Perception on Anxiety among Adolescents and Mental Health Professionals and Development of Comic Strip.

Background: Adolescence is a phase of many physical and psychological developments. This stage is also vulnerable to the development of mental illness. Anxiety is one of the most prevalent disorders among adolescents, which mostly onsets before puberty. This study aimed to understand the perception on anxiety among adolescents and mental health professionals and develop a comic strip on anxiety. Methods: This qualitative study involved focus group discussions (FGDs) among mental health professionals and pre-university college students. The FGD data were analyzed through direct content analysis according to the predetermined broad themes. Results: Awareness regarding anxiety was poor among adolescents, and in certain situations they were found to be reluctant to seek professional help. The comic strip on anxiety was developed based on the findings of FGD based on the themes, general awareness, factors responsible, symptoms and coping, management, and barriers to seeking professional help. Conclusions: Adolescents perceived mental health as very important for their overall health. The adolescents did not know that they were going through anxiety, but they noticed that they had some mental health issues and wanted to get out of it. The development of comic strip on anxiety for adolescents was an attempt to overcome these barriers.

Lipoprotein Insulin Resistance Index: A Simple, Accurate Method for Assessing Insulin Resistance in South Asians.

Context: Identification of insulin resistance (IR) in South Asians, who are at a higher risk for type 2 diabetes, is important. Lack of standardization of insulin assays limits the clinical use of insulin-based surrogate indices. The lipoprotein insulin resistance index (LP-IR), a metabolomic marker, reflects the lipoprotein abnormalities observed in IR. The reliability of the LP-IR index in South Asians is unknown. Objective: We evaluated the predictive accuracy of LP-IR compared with other IR surrogate indices in South Asians. Methods: In a cross-sectional study (n = 55), we used calibration model analysis to assess the ability of the LP-IR score and other simple surrogate indices (Homeostatic Model Assessment of Insulin Resistance, Quantitative insulin sensitivity check index, Adipose insulin resistance index, and Matsuda Index) to predict insulin sensitivity (SI) derived from the reference frequently sampled intravenous glucose tolerance test. LP-IR index was derived from lipoprotein particle concentrations and sizes measured by nuclear magnetic resonance spectroscopy. Predictive accuracy was determined by root mean squared error (RMSE) of prediction and leave-one-out cross-validation type RMSE of prediction (CVPE). The optimal cut-off of the LP-IR index was determined by the area under the receiver operating characteristic curve (AUROC) and the Youden index. Results: The simple surrogate indices showed moderate correlations with SI (r = 0.53-0.69, P < .0001). CVPE and RMSE were not different in any of the surrogate indices when compared with LP-IR. The AUROC was 0.77 (95% CI 0.64-0.89). The optimal cut-off for IR in South Asians was LP-IR >48 (sensitivity: 75%, specificity: 70%). Conclusion: The LP-IR index is a simple, accurate, and clinically useful test to assess IR in South Asians.

Potential for Increased Epilepsy Awareness: Impact of Health Education Program in Schools for Teachers and Children.

Background Epilepsy, although a common disorder, yet is highly stigmatized. Under this condition, children with epilepsy are more vulnerable to stigmatization, social isolation, lack of support, and psychological and emotional problems. Thus, there is an immediate need of literature focusing on intervention studies to change the attitudes of school teachers and children. Methods The study was conducted with the objectives to evaluate knowledge, attitude, and practices (KAP) about epilepsy among school teachers and children; provide educational training program to teachers; and check the efficacy of training program imparted by teacher and trainer. Results Repeated measure of analysis of variance shows that knowledge ( F = 1,134.875, p < 0.001), attitude ( F = 2,429.909, p < 0.001), and practice ( F = 2,205.122, p < 0.001) are significantly different between pre- and posttests indicated by Pillai's trace test. Similarly, from the Pillai's test, knowledge ( F = 49.317, p < 0.001), attitude ( F = 125.304, p < 0.001), and practice ( F = 178.697, p < 0.001) are significantly different among teachers, trainer imparting training to children, and teachers imparting training to children. It is seen that KAP scores significantly differ between two time points and across the three groups. Among all the groups, teachers imparting training to children had high level of practice. Conclusion Inclusion of health education programs in the textbooks and health education schemes for teachers and school children are crucial ways to bring a change in their attitude, behavior, and practices toward epilepsy.

