to the first of the three binding sites causes a conformational change to an intermediate closed state that shows increased effectiveness of pyrimidine and diphosphate nucleotide analogs.

Angiogenic factors combined with clinical risk factors to predict preterm pre-eclampsia in nulliparous women: a predictive test accuracy study.

OBJECTIVES: To assess the performance of clinical risk factors, uterine artery Doppler and angiogenic markers to predict preterm pre-eclampsia in nulliparous women. DESIGN: Predictive test accuracy study. SETTING: Prospective multicentre cohort study Screening for Pregnancy Endpoints (SCOPE). METHODS: Low-risk nulliparous women with a singleton pregnancy were recruited. Clinical risk factor data were obtained and plasma placental growth factor (PlGF), soluble endoglin and soluble fms-like tyrosine kinase-1 (sFlt-1) were measured at 14-16 weeks of gestation. Prediction models were developed using multivariable stepwise logistic regression. MAIN OUTCOME MEASURE: Preterm pre-eclampsia (delivered before 37(+0)  weeks of gestation). RESULTS: Of the 3529 women recruited, 187 (5.3%) developed pre-eclampsia of whom 47 (1.3%) delivered preterm. Controls (n = 188) were randomly selected from women without preterm pre-eclampsia and included women who developed other pregnancy complications. An area under a receiver operating characteristic curve (AUC) of 0.76 (95% CI 0.67-0.84) was observed using previously reported clinical risk variables. The AUC improved following the addition of PlGF measured at 14-16 weeks (0.84; 95% CI 0.77-0.91), but no further improvement was observed with the addition of uterine artery Doppler or the other angiogenic markers. A sensitivity of 45% (95% CI 0.31-0.59) (5% false-positive rate) and post-test probability of 11% (95% CI 9-13) were observed using clinical risk variables and PlGF measurement. CONCLUSIONS: Addition of plasma PlGF at 14-16 weeks of gestation to clinical risk assessment improved the identification of nulliparous women at increased risk of developing preterm pre-eclampsia, but the performance is not sufficient to warrant introduction as a clinical screening test. These findings are marker dependent, not assay dependent; additional markers are needed to achieve clinical utility.

Functional properties of five Dictyostelium discoideum P2X receptors.

The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. Extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.

P2X7 receptor channels allow direct permeation of nanometer-sized dyes.

P2X receptors are widely distributed in the nervous system, and P2X7 receptors have roles in neuropathic pain and in the release of cytokines from microglia. They are trimeric membrane proteins, which open an integral ion channel when ligated by extracellular ATP. This channel is preferentially permeable to small cations (sodium, potassium, calcium) but also allows permeation of larger cations such as N-methyl-d-glucamine. ATP also leads to entry of fluorescent dyes in many cells expressing P2X7 receptors, but controversy persists as to whether such large molecules pass directly through the open ion channel or enter the cell by a different route. We measured cellular fluorescence and membrane currents in individual human embryonic kidney cells expressing rat P2X7 receptors. Introduction of positive side chains by mutagenesis into the inner half of the pore-forming second transmembrane domain of the receptor (T348K, D352N, D352K) increased relative permeability of chloride ions. It also promoted entry of the large (>1 nm) negative dye fluorescein-5-isothiocyanate while decreasing entry of the structurally similar but positive dye ethidium. Furthermore, larger cysteine-reactive methanethiosulfonates [sulforhodamine-methanethiosulfonate and 2-((biotinoyl)amino)ethyl methanethiosulfonate] reduced both ATP-evoked currents and dye entry when applied to open P2X7[G345C] receptors. The results demonstrate that the open channel of the P2X7 receptor can allow passage of molecules with sizes up to 1.4 nm.