Routine germline BRCA1 and BRCA2 testing in patients with ovarian carcinoma: analysis of the Scottish real-life experience.

OBJECTIVE: To determine the rates of germline BRCA1 and BRCA2 mutations in Scottish patients with ovarian cancer, before and after a change in testing policy. DESIGN: Retrospective cohort study. SETTING: Four cancer/genetics centres in Scotland. POPULATION: Patients with ovarian cancer undergoing germline BRCA1 and BRCA2 (gBRCA1/2) sequencing before 2013 (under the 'old criteria', with selection based solely on family history), after 2013 (under the 'new criteria', with sequencing offered to newly presenting patients with non-mucinous ovarian cancer), and in the 'prevalent population' (who presented before 2013, but were not eligible for sequencing under the old criteria but were sequenced under the new criteria). METHODS: Clinicopathological and sequence data were collected before and for 18 months after this change in selection criteria. MAIN OUTCOME MEASURES: Frequency of germline BRCA1, BRCA2, RAD51C, and RAD51D mutations. RESULTS: Of 599 patients sequenced, 205, 236, and 158 were in the 'old criteria', 'new criteria', and 'prevalent' populations, respectively. The frequency of gBRCA1/2 mutations was 30.7, 13.1, and 12.7%, respectively. The annual rate of gBRCA1/2 mutation detection was 4.2 before and 20.7 after the policy change. A total of 48% (15/31) 'new criteria' patients with gBRCA1/2 mutations had a Manchester score of <15 and would not have been offered sequencing based on family history criteria. In addition, 20 patients with gBRCA1/2 were identified in the prevalent population. The prevalence of gBRCA1/2 mutations in patients aged >70 years was 8.2%. CONCLUSIONS: Sequencing all patients with non-mucinous ovarian cancer gives a much higher annual gBRCA1/2 mutation detection rate, with the frequency of positive tests still exceeding the 10% threshold upon which many family history-based models operate. TWEETABLE ABSTRACT: BRCA sequencing all non-mucinous cancer patients increases mutation detection five fold.

[Safety and efficacy of Cox-maze â ¢ procedure for patients with atrial fibrillation associated with rheumatic mitral valve disease].

OBJECTIVE: To evaluate the safety and efficacy of cut-and-sew Cox-Maze (CM) Ⅲ procedure for patients with atrial fibrillation associated with rheumatic mitral valve disease. METHODS: Patients received mitral valve replacement and CM procedure between January 2007 and July 2015, were 1∶1 matched and assigned to CM Ⅳ group (undergoing surgical ablation) and CM Ⅲ group (undergoing cut-and-sew CM Ⅲ). Safety indices, mortality rate, complications and recovery rate of sinus rhythm were compared between the two groups. RESULTS: Mean extracorporeal circulation time was (154±22) min in the CM Ⅲ group and (136±21) min in the CM Ⅳ group (P<0.001), and aortic clamp time was (85±9) min in the CM Ⅲ group and (74±12) min in the CM Ⅳ group (P<0.001). One patient (1.18%) in the CM Ⅲ group died of multi-organ failure 4 day after left ventricular rupture being sucessfully repaired, and 2 patients died (2.35%) in the CM Ⅳ group ( 1 due to left ventricular rupture and 1 due to low cardiac output syndrome). There was no significant difference in mortality between the two groups. Temporary pacemaker were applied for 76 cases (89.41%) in the CM Ⅲ and 70 cases (82.35%) in the CM Ⅳ group, without a statistical difference (P=0.186). There were also no statistical difference between the two groups in intraoperative urinary volume, postoperative drainage of fluid volume, hemoglobinuria, ventilator time, ICU time, hospital stay and the incidence of the complications (all P>0.05). Sinus rhythm recovery rate was 62.35 % in the CM Ⅲ group and 57.65 % in the CM Ⅳ group, without a statistical difference (P=0.531) at the operation day, but at discharge a statistical difference was found (97.62% in the CM Ⅲ group vs 81.93% in the CM Ⅳ group, P<0.001). All patients were followed up for three months, and no deaths occurred. Sinus rhythm recovery rate was 96.43 % in the CM Ⅲ group and 86.75% in the CM Ⅳ group, with a statistical difference (P=0.024). CONCLUSION: The cut-and-sew Cox-Maze Ⅲ procedure is a safe and effective method for patients with atrial fibrillation associated with rheumatic mitral valve disease.