Exploring the knowledge and attitude of public about mental health problems: A pilot intervention for effective mental health promotion.

CONTEXT: The knowledge about mental health problems among the general public is comparatively quite low. The pilot study was conducted with an aim to increase the knowledge of the day-to-day mental health problems which people can have among the mass. AIMS: The aims of this study are (1) to assess the knowledge and attitude about mental health problems of the selected sample and (2) to build the capacity in providing first aid for mental health to the selected sample through training. SETTINGS AND DESIGN: A cross-sectional study was used in assessing the knowledge and skills of the participants of the first aid for mental health problems. It was conducted in the institute itself. SUBJECTS AND METHODS: A total of 89 participants were taken to participate in the cross-sectional study. Using a semi-structured self-administered questionnaire, a brief training, and a feedback form, capacity building for first aid for mental health problems was provided. STATISTICAL ANALYSIS USED: The questionnaires were analyzed using descriptive statistics. RESULTS: The capacity building program for first aid for mental health problems appears to be effective in improving the knowledge and attitude with regard to the mental health problems. The findings from the program indicated lack of knowledge in understanding of mental health, knowledge of causation, and treatment of mental health problems. Feedback of the training program indicated that it improved the ability of the participants in recognizing persons undergoing mental health problems and brought about a change in their beliefs about mental health, attitudes, and need for prompt referral. CONCLUSIONS: The program was successful in increasing the confidence of the participants in providing help to someone with a mental health problem and referring to appropriate mental health professional. This shows that there is an immediate need for empowering general public with knowledge and skills to provide support to people with mental health problems.

Potential for Increased Epilepsy Awareness: Impact of Health Education Program in School on Teachers and Children.

Background Although epilepsy is a common disorder, it is highly stigmatized. Not only public but even teachers are not free from stigmatization and cultural barriers. Under this condition, children with epilepsy are more vulnerable for stigmatization, social isolation, lack of support, and psychological and emotional problems. At the same time, there is an immediate need of literature focusing on intervention studies to change the attitudes of school teachers and children. Materials and Methods The study was conducted with the objectives to evaluate knowledge, attitude, and practices about epilepsy among school teachers and children; provide educational training program to teachers; and check the efficacy of training program imparted by teacher and trainer. Results Repeated measure of analysis of variance shows that knowledge ( F = 1134.875, p < 0.001), attitude ( F = 2429.909, p < 0.001), and practice ( F = 2205.122, p < 0.001) are significantly different between pre- and posttest indicated by Pillai's trace test. Similarly, from the Pillai's test knowledge ( F = 49.317, p < 0.001), attitude ( F = 125.304, p < 0.001) and practice ( F = 178.697, p < 0.001) are significantly different among the teachers, trainer imparted training group of children, and teachers imparted training group of children groups. It is seen that knowledge, attitude, and practice scores significantly differ between two time points and across three groups. Among all the groups, teachers imparted training group children had high level of practice. Conclusion Inclusion of health education programs in the textbooks and health education schemes for teachers and school children is a crucial way to bring a change in their attitude, behavior, and practices.

Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K.

A potassium (K+ ) rich diet is known to have an antihypertensive effect that has been embodied by the NHLBI in the DASH diet. However, the molecular basis for this blood pressure-lowering effect has been unclear, until a recent study proposed a model in which the DCT cells of the kidney regulate their salt transport in response to variations in intracellular chloride ([Cl- ]i ), which are directly regulated by serum K+ . With the knowledge that WNK proteins are Cl- sensors, and are a part of the WNK/SPAK/NCC signaling cascade which regulates the NCC, the main salt transporter in the distal nephron, we examined the effect of serum K+ on the ([Cl- ]i ) and, in turn its effect on the WNK4 signaling pathway in a "modified HEK 293T" cell line. Using a fluorescence-based approach in this cell line, we have shown that the membrane potential of the cell membrane is sensitive to the small changes in external KCl within the physiological range (2-5 mM), thus functioning as a K+ electrode. When the extracellular K+ was progressively increased (2-5 mM), the membrane depolarization lead to a subsequent increase in [Cl- ]i measured by fluorescence quenching of an intracellular chloride sensor. Increase in extracellular [K] resulted in a decrease in the phosphorylation of the WNK4 protein and its downstream targets, SPAK and NCC. This confirms that small changes in serum K can affect WNK4/SPAK/NCC signaling and transcellular Na+ flux through the DCT and provide a possible mechanism by which a K-rich DASH diet could reduce blood pressure.

