A survey of clinical empathy training at UK medical schools.

BACKGROUND: The benefits of enhancing practitioner empathy include better patient outcomes and improved job satisfaction for practitioners. Evidence suggests empathy can be taught and empathy is listed as an outcome for graduates in the General Medical Council requirements. Despite this, empathy training is not mandatory on medical school curricula and the extent to which medical students are given empathy-specific training is unknown. AIM: To conduct a survey of empathy training currently offered to medical students in UK medical schools. METHODS: An invitation to participate in an online survey was sent to all UK medical schools (n = 40). The survey was developed through a consultancy and pilot process to ensure validity and reliability. Questions explored what empathy-focused training is offered, and asked educators whether or not they believed that current provision of empathy training is sufficient. In parallel, medical school websites were searched to identify what information regarding empathy-focused training is described as being part of the degree course. Descriptive statistics were used to describe empathy training delivery from the results of the online materials survey and closed survey questions. Thematic analysis was used to explore free text comments. RESULTS: Response rate was 70% (28/40), with 28 medical schools included in the analysis. Twenty-six schools reported that their undergraduate curriculum included some form of empathy-focused training with variation in what, when and how this is delivered. Thematic analysis revealed two overarching themes with associated sub-themes: (i) empathy-focused training and development (considering where, when and how empathy training should be integrated); (ii) challenges presented by including empathy on the curriculum (considering the system, students and faculty). All schools agreed empathy training should be on the undergraduate curriculum. CONCLUSION: This is the first nationwide survey of empathy-focused training at UK medical schools. While some form of empathy-focused training appears to be provided on the undergraduate curriculum at most UK medical schools, empathy is rarely specifically assessed. Most medical educators do not feel their school does enough to promote empathy and the majority would like to offer more.

Kir6.2-D323 and SUR2A-Q1336: an intersubunit interaction pairing for allosteric information transfer in the KATP channel complex.

ATP-sensitive potassium (KATP) channels are widely expressed and play key roles in many tissues by coupling metabolic state to membrane excitability. The SUR subunits confer drug and enhanced nucleotide sensitivity to the pore-forming Kir6 subunit, and so information transfer between the subunits must occur. In our previous study, we identified an electrostatic interaction between Kir6 and SUR2 subunits that was key for allosteric information transfer between the regulatory and pore-forming subunit. In this study, we demonstrate a second putative interaction between Kir6.2-D323 and SUR2A-Q1336 using patch clamp electrophysiological recording, where charge swap mutation of the residues on either side of the potential interaction compromise normal channel function. The Kir6.2-D323K mutation gave rise to a constitutively active, glibenclamide and ATP-insensitive KATP complex, further confirming the importance of information transfer between the Kir6 and SUR2 subunits. Sensitivity to modulators was restored when Kir6.2-D323K was co-expressed with a reciprocal charge swap mutant, SUR-Q1336E. Importantly, equivalent interactions have been identified in both Kir6.1 and Kir6.2 suggesting this is a second important interaction between Kir6 and the proximal C terminus of SUR.

A qualitative exploration of the lived experience of GP trainees failing to progress in training.

Challenges facing general practice are multiple and extreme. Amongst them is the increasing difficulty of recruiting and retaining General Practitioners (GPs). GPs cite heavy workload, work-related stress, little family time and psychological ill-health as factors influencing their decisions to leave or reduce working hours. Analysis of the literature suggests that these factors, amongst others, are present in GP training and trainees have similar experiences. An in-depth understanding of the challenges trainees in difficulty face is lacking.Our research aim was to better understand the factors that trainees perceive contribute to their failure to progress in training. A qualitative approach was adopted using semi-structured interviews with GP trainees identified as failing to progress satisfactorily or failing the MRCGP examinations. Interviews were audio-recorded and transcribed. Thematic analysis was used to understand the unique experiences of GP trainees and find common themes.Twenty-three interview transcripts were analysed. Emergent themes were presented using a framework of three distinct categories to aid data organisation and allocating themes and sub-themes: professional factors, personal factors, and social factors. Difficulties with managing work-load, poor motivation, lack of family time and psychological ill-health were significant themes for many. This study supports the evidence that difficulties facing GPs take root in training. Failure to fully understand trainees' journeys and associated challenges reduces opportunities to provide bespoke packages of care and remediation that fully address their needs.