Clinical and immunochemical profiles of food challenge proven or anaphylactic shrimp allergy in tropical Singapore.

BACKGROUND: Shellfish allergy in Singapore is highly prevalent, and shrimp allergy is the most common. OBJECTIVE: This study aims to evaluate the clinical characteristics and immunological phenotype of shellfish allergy in this population. METHODS: Patients with self-reported shellfish allergy were recruited from outpatient clinics of three large hospitals and from a population survey. Open oral food challenges (OFC) to glass prawn (Litopenaeus vannamei) and tiger prawn (Penaeus monodon) were carried out on all patients except for those who had a history of severe anaphylaxis. Skin prick tests (SPT) and specific IgE to crude and recombinant allergens were carried out to evaluate shrimp and dust mite sensitization. Immunoblots were used to assess IgE-binding proteins. RESULTS: The 104 patients recruited were categorized into shellfish allergic (SA) when OFC was positive or had a history of severe anaphylaxis (n = 39), shellfish tolerant (ST) when OFC was negative (n = 27), and house dust mite positive controls (HDM(+) ) who were ST (n = 38). Oral symptoms (87.1%) were the predominant clinical manifestation. Positive challenge doses ranged from 2 to 80 g of cooked shrimp, with 25/52 patients reacting to either one or both shrimps challenged. The presence of specific IgE to shrimp either by SPT and/or ImmunoCAP(®) assay provided diagnostic test sensitivity of 82% and specificity of 22.2%. The inclusion of specific IgE to shrimp tropomyosin and IgE immunoblots with shrimp extracts did not improve the diagnostic proficiency substantially. CONCLUSIONS AND CLINICAL RELEVANCE: This study highlights the predominance of oral symptoms in shrimp allergy in tropical Asia and that a high provocation dose may be necessary to reveal shrimp allergy. Furthermore, specific IgE diagnostic tests and immunoblots were of limited use in this population.

An energy adjustable linearly polarized passively Q-switched bulk laser with a wedged diffusion-bonded Nd:YAG/Cr4⁺:YAG crystal.

An energy adjustable passively Q-switched laser is demonstrated with a composite Nd:YAG/Cr4⁺:YAG crystal by applying a wedged interface inside the crystal. The theoretical model of the monolithic laser resonator is explored to show the energy adjustable feature with different initial transmissions of the saturable absorber at the horizontal axis. By adjusting the pump beam location across the Nd:YAG crystal, the output pulse energy can be flexibly changed from 10.9 µJ to 17.6 µJ while maintaining the same output efficiency. The polarization state of the laser output is found to be along with the polarization of the C-mount pump diode. Finally, the behavior of the multi-transverse-mode oscillation is also discussed for eliminating the instability of the pulse train.

First Report of Acrostalagmus luteo-albus Causing Red Rust of Needle Mushroom (Flammulina velutipes) in China.