Potential for a comprehensive stroke education: Assessing awareness about stroke among community health workers - A qualitative study from Urban Bangalore, Karnataka, India.

CONTEXT: The incidence of stroke is evolving to be a major public health issue in recent years. The situation is aggravated by the limited public awareness about its risk factors and treatment procedures. It is important to explore the level of awareness of the Accredited Social Health Activists (ASHAs) about stroke as they work in the community as part of the public healthcare delivery systems. AIMS: The aim of this study was to explore the awareness about stroke among the ASHAs through a qualitative study. SETTINGS AND DESIGN: The study was conducted in the institution using a cross-sectional qualitative approach. METHODS AND MATERIAL: A focus group discussion with 12 ASHAs from Bengaluru district of Karnataka was conducted. STATISTICAL ANALYSIS USED: Data were analyzed manually through direct content analysis. RESULTS: It was revealed that the ASHAs did not have adequate general awareness about stroke, its causes, and related treatment procedures. The ASHAs overgeneralized the causes to lifestyle factors, such as smoking, drinking, and so on. CONCLUSIONS: The study showed the level of awareness of stroke among the ASHAs is inadequate at present, and they would benefit from systematic sensitisation programmes. Limited awareness could lead to failure to identify the early warning signs and appropriate, timely help. A need of a comprehensive educational program to increase their awareness is emphasized.

Exploring knowledge, attitude, and practices in relation to epilepsy among undergraduates for effective health promotion: Initial evaluation.

BACKGROUND: The condition of epilepsy has a considerable effect on a person's social and personal life. Currently, a knowledge gap exists regarding the knowledge, attitude, and perception towards epilepsy among graduate students. The objectives of the study were to initially explore the knowledge, attitude, practices and to examine their inter-relationship among graduate students. MATERIALS AND METHODS: A total of 300 3rd year Bachelor of Science graduate students from colleges near Hombegowda nagar, Bangalore, Karnataka were included from three colleges. To achieve the objective mentioned, an instrument was framed and approved by specialists in the field. RESULTS: About 26% believed that epilepsy is a mental illness, 64% reported that it is not a disease of the brain, 96% believed it to be a hereditary disease. Attitude shows that 29%, 33%, and 49% believed that Epilepsy can disturb anybody's normal life, education, and occupation, respectively. About 31% reported that if they see a person with epileptic attack they ran away. Knowledge score have significantly (P < 0.001) positive correlation of r = 0.810 and r = 0.794 with both attitude and practice, respectively. Attitude and practice also have significantly (P < 0.001) positive correlation (0.856) with practice. This clearly shows that if knowledge increases, persons will have positive attitude and good practices whereas less knowledge leads to faulty attitude and practices. CONCLUSIONS: There is a need to include health education programs for school children and college students irrespective of streams, as it is crucial to bring an alteration in the presently observed perspective, behavior, and practice.

Mental Health and Stress Among South Asians.

Addressing mental illness requires a culturally sensitive approach. As detailed in this literature review, treating mental illness in the South Asian immigrant community necessitates a thorough understanding of the South Asian conceptualization of mental illness. Past research, though limited, has described the different reasons the South Asian community attributes to causing mental illness, as well as the stigma associated with acknowledging the disease. Acculturation of the community also plays a significant role in cultural acceptability and the receipt of quality care. Lessons from local organizations can be applied at the national level to promote cultural responsiveness in treating mental illness in the South Asian immigrant community.

