Molecular mechanism for 3:1 subunit stoichiometry of rod cyclic nucleotide-gated ion channels

Molecular determinants of ion channel tetramerization are well characterized, but those involved in heteromeric channel assembly are less clearly understood. The heteromeric composition of native channels is often precisely controlled. Cyclic nucleotide-gated (CNG) channels from rod photoreceptors exhibit a 3:1 stoichiometry of CNGA1 and CNGB1 subunits that tunes the channels for their specialized role in phototransduction. Here we show, using electrophysiology, fluorescence, biochemistry, and X-ray crystallography, that the mechanism for this controlled assembly is the formation of a parallel 3-helix coiled-coil domain of the carboxy-terminal leucine zipper region of CNGA1 subunits, constraining the channel to contain three CNGA1 subunits, followed by preferential incorporation of a single CNGB1 subunit. Deletion of the carboxy-terminal leucine zipper domain relaxed the constraint and permitted multiple CNGB1 subunits in the channel. The X-ray crystal structures of the parallel 3-helix coiled-coil domains of CNGA1 and CNGA3 subunits were similar, suggesting that a similar mechanism controls the stoichiometry of cone CNG channels

Contribution of the cyclic nucleotide gated channel subunit, CNG-3, to olfactory plasticity in Caenorhabditis elegans.

In Caenorhabditis elegans, the AWC neurons are thought to deploy a cGMP signaling cascade in the detection of and response to AWC sensed odors. Prolonged exposure to an AWC sensed odor in the absence of food leads to reversible decreases in the animal's attraction to that odor. This adaptation exhibits two stages referred to as short-term and long-term adaptation. Previously, the protein kinase G (PKG), EGL-4/PKG-1, was shown necessary for both stages of adaptation and phosphorylation of its target, the beta-type cyclic nucleotide gated (CNG) channel subunit, TAX-2, was implicated in the short term stage. Here we uncover a novel role for the CNG channel subunit, CNG-3, in short term adaptation. We demonstrate that CNG-3 is required in the AWC for adaptation to short (thirty minute) exposures of odor, and contains a candidate PKG phosphorylation site required to tune odor sensitivity. We also provide in vivo data suggesting that CNG-3 forms a complex with both TAX-2 and TAX-4 CNG channel subunits in AWC. Finally, we examine the physiology of different CNG channel subunit combinations

Trafficking-Deficient G572R-hERG and E637K-hERG Activate Stress and Clearance Pathways in Endoplasmic Reticulum

Background: Long QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human ether-a-go-go-related gene (hERG) proteins are often involved in LQT2. Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins (Calnexin and Calreticulin) in the processing of G572R-hERG and E637K-hERG mutant proteins. Methods: pcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined. Conclusion: Our results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin wa

How does gender influence the recognition of cardiovascular risk and adherence to self-care recommendations? : a study in polish primary care

Background: Studies have shown a correlation between gender and an ability to change lifestyle to reduce the risk of disease. However, the results of these studies are ambiguous, especially where a healthy lifestyle is concerned. Additionally, health behaviors are strongly modified by culture and the environment. Psychological factors also substantially affect engagement with disease-related lifestyle interventions. This study aimed to examine whether there are differences between men and women in the frequency of health care behavior for the purpose of reducing cardiovascular risk (CVR), as well as cognitive appraisal of this type of risk. We also aimed to identify the psychological predictors of engaging in recommended behavior for reducing the risk of cardiovascular disease after providing information about this risk in men and women. Methods: A total of 134 consecutive eligible patients in a family practice entered a longitudinal study. At initial consultation, the individual’s CVR and associated health burden was examined, and preventive measures were recommended by the physician. Self-care behavior, cognitive appraisal of risk, and coping styles were then assessed using psychological questionnaires. Six months after the initial data collection, the frequency of subjects’ self-care behavior was examined. Results: We found an increase in health care behavior after providing information regarding the rate of CVR in both sexes; this increase was greater for women than for men. Women followed self-care guidelines more often than men, particularly for preventive measures and dietary advice. Women were more inclined to recognize their CVR as a challenge. Coping style, cognitive appraisal, age, level of health behaviors at baseline and CVR values accounted for 48% of the variance in adherence to self-care guidelines in women and it was 52% in men. In women, total risk of CVD values were most important, while in men, cognitive appraisal of harm/loss was most important. Conclusions: Different predictors of acquisition of health behavior are encountered in men and women. Our results suggest that gender-adjusted motivation models influencing the recognition process need to be considered to optimize compliance in patients with CVR

Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel

Inherited human long-QT2 syndrome (LQTS) results from mutations in the gene encoding the HERG channel. Several LQT2-associated mutations have been mapped to the amino terminal cytoplasmic Per-Arnt-Sim (PAS) domain of the HERG1a channel subunit. Here we have characterized the trafficking properties of some LQT2-associated PAS domain mutants and analyzed rescue of the trafficking mutants by low temperature (27°C) or by the pore blocker drug E4031. We show that the LQT2-associated mutations in the PAS domain of the HERG channel display molecular properties that are distinct from the properties of LQT2-associated mutations in the trans-membrane region. Unlike the latter, many of the tested PAS domain LQT2-associated mutations do not result in trafficking deficiency of the channel. Moreover, the majority of the PAS domain mutations that cause trafficking deficiencies are not rescued by a pore blocking drug. We have also explored the in vitro folding stability properties of isolated mutant PAS domain proteins using a thermal unfolding fluorescence assay and a chemical unfolding assay