A Qualitative Exploration of the Help-Seeking Behaviors of Students Who Experience Psychological Distress Around Assessment at Medical School.

OBJECTIVE: Medical students are at high risk of experiencing psychological distress at medical school and developing mental ill-health during professional practice. Despite efforts by faculty to raise awareness about this risk, many students choose to suffer in silence in the face of psychological distress. The aim of this study was to explore drivers that prompted help-seeking behavior and barriers that prevented individuals prioritizing their well-being around the time of high-stakes assessment at medical school. METHODS: Semi-structured interviews were conducted with fifty-seven students who failed high-stakes assessment at two UK medical schools, exploring their experience of academic difficulty and perceptions about causes. A thematic analysis of twenty transcripts that met inclusion criteria was completed to identify key factors that influenced participants' decisions around seeking help for their psychological distress, and in some cases, mental health problems. Twenty participants who specifically described a deterioration in their mental health around the time of assessment were included in this study. RESULTS: Barriers to seeking help in these instances included: normalization of symptoms or situation; failure to recognize a problem existed; fear of stigmatisation; overt symptoms of mental distress; and misconceptions about the true nature of the medical school, for example beliefs about a punitive response from the school if they failed. Drivers for seeking help appropriately included: building trust with someone in order to confide in them later on, and self-awareness about the need to maintain good mental health. CONCLUSION: There are various drivers and barriers for students' help seeking behaviors when experiencing psychological distress around the time of assessment, particularly self-awareness about the problem and prioritisation of well-being. Students who fail to recognize their own deteriorating mental health are at risk of academic failure and medical schools need to develop strategies to tackle this problem in order to protect these students from harm.

Exploring the climates of undergraduate professionalism in a Saudi and a UK medical school.

AIM: To explore the usefulness of an online inventory for tracking medical students' understanding of the importance of lapses in academic integrity. METHOD: Respondents were asked to recommend sanctions for lapses as a proxy of their understanding of the importance of the 34 types of poor professionalism. RESULTS: The data suggest that while there is congruence, there are also substantial differences between ratings of the importance of poor professionalism, particularly in relation to data integrity, between a cohort in Saudi Arabia and one in the UK. CONCLUSION: This resource may be useful both for teaching and learning in individual schools, and particularly for the induction of doctors into organisational environments different from the one they were trained in.

Publish or Perish mantra in the medical field: A systematic review of the reasons, consequences and remedies.

OBJECTIVES: Generally, academic promotions, job retention, job mobility, and professional development of a medical faculty members are judged primarily by the growth in publication outputs. Universities and research institutions are more likely to recruit and promote those academics carrying voluminous résumés with larger number of published articles. This review elaborates the causes and consequences of the pressure to publish and the ways and means to cope with this paradigm. METHODS: In 2015, database of Abstracts of Reviews of Effects, LISTA (EBSCO), Medline and Oxford University Library were searched for the English language full-text articles published during 2000-2015, by using MeSH terms "pressure to publish", "urge to publish", "research ethics", "plagiarism", "article retraction", "medical field". This search was further refined by selecting the articles in terms of relevancy and contents. RESULTS: This research showed that some universities offer generous grants to researchers with a high h-index and with more publications in elite journals, which promise an enhanced prospect of citations and elevation in the scientific rankings of the funding institutions. This generates an involuntary obsession to publish with the primary intention to obtain promotions, high scientific rankings, and improved job security. This compelling pressure to publish results in widespread publication of non-significant research with a high index of plagiarism that eventually leads to an increased frequency of retractions. CONCLUSION: Research centers and academic institutions have an obligation to train their academics in sound scientific writing and to apprise them of the publication ethics and the grave consequences of plagiarism and research misconduct.