Perception of employees on mental health at a leading botanical garden, India: A qualitative study.

BACKGROUND: The prevalence of mental health issues in India is increasing. The lack of knowledge about mental illnesses and negative beliefs about treatment usually lead to delayed treatment. Mental health literacy is a prerequisite for early recognition and intervention in mental disorders. AIMS: The study was undertaken with the aim to explore the awareness of mental health among the employees of the botanical gardens to address this massive issue. MATERIALS AND METHODS: Two focus group discussions were carried out with five and ten members from two diverse groups of population respectively. RESULTS: The study highlights that the signs and symptoms of mental illness were not well recognized by the participants except few.The study also shows the presence of misconceptions among the sample regarding mental health and the role of mental health professionals. This indicates the need for mental health literacy among the community. CONCLUSION: The study shows that the knowledge of the employees of the botanical garden regarding mental health was limited. This limited knowledge may be a prominent cause of poor treatment turn up. Therefore a need of systematic sensitization program to increase mental health literacy is highlighted through the study.

Pathways to care among persons with dementia: Study from a tertiary care center.

The prevalence of dementia is increasing rapidly, specifically in low and middle income countries (LAMIC) due to demographic aging. Help seeking is delayed and usually sought at an advanced stage of illness and many are yet to receive a formal diagnosis. We interviewed 35 caregivers of persons with Dementia (as per ICD-10) using a semi-structured questionnaire, the Short Explanatory Model Interview (SEMI). We explored the pathways taken by caregivers of people with dementia en route to a tertiary care centre and the interactions they had with different health care providers. Qualitative data analysis was done using ATLAS.ti. We identified three major pathways: I) The Neuropsychiatric pathway II) The General Practitioner pathway and III) The Non-cohesive pathway. In general, the caregivers were poorly informed about the illness details such as diagnosis, course and outcome. Neurologists communicated the diagnosis of 'Dementia' more frequently. When information was made available, the caregivers were satisfied with proper information about illness and with at least, partial improvement of symptoms. There is a need for increasing the awareness of dementia in community at large. Health services and systems that address this important public health problem need strengthening. Sensitization and training of primary care physician and staff to identify dementia at an early stage are the need of the hour.

WNK signalling pathways in blood pressure regulation.

Hypertension (high blood pressure) is a major public health problem affecting more than a billion people worldwide with complications, including stroke, heart failure and kidney failure. The regulation of blood pressure is multifactorial reflecting genetic susceptibility, in utero environment and external factors such as obesity and salt intake. In keeping with Arthur Guyton's hypothesis, the kidney plays a key role in blood pressure control and data from clinical studies; physiology and genetics have shown that hypertension is driven a failure of the kidney to excrete excess salt at normal levels of blood pressure. There is a number of rare Mendelian blood pressure syndromes, which have shed light on the molecular mechanisms involved in dysregulated ion transport in the distal kidney. One in particular is Familial hyperkalemic hypertension (FHHt), an autosomal dominant monogenic form of hypertension characterised by high blood pressure, hyperkalemia, hyperchloremic metabolic acidosis, and hypercalciuria. The clinical signs of FHHt are treated by low doses of thiazide diuretic, and it mirrors Gitelman syndrome which features the inverse phenotype of hypotension, hypokalemic metabolic alkalosis, and hypocalciuria. Gitelman syndrome is caused by loss of function mutations in the thiazide-sensitive Na/Cl cotransporter (NCC); however, FHHt patients do not have mutations in the SCL12A3 locus encoding NCC. Instead, mutations have been identified in genes that have revealed a key signalling pathway that regulates NCC and several other key transporters and ion channels in the kidney that are critical for BP regulation. This is the WNK kinase signalling pathway that is the subject of this review.

ROMK expression remains unaltered in a mouse model of familial hyperkalemic hypertension caused by the CUL3Δ403-459 mutation.

Familial hyperkalemic hypertension (FHHt) is a rare inherited form of salt-dependent hypertension caused by mutations in proteins that regulate the renal Na(+)-Cl(-) cotransporter NCC Mutations in four genes have been reported to cause FHHt including CUL3 (Cullin3) that encodes a component of a RING E3 ligase. Cullin-3 binds to WNK kinase-bound KLHL3 (the substrate recognition subunit of the ubiquitin ligase complex) to promote ubiquitination and proteasomal degradation of WNK kinases. Deletion of exon 9 from CUL3 (affecting residues 403-459, CUL3(Δ403-459)) causes a severe form of FHHt (PHA2E) that is recapitulated closely in a knock-in mouse model. The loss of functionality of CUL3(Δ403-459) and secondary accumulation of WNK kinases causes substantial NCC activation. This accounts for the hypertension in FHHt but the origin of the hyperkalemia is less clear. Hence, we explored the impact of CUL3(Δ403-459) on expression of the distal secretory K channel, ROMK, both in vitro and in vivo. We found that expressing wild-type but not the CUL3(Δ403-459) mutant form of CUL3 prevented the suppression of ROMK currents by WNK4 expressed in Xenopus oocytes. The mutant CUL3 protein was also unable to affect ROMK-EGFP protein expression at the surface of mouse M-1 cortical collecting duct (CCD) cells. The effects of CUL3 on ROMK expression in both oocytes and M-1 CCD cells was reduced by addition of the neddylation inhibitor, MLN4924. This confirms that neddylation is important for CUL3 activity. Nevertheless, in our knock-in mouse model expressing CUL3(Δ403-459) we could not show any alteration in ROMK expression by either western blotting whole kidney lysates or confocal microscopy of kidney sections. This suggests that the hyperkalemia in our knock-in mouse and human PHA2E subjects with the CUL3(Δ403-459) mutation is not caused by reduced ROMK expression in the distal nephron.

Outcome of a school-based health education program for epilepsy awareness among schoolchildren.

BACKGROUND: A diagnosis of epilepsy has a major effect on children; especially among schoolchildren. Studies have shown that a significant proportion of teachers and students have negative attitude and misunderstanding towards epilepsy making it difficult for a child with epilepsy. At the same time, there is a dearth is dearth of literature regarding interventions to bring about a change in the attitudes of children. METHODOLOGY: The aim of the present study was to study the outcome of a school-based health education program for epilepsy awareness among schoolchildren. The objectives were to assess the level of knowledge, attitude, and practices about epilepsy and relationship among these variables. A total of 70 children, from 8th-10th grades were selected randomly for the study. For the study purpose, knowledge, attitude, and practices of epilepsy instrument were developed and face validated by experts. RESULTS: Results show that the mean, median, and mode age of the respondents in the study were 14.55 (±1.33), 15, and 14 years, respectively. The gender distribution of the respondents was 54.9% boys and 45.1% girls. With regard to knowledge, attitude, and practices in epilepsy, knowledge strongly and positively correlated with attitude (p=0.001, r=.423) and practice domains (p=0.001, r=.486). Postattitude and (p=0.001, r=.338) practice were positively correlated, which shows that positive attitude brings positive practice. Hence, it is concluded that a training program brings desirable change in the knowledge, attitude, and practice domains among children. CONCLUSION: Health education programs for schoolchildren are very important to bring changes in their attitude, behavior, and practices.

Neural control of submucosal gland and apical membrane secretions in airways.

The mechanisms that lay behind the low-level secretions from airway submucosal glands and the surface epithelium in the absence of external innervation have been investigated in small areas (1.0-1.5 cm(2)) of mucosa from sheep tracheas, freshly collected from a local abattoir. Glandular secretion was measured by an optical method while short circuit current was used as a measure of surface secretion. Activation of neurones in the intrinsic nerve net by veratrine alkaloids caused an immediate increase in both glandular secretion and short circuit current, both effects being blocked by the addition of tetrodotoxin. However, agents known to be acting directly on the glands, such as muscarinic agonists (e.g., carbachol) or adenylate cyclase activators (e.g., forskolin) were not influenced by tetrodotoxin. The toxin alone had no discernable effect on the low-level basal secretion shown by unstimulated glands. Calu-3 cell monolayers, generally agreed to be a surrogate for the secretory cells of submucosal glands, showed no sensitivity to veratrine alkaloids, strengthening the view that the veratrine-like drugs acted exclusively on the intrinsic nerve net. The data are discussed in relation way in which transplanted lungs can maintain mucociliary clearance and hence a sterile environment in the absence of external innervation, as in transplanted lungs.

Role for germline mutations and a rare coding single nucleotide polymorphism within the KCNJ5 potassium channel in a large cohort of sporadic cases of primary aldosteronism.

Primary aldosteronism (autonomous aldosterone production with suppressed renin) plays an important pathophysiological role in what has been previously labeled as essential hypertension. Besides the recently described germline mutations in the KCNJ5 potassium channel associated with familial primary aldosteronism, somatic mutations in the same channel have been identified within aldosterone-producing adenomas. In this study, we have resequenced the flanking and coding region of KCNJ5 in peripheral blood DNA from 251 white subjects with primary aldosteronism to look for rare variants that might be important for the pathophysiology of sporadic primary aldosteronism. We have identified 3 heterozygous missense mutations (R52H, E246K, and G247R) in the cohort and found that 12 (5% of the cohort) were carriers for the rare nonsynonymous single nucleotide polymorphism rs7102584 causing E282Q substitution of KCNJ5. By expressing the channels in Xenopus oocytes and human adrenal H295R cells, we have shown that the R52H, E246K, and E282Q substitutions are functional, but the G247R mutation is indistinguishable from wild type. Although the functional substitutions are remote from the selectivity filter, they affect the inward-rectification, the ability of the KCNJ5 channels to conduct Na(+) currents and ATII-induced aldosterone release from the H295R cell line. Together these data suggest that germline variation in the KCNJ5 gene has a role to play in the common sporadic form as well as the much rarer syndromic forms of primary aldosteronism.

Characterization of a novel somatic KCNJ5 mutation delI157 in an aldosterone-producing adenoma.

OBJECTIVE: Adrenal aldosterone-producing adenomas (APAs) are an increasingly recognized cause of primary aldosteronism, and somatic mutations within the KCNJ5 gene encoding an inwardly rectifying K(+) channel (also called GIRK4 or Kir3.4) have been identified by several groups including our own. We identified the previously noted G151R and L168R mutations in the region of a selectivity filter of the channel as well as a previously unreported 3-base deletion, delI157. Here, we report the functional properties of KCNJ5 channels carrying this novel delI157 mutation. METHODS: The delI157 mutation was introduced into wild-type KCNJ5 sequences to allow its expression in both H295R cells and Xenopus oocytes to study its expression and electrophysiology, respectively. RESULTS: In the adrenal cell line H295R, the delI157 mutant expresses and traffics normally to the cell surface. However, the current-voltage behavior of the mutant in oocytes is distinct from wild-type channels and mimics closely other selectivity filter mutations. In particular, its ability to support substantial current when extracellular K(+) is replaced by Na(+). We also report for the first time that the mutants have reduced sensitivity to the KCNJ5 inhibitor tertiapin-Q that binds to the external vestibule of the channel pore. CONCLUSION: This novel KCNJ5 mutation behaves like the three selectivity filter mutations previously reported in APAs depolarizing the cell and showing reduced cation selectivity. The reduced sensitivity to tertiapin-Q suggests that the abnormal Na(+) permeability of these selectivity mutations does indeed reflect structural changes around the mouth of the ion channel.

Somatic mutations affecting the selectivity filter of KCNJ5 are frequent in 2 large unselected collections of adrenal aldosteronomas.

Primary hyperaldosteronism, one cause of which is aldosterone-producing adenomas (APAs), may account for ≤5% to 10% of cases of essential hypertension. Germline mutations have been identified in 2 rare familial forms of primary hyperaldosteronism, but it has been reported recently that somatic mutations of the KCNJ5 gene, which encodes a potassium channel, are present in some sporadic nonsyndromic APAs. To address this further we screened 2 large collections of sporadic APAs from the United Kingdom and Australia (totalling 73) and found somatic mutations in the selectivity filter of KCNJ5 in 41% (95% CI: 31% to 53%) of the APAs (30 of 73). These included the previously noted nonsynonymous mutations, G151R and L158R, and an unreported 3-base deletion, delI157, in the region of the selectivity filter. APAs containing a somatic KCNJ5 mutation were significantly larger than those without (1.61 cm [95% CI: 1.39-1.83 cm] versus 1.04 cm [95% CI: 0.91-1.17 cm]; P<0.0001) but with substantial overlap in size between genotypes. The APAs carrying a mutation, but not those without, also consistently lacked a postural aldosterone response, suggesting a physiologically distinct subtype. Hence, somatic KCNJ5 mutations are not restricted to large APAs (>2 cm), and their frequency in our unselected series suggests they are common and could be important in the molecular pathogenesis of many sporadic cases of APA.

The acidic motif of WNK4 is crucial for its interaction with the K channel ROMK.

WNK kinases have rapidly emerged as important regulators of Na+ and K+ homoeostasis in the mammalian kidney where they regulate the trafficking of proteins such as the NaCl-cotransporter (NCCT) and K+ channel, ROMK. However, an increasing number of WNK effects are kinase-independent, including their interaction with ROMK, and involve instead protein-protein interactions. Outside of their kinase domain all WNKs contain a unique run of predominantly negatively charged amino acids dubbed the acidic motif, where the WNK4 disease mutations causing Gordon's syndrome also cluster. To look further at the role of this motif we studied the effects of WNK4 fragments, including one with a deleted acidic motif (DeltaAM) and a 10-mer acidic motif peptide on ROMK expression in Xenopus oocytes. We found that an N-terminal fragment of WNK4 (1-620 WNK4) containing the acidic motif retains full activity in inhibiting ROMK currents. However, DeltaAM WNK4 is completely inactive and the effect of WNK4 or 1-620 WNK4 can be completely blocked by co-injection of the 10-mer acidic motif peptide. The blocking action of the peptide was sequence specific as a peptide with a randomised sequence was inactive. These results on ROMK currents were paralleled by changes in membrane expression of fluorescent EGFP-ROMK. Finally, we show that 1-620 WNK4 can pull down ROMK and this interaction can be blocked with the acidic motif peptide. These results confirm the important role of the acidic motif of WNK4 in its protein-protein interaction with the ROMK channel.

Regulation of the expression of the Na/Cl cotransporter by WNK4 and WNK1: evidence that accelerated dynamin-dependent endocytosis is not involved.

The novel serine/threonine kinases (with no lysine kinases or WNKs), WNK1 and WNK4, are encoded by the disease genes for Gordon syndrome (PRKWNK1 and PRKWNK4), a rare monogenic syndrome of hypertension and hyperkalemia. These proteins alter the expression of the thiazide-sensitive Na/Cl cotransporter (NCCT) in Xenopus laevis oocytes, although the details are controversial. We describe here our own experience and confirm that kinase-dead WNK4 (318D>A) is unable to affect Na+ fluxes through the thiazide-sensitive Na/Cl transporter (NCCT) or its membrane expression as an ECFP-NCCT fusion protein. However, the kinase domain is not sufficient for a functional WNK4 since deletion of the acidic motif (a motif unique to WNK family members) completely abolishes functional activity. Indeed, the NH2 terminal of WNK4 (1-620) containing the kinase domain and acidic motif retains full activity, but does not interact directly with NCCT in pull-down assays. Coexpression of WNK1 antagonizes the action of WNK4, and kinase-dead WNK1 (368D>A) or WNK1 carrying a WNK4 disease mutation (565Q>E) behaves in the same way as wild-type WNK1. This suggests kinase activity and charge conservation within the acidic motif are not essential for the WNK1-WNK4 interaction. We also report that WNK4 probably reduces surface expression largely through an effect on forward trafficking. Hence, the effect of WNK4 on NCCT expression is mimicked by dynamin, but the dominant-negative K44A dynamin mutant does not block the action of WNK4 itself. These results further highlight important differences in the mechanism by which WNK kinases affect expression of NCCT vs. other membrane proteins such as ROMK.