Needle mushroom (Flammulina velutipes) is an edible and medicinal mushroom with annual production yields for China exceeding 2 million metric tons. Hebei province is one of the biggest producers of needle mushrooms in China. From 2009 to 2010, red rust-colored sporulation (fungal mycelia and conidia) was found on the substrate surface of white needle mushroom bags in more than 10 mushroom cultivation operations in Hebei. The rust-colored sporulation covered the substrate surface, where the development of the fruiting bodies was inhibited; the stipes were sparse and became light brown when the substrate was slightly affected. The fruiting bodies on severely affected substrate were unable to complete development or shriveled and died off on the substrate. More than 30 to 40% substrate bags were contaminated with the red rust mold, with approximately a 40% yield reduction in each production house. Single conidia were isolated from the red rust mold and cultured on potato dextrose agar at 25°C in the dark. The colony was round, compact, reddish-brown, and slow-growing (2 to 3 mm/d). Main conidiophore axes were erect, often branched in five to seven layers, tapered from the base to the upper part, pale reddish brown at the base, and almost hyaline at the apex. Two to five phialides usually verticillated at each level along the main stipe and its branches. Phialides were narrowly flask-shaped and only very slightly swollen at the base. Conidia were oval, measured 3.5 to 5.4 (4.5) × 2.3 to 3.6 (3.0) µm, L/W = 1.2 to 2.0 (1.5), and formed pale reddish brown, slimy heads. The isolate was tentatively identified as Acrostalagmus luteo-albus based on its morphological characteristics (2). For molecular analysis, the internal transcribed spacer (ITS) regions of ribosomal DNA from the isolate were PCR-amplified using universal primers ITS1 and ITS4. The resulting sequence (Accession No. KC127681) submitted to GenBank had a 99% identity to that of A. luteo-albus (JN545827) isolated from vineyard soil and Accession No. JQ387575 isolated from twig of declining persimmon tree. To confirm the pathogenicity of the isolate to needle mushroom, five bags of needle mushroom with stipes of 1 to 2 cm long were inoculated with a conidia suspension (105 conidia/ml) of the isolate and incubated at 13 to 15°C in the dark, while five non-inoculated bags (sprayed with sterile water only) were used as a control. Five days after inoculation, the development of the fruiting body was obviously inhibited, and a few growing fruiting bodies became light brown, compared with the non-inoculated fruiting bodies, which were growing healthily and fast and were white with no symptoms. The pathogen was re-isolated from the inoculated fruiting bodies. A. luteo-albus is primarily saprophytic but is sometimes pathogenic. Indeed, it has been associated with post-harvest rot of ginger rhizomes in Brazil (1). To our knowledge, this is the first report of A. luteo-albus causing damage to needle mushroom in China. References: (1) S. I. Moreira et al. Trop. Plant Pathol. 38:218, 2013. (2) R. Zare et al. Mycol. Res. 108:576, 2004.

Efficient high-power terahertz beating in a dual-wavelength synchronously mode-locked laser with dual gain media.

We originally present a novel tactic to accomplish a compact efficient dual-wavelength synchronously mode-locked laser by physically combining the Nd:YVO4 crystal to the Nd:GdVO4 crystal as a composite gain medium. With the developed method, the total output power at 1.06 µm could be effectually produced to reach 1.3 W under the optimally balanced two-color intensities. The corresponding mode-locked pulse width and repetition rate are measured to be 47 ps and 2.86 GHz, respectively. Through the optical beating between two carrier frequencies of dual-color synchronous pulses, a train of 0.32 THz ultrashort pulses is further generated with the effective duration of down to 1.6 ps.

High-power tunable single- and multi-wavelength diode-pumped Nd:YAP laser in the (4)F3/2 → (4)I11/2 transition.

We experimentally explore the fluorescent spectrum of the Nd:YAP crystal to manifest the feasibility of tunable single- and multi-wavelength operations in the (4)F3/2 → (4)I11/2 transition. An intracavity etalon is subsequently exploited to effectively select spectral lines at 1073, 1080, and 1084 nm with the tunabilities of 0.56, 1.13, and 0.1 nm, respectively. We also experimentally obtain multi-wavelength oscillations among various intermanifold lines in the Nd:YAP crystal with the output powers on the order of several watts for each group. Employing the Cr(4+):YAG crystal to realize the passively Q-switched operation, the maximum average output powers as high as 2.3 and 3.5 W for 1073 and 1080 nm are obtained. The corresponding pulse energies at 1073 and 1080 nm are up to 177 and 159 µJ, respectively.

Efficient high-energy passively Q-switched Nd:YLF/Cr4+:YAG UV laser at 351 nm with pulsed pumping in a nearly hemispherical cavity.