Early opening of sarcolemmal ATP-sensitive potassium channels is not a key step in PKC-mediated cardioprotection.

ATP-sensitive potassium (KATP) channels are abundantly expressed in the myocardium. Although a definitive role for the channel remains elusive they have been implicated in the phenomenon of cardioprotection, but the precise mechanism is unclear. We set out to test the hypothesis that the channel protects by opening early during ischemia to shorten action potential duration and reduce electrical excitability thus sparing intracellular ATP. This could reduce reperfusion injury by improving calcium homeostasis. Using a combination of contractile function analysis, calcium fluorescence imaging and patch clamp electrophysiology in cardiomyocytes isolated from adult male Wistar rats, we demonstrated that the opening of sarcolemmal KATP channels was markedly delayed after cardioprotective treatments: ischemic preconditioning, adenosine and PMA. This was due to the preservation of intracellular ATP for longer during simulated ischemia therefore maintaining sarcolemmal KATP channels in the closed state for longer. As the simulated ischemia progressed, KATP channels opened to cause contractile, calcium transient and action potential failure; however there was no indication of any channel activity early during simulated ischemia to impart an energy sparing hyperpolarization or action potential shortening. We present compelling evidence to demonstrate that an early opening of sarcolemmal KATP channels during simulated ischemia is not part of the protective mechanism imparted by ischemic preconditioning or other PKC-dependent cardioprotective stimuli. On the contrary, channel opening was actually delayed. We conclude that sarcolemmal KATP channel opening is a consequence of ATP depletion, not a primary mechanism of ATP preservation in these cells.

Sulfonylurea receptors regulate the channel pore in ATP-sensitive potassium channels via an intersubunit salt bridge.

ATP-sensitive potassium channels play key roles in many tissues by coupling metabolic status to membrane potential. In contrast with other potassium channels, the pore-forming Kir6 subunits must co-assemble in hetero-octameric complexes with ATP-binding cassette (ABC) family sulfonylurea receptor (SUR) subunits to facilitate cell surface expression. Binding of nucleotides and drugs to SUR regulates channel gating but how these responses are communicated within the complex has remained elusive to date. We have now identified an electrostatic interaction, forming part of a functional interface between the cytoplasmic nucleotide-binding domain-2 of SUR2 subunits and the distal C-terminus of Kir6 polypeptides that determines channel response to nucleotide, potassium channel opener and antagonist. Mutation of participating residues disrupted physical interaction and regulation of expressed channels, properties that were restored in paired charge-swap mutants. Equivalent interactions were identified in Kir6.1- and Kir6.2-containing channels suggesting a conserved mechanism of allosteric regulation.

PKC-mediated toxicity of elevated glucose concentration on cardiomyocyte function.

While it is well established that mortality risk after myocardial infarction (MI) increases in proportion to blood glucose concentration at the time of admission, it is unclear whether there is a direct, causal relationship. We investigated potential mechanisms by which increased blood glucose may exert cardiotoxicity. Using a Wistar rat or guinea-pig isolated cardiomyocyte model, we investigated the effects on cardiomyocyte function and electrical stability of alterations in extracellular glucose concentration. Contractile function studies using electric field stimulation (EFS), patch-clamp recording, and Ca2+ imaging were used to determine the effects of increased extracellular glucose concentration on cardiomyocyte function. Increasing glucose from 5 to 20 mM caused prolongation of the action potential and increased both basal Ca2+ and variability of the Ca2+ transient amplitude. Elevated extracellular glucose concentration also attenuated the protection afforded by ischemic preconditioning (IPC), as assessed using a simulated ischemia and reperfusion model. Inhibition of PKCα and ß, using Gö6976 or specific inhibitor peptides, attenuated the detrimental effects of glucose and restored the cardioprotected phenotype to IPC cells. Increased glucose concentration did not attenuate the cardioprotective role of PKCε, but rather activation of PKCα and ß masked its beneficial effect. Elevated extracellular glucose concentration exerts acute cardiotoxicity mediated via PKCα and ß. Inhibition of these PKC isoenzymes abolishes the cardiotoxic effects and restores IPC-mediated cardioprotection. These data support a direct link between hyperglycemia and adverse outcome after MI. Cardiac-specific PKCα and ß inhibition may be of clinical benefit in this setting.