The S4–S5 Linker Acts as a Signal Integrator for hERG K+ Channel Activation and Deactivation Gating

Human ether-à-go-go-related gene (hERG) K+ channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4–S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4–S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4–S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4–S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4–S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4–S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel

The N–Terminal Tail of hERG Contains an Amphipathic α–Helix That Regulates Channel Deactivation

The cytoplasmic N–terminal domain of the human ether–a–go–go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N–terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N–terminal 135 residues of hERG contains a previously described Per–Arnt–Sim (PAS) domain (residues 26–135) as well as an amphipathic α–helix (residues 13–23) and an initial unstructured segment (residues 2–9). Deletion of residues 2–25, only the unstructured segment (residues 2–9) or replacement of the α–helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α–helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N–terminal α–helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel

Performance in Physical Education and Health Impairment 30 Years Later—A Community Based Cohort Study

Objective: A main purpose of physical education (PE) in school is to promote future health. However, there is very limited evidence of the effects of PE on the adult health. We hypothesized that a low performance in PE was associated with an increased risk of health impairment by middle age. Methods: We performed a cohort study in a community-based setting in Sweden spanning over three decades. We followed up on 1712 of 2225 students (76.9%) who in 1974–1976 graduated with a grade in PE after 9 years of education (mean subject age 16 years). The grade in PE (compulsory subject) was retrieved from municipal archives. We defined three proxies for health impairment: total number of visits to primary care physicians in 2003–2007, having been hospitalized 2003–2007, and total number of days with sick leave in 2004–2007. Using binomial regression models, we adjusted the risk estimates for level of education and occupation. Subjects with an average grade in PE served as reference category. Results: In both the crude and adjusted model, women with a low grade in PE had more physician visits (adjusted IRR 1.30, 95 % confidence interval 1.06–1.60) and an increased number of days with sick leave (adjusted IRR 1.44, 1.05–1.95). An increased, although not significant, risk was also observed for having received in-patient care (adjusted RR 1.26; 0.88–1.80). No significant results or similar pattern were observed in men. Conclusion: Women with a low grade in PE in adolescence seem to have an increased risk of health impairment by middl

Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy

BACKGROUND: Ca(2+)-mediated regulation of ion channels provides a link between intracellular signaling pathways and membrane electrical activity. Intracellular Ca(2+) inhibits the voltage-gated potassium channel EAG1 through the direct binding of calmodulin (CaM). Three CaM binding sites (BD-C1: 674-683, BD-C2: 711-721, BD-N: 151-165) have been identified in a peptide screen and were proposed to mediate binding. The participation of the three sites in CaM binding to the native channel, however, remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we studied the binding of Ca(2+)/CaM to the EAG channel by visualizing the interaction between YFP-labeled CaM and Cerulean-labeled hEAG1 in mammalian cells by FRET. The results of our cellular approach substantiate that two CaM binding sites are predominantly involved; the high-affinity 1-8-14 based CaM binding domain in the N-terminus and the second C-terminal binding domain BD-C2. Mutations at these sites completely abolished CaM binding to hEAG1. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the BD-N and BD-C2 binding domains are sufficient for CaM binding to the native channel, and, therefore, that BD-C1 is unable to bind CaM independently

Effects of social approval bias on self-reported fruit and vegetable consumption: a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Self-reports of dietary intake in the context of nutrition intervention research can be biased by the tendency of respondents to answer consistent with expected norms (social approval bias). The objective of this study was to assess the potential influence of social approval bias on self-reports of fruit and vegetable intake obtained using both food frequency questionnaire (FFQ) and 24-hour recall methods.</p> <p>Methods</p> <p>A randomized blinded trial compared reported fruit and vegetable intake among subjects exposed to a potentially biasing prompt to that from control subjects. Subjects included 163 women residing in Colorado between 35 and 65 years of age who were randomly selected and recruited by telephone to complete what they were told would be a future telephone survey about health. Randomly half of the subjects then received a letter prior to the interview describing this as a study of fruit and vegetable intake. The letter included a brief statement of the benefits of fruits and vegetables, a 5-A-Day sticker, and a 5-a-Day refrigerator magnet. The remainder received the same letter, but describing the study purpose only as a more general nutrition survey, with neither the fruit and vegetable message nor the 5-A-Day materials. Subjects were then interviewed on the telephone within 10 days following the letters using an eight-item FFQ and a limited 24-hour recall to estimate fruit and vegetable intake. All interviewers were blinded to the treatment condition.</p> <p>Results</p> <p>By the FFQ method, subjects who viewed the potentially biasing prompts reported consuming more fruits and vegetables than did control subjects (5.2 vs. 3.7 servings per day, p < 0.001). By the 24-hour recall method, 61% of the intervention group but only 32% of the control reported eating fruits and vegetables on 3 or more occasions the prior day (p = 0.002). These associations were independent of age, race/ethnicity, education level, self-perceived health status, and time since last medical check-up.</p> <p>Conclusion</p> <p>Self-reports of fruit and vegetable intake using either a food frequency questionnaire or a limited 24-hour recall are both susceptible to substantial social approval bias. Valid assessments of intervention effects in nutritional intervention trials may require objective measures of dietary change.</p