We originally utilize a nearly hemispherical cavity to accomplish the energy scale-up for a high-repetition-rate nanosecond pulsed pumped Nd:YLF laser passively Q-switched by the Cr(4+):YAG saturable absorber. This compact laser is able to efficiently generate pulse energy as large as 1.38 mJ and pulse width as short as 5 ns under a pulse repetition rate of 100 Hz. Further employing the developed Nd:YLF laser to perform extracavity harmonic generations, the maximum pulse energies of 490 µJ at 527 nm and 360 µJ at 351 nm are achieved with the shortest pulse duration of 4 ns.

Tunable GHz pulse repetition rate operation in high-power TEM(00)-mode Nd:YLF lasers at 1047 nm and 1053 nm with self mode locking.

We report on a high-power diode-pumped self-mode-locked Nd:YLF laser with the pulse repetition rate up to several GHz. A novel tactic is developed to efficiently select the output polarization state for achieving the stable TEM(00)-mode self-mode-locked operations at 1053 nm and 1047 nm, respectively. At an incident pump power of 6.93 W and a pulse repetition rate of 2.717 GHz, output powers as high as 2.15 W and 1.35 W are generated for the σ- and π-polarization, respectively. We experimentally find that decreasing the separation between the gain medium and the input mirror not only brings in the pulse shortening thanks to the enhanced effect of the spatial hole burning, but also effectively introduces the effect of the spectral filtering to lead the Nd:YLF laser to be in a second harmonic mode-locked status. Consequently, pulse durations as short as 8 ps and 8.5 ps are obtained at 1053 nm and 1047 nm with a pulse repetition rate of 5.434 GHz.

Alterations of white matter tracts following neurotoxic hippocampal lesions in macaque monkeys: a diffusion tensor imaging study.

Diffusion tensor imaging (DTI) is a valuable tool for assessing presumptive white matter alterations in human disease and animal models. The current study used DTI to examine the effects of selective neurotoxic lesions of the hippocampus on major white matter tracts and anatomically related brain regions in macaque monkeys. Two years postlesion, structural MRI, and DTI sequences were acquired for each subject. Volumetric assessment revealed a substantial reduction in the size of the hippocampus in experimental subjects, averaging 72% relative to controls, without apparent damage to adjacent regions. DTI images were processed to yield measures of fractional anisotropy (FA), apparent diffusion coefficient (ADC), parallel diffusivity (lADC), and perpendicular diffusivity (tADC), as well as directional color maps. To evaluate potential changes in major projection systems, a region of interest (ROI) analysis was conducted including the corpus callosum, fornix, temporal stem, cingulum bundle, ventromedial prefrontal white matter, and optic radiations. Lesion-related abnormalities in the integrity of the fiber tracts examined were limited to known hippocampal circuitry, including the fornix and ventromedial prefrontal white matter. These findings are consistent with the notion that hippocampal damage results in altered interactions with multiple memory-related brain regions, including portions of the prefrontal cortex.

Water transport properties of boron nitride nanosheets mixed matrix membranes for humic acid removal.

Ultrafiltration grade polysulfone-based mixed matrix membranes (MMMs) incorporated with two-dimensional boron nitride nanosheet (BNNS) was prepared via phase inversion method. The amount of BN incorporated was varied and the influence on membrane morphology, contact angle, surface charge, as well as water permeability and humic acid rejection were investigated. Results revealed that the addition of BN to the membrane matrix resulted in profound increase in water permeability (almost tripled to that of neat PSf) and humic acid rejection due to the increase in pore size and surface negative charge. Beyond the morphological changes imparted by the inclusion of BNNS, we postulated that the presence of BNNS within the membrane matrix also contribute to the enhancement in flux and rejection based on surface-slip and selective interlayer transport. Despite the favourable augmentation of water transport and filtration performance, the MMMs suffered with fouling problem due to the entrapment of foulant within the enlarged pores and the membrane valleys. Its inherent adsorptive character could be a disadvantage when utilized as membrane filler.

NLRP3 inflammasome pathway has a critical role in the host immunity against clinically relevant Acinetobacter baumannii pulmonary infection.

The opportunistic Gram-negative bacterium Acinetobacter baumannii (AB) is a leading cause of life-threatening nosocomial pneumonia. Outbreaks of multidrug resistant (MDR)-AB belonging to international clones (ICs) I and II with limited treatment options are major global health threats. However, the pathogenesis mechanisms of various AB clonal groups are understudied. Although inflammation-associated interleukin-1ß (IL-1ß) levels and IL-1 receptor antagonist polymorphisms were previously implicated in MDR-AB-related pneumonia in patients, whether inflammasomes has any role in the host defense and/or pathogenesis of clinically relevant A. baumannii infection is unknown. Using a sublethal mouse pneumonia model, we demonstrate that an extensively drug-resistant clinical isolate (ICII) of A. baumannii exhibits reduced/delayed early pulmonary neutrophil recruitment, higher lung persistence, and, most importantly, elicits enhanced IL-1ß/IL-18 production and lung damage through NLRP3 inflammasome, in comparison with A. baumannii-type strain. A. baumannii infection-induced IL-1ß/IL-18 production is entirely dependent on NLRP3-ASC-caspase-1/caspase-11 pathway. Using Nlrp3-/- mice infection models, we further show that while NLRP3 inflammasome pathway contributes to host defense against A. baumannii clinical isolate, it is dispensable for protection against A. baumannii-type strain. Our study reveals a novel differential role for NLRP3 inflammasome pathway in the immunity against clinically relevant A. baumannii infections, and highlights inflammasome pathway as a potential immunomodulatory target.

Involvement of LeMRP, an ATP-binding cassette transporter, in shikonin transport and biosynthesis in Lithospermum erythrorhizon.

Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown. In this study, we isolated a cDNA encoding LeMRP, an ATP-binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real-time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes. Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real-time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up-regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down-regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots. Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin.

Image deconvolution in spherical aberration-corrected high-resolution transmission electron microscopy.

The method of image deconvolution developed previously for FEG high-resolution transmission electron microscope (HRTEM) without a spherical aberration (C(s)) corrector was for the first time applied to FEG HRTEM with a C(s)-corrector. The principle and the procedure of image deconvolution are briefly described. Four qualified [1 1 0] images of Si were selected from a through-focus series to perform image deconvolution. The projected potential is successfully derived from all the images, and the obtained "dumbbell" structure maps of Si [1 1 0] are in good agreement with the calculated potential map. The criterion of selecting qualified images for performing image deconvolution is indicated. The possibility of applying image deconvolution to defect study and to ab initio crystal structure determination is discussed.

Cerebral [18 F]T807/AV1451 retention pattern in clinically probable CTE resembles pathognomonic distribution of CTE tauopathy.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disorder most commonly associated with repetitive traumatic brain injury (TBI) and characterized by the presence of neurofibrillary tangles of tau protein, known as a tauopathy. Currently, the diagnosis of CTE can only be definitively established postmortem. However, a new positron emission tomography (PET) ligand, [18F]T807/AV1451, may provide the antemortem detection of tau aggregates, and thus various tauopathies, including CTE. Our goal was to examine [18F]T807/AV1451 retention in athletes with neuropsychiatric symptoms associated with a history of multiple concussions. Here we report a 39-year-old retired National Football League player who suffered 22 concussions and manifested progressive neuropsychiatric symptoms. Emotional lability and irritability were the chief complaints. Serial neuropsychological exams revealed a decline in executive functioning, processing speed and fine motor skills. Naming was below average but other cognitive functions were preserved. Structural analysis of longitudinally acquired magenetic resonance imaging scans revealed cortical thinning in the left frontal and lateral temporal areas, as well as volume loss in the basal ganglia. PET with [18F]florbetapir was negative for amyloidosis. The [18F]T807/AV1451 PET showed multifocal areas of retention at the cortical gray matter-white matter junction, a distribution considered pathognomonic for CTE. [18F]T807/AV1451 standard uptake value (SUV) analysis showed increased uptake (SUVr⩾1.1) in bilateral cingulate, occipital, and orbitofrontal cortices, and several temporal areas. Although definitive identification of the neuropathological underpinnings basis for [18F]T807/AV1451 retention requires postmortem correlation, our data suggest that [18F]T807/AV1451 tauopathy imaging may be a promising tool to detect and diagnose CTE-related tauopathy in living subjects.

Transfer of voltage independence from a rat olfactory channel to the Drosophila ether-à-go-go K+ channel.

The S4 segment is an important part of the voltage sensor in voltage-gated ion channels. Cyclic nucleotide-gated channels, which are members of the superfamily of voltage-gated channels, have little inherent sensitivity to voltage despite the presence of an S4 segment. We made chimeras between a voltage-independent rat olfactory channel (rolf) and the voltage-dependent ether-à-go-go K+ channel (eag) to determine the basis of their divergent gating properties. We found that the rolf S4 segment can support a voltage-dependent mechanism of activation in eag, suggesting that rolf has a potentially functional voltage sensor that is silent during gating. In addition, we found that the S3-S4 loop of rolf increases the relative stability of the open conformation of eag, effectively converting eag into a voltage-independent channel. A single charged residue in the loop makes a significant contribution to the relative stabilization of the open stage in eag. Our data suggest that cyclic nucleotide-gated channels such as rolf contain a voltage sensor which, in the physiological voltage range, is stabilized in an activated conformation that is permissive for pore opening.

Extracellular Mg(2+) modulates slow gating transitions and the opening of Drosophila ether-à-Go-Go potassium channels.

We have characterized the effects of prepulse hyperpolarization and extracellular Mg(2+) on the ionic and gating currents of the Drosophila ether-à-go-go K(+) channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg(2+) dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg(2+) modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg(2+) slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg(2+) modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg(2+). We have also identified mutations in the S3-S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg(2+), indicating an important role for this region in the voltage-dependent activation of eag.

Mg(2+) modulates voltage-dependent activation in ether-à-go-go potassium channels by binding between transmembrane segments S2 and S3.

Extracellular Mg(2+) directly modulates voltage-dependent activation in ether-à-go-go (eag) potassium channels, slowing the kinetics of ionic and gating currents (Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 2000. J. Gen. Physiol. 115:319-337). To exert its effect, Mg(2+) presumably binds to a site in or near the eag voltage sensor. We have tested the hypothesis that acidic residues unique to eag family members, located in transmembrane segments S2 and S3, contribute to the Mg(2+)-binding site. Two eag-specific acidic residues and three acidic residues found in the S2 and S3 segments of all voltage-dependent K(+) channels were individually mutated in Drosophila eag, mutant channels were expressed in Xenopus oocytes, and the effect of Mg(2+) on ionic current kinetics was measured using a two electrode voltage clamp. Neutralization of eag-specific residues D278 in S2 and D327 in S3 eliminated Mg(2+)-sensitivity and mimicked the slowing of activation kinetics caused by Mg(2+) binding to the wild-type channel. These results suggest that Mg(2+) modulates activation kinetics in wild-type eag by screening the negatively charged side chains of D278 and D327. Therefore, these residues are likely to coordinate the bound ion. In contrast, neutralization of the widely conserved residues D284 in S2 and D319 in S3 preserved the fast kinetics seen in wild-type eag in the absence of Mg(2+), indicating that D284 and D319 do not mediate the slowing of activation caused by Mg(2+) binding. Mutations at D284 affected the eag gating pathway, shifting the voltage dependence of Mg(2+)-sensitive, rate limiting transitions in the hyperpolarized direction. Another widely conserved residue, D274 in S2, is not required for Mg(2+) sensitivity but is in the vicinity of the binding site. We conclude that Mg(2+) binds in a water-filled pocket between S2 and S3 and thereby modulates voltage-dependent gating. The identification of this site constrains the packing of transmembrane segments in the voltage sensor of K(+) channels, and suggests a molecular mechanism by which extracellular cations modulate eag activation kinetics.