A survey of the practice and experience of clinical educators in UK secondary care.

BACKGROUND: Experiences and attitudes of clinical trainers of undergraduate medical students and postgraduate medical trainees in secondary care have received limited attention. Anecdotally, clinical teaching is becoming increasingly restricted by clinical service pressures, thereby presenting a risk to the quality of training provision. METHODS: To explore the commitment, experience and attitudes of clinical teachers and trainers of undergraduate medical students and postgraduate trainees, respectively, amongst secondary care providers across a UK Healthcare Workforce Deanery, an invitation to complete a study-specific, on-line survey, comprising predominantly yes/no response and 5-point Likert scale statements with some open questions, was sent to all registered secondary care trainers/supervisors working in the East Midlands Strategic Health Authority. The survey was open between February and June 2012, with two reminders to complete. Responses were anonymised and the frequency of responses to questions was analysed. Data were analysed for the whole study population and for the relationship between frequency of responses and gender. RESULTS: The majority of teachers/trainers considered that they were well prepared and fulfilled their clinical teaching responsibilities. Many reported having restricted time for preparation and delivery and that teaching activities were often completed in their own time. Despite reported poor support and low incentives, many respondents felt valued for their clinical teaching by their Medical Schools and the Deanery, but less so by hospital Trusts. CONCLUSIONS: Respondents indicated that some faculty like and enjoy clinical teaching despite lack of allocated time, resources and recognition. The majority indicated that they feel confident and competent in their clinical teaching roles. Insufficient dedicated time due to competing clinical service pressures was reported as the major barrier to clinical teaching provision.

Experiences of empathy training in healthcare: A systematic review of qualitative studies.

OBJECTIVE: To synthesise qualitative studies reporting student, practitioner, or patient experiences of empathy-training in healthcare. METHODS: We included qualitative studies exploring (i) student or practitioner experiences of empathy training, or (ii) patient experiences of being treated by someone who has undergone empathy training. We used the Critical Appraisal Skills Programme (CASP) tool to assess study quality. Thematic synthesis was used to integrate findings from studies and to generate new insights. RESULTS: Our search yielded 2768 citations, of which 23 (1487 participants) met inclusion criteria. Two clusters of themes were identified from included studies. Firstly, themes related to practitioner/trainee professionalism and wellbeing, where the main finding was that participants experienced benefit from therapeutic empathy training. Secondly, themes related to the understanding and treatment of patients, where the main finding was practitioners' deeper recognition of the positive impact of empathic care. CONCLUSIONS: This review found that taking part in empathy-focused training can benefit practitioner/student personal growth and professional development, and benefits patient care. This review is limited by the difficulty in defining empathy and heterogeneity amongst included studies. PRACTICE IMPLICATIONS: These results support a rationale for empathy training and the development of a framework to ensure training is having the desired effect.

Phenylephrine preconditioning involves modulation of cardiac sarcolemmal K(ATP) current by PKC delta, AMPK and p38 MAPK.

Preconditioning of hearts with the α(1)-adrenoceptor agonist phenylephrine decreases infarct size and increases the functional recovery of the heart following ischaemia-reperfusion. However, the cellular mechanisms responsible for this protection are not known. We investigated the role of protein kinase C ε and δ (PKCε and PKCδ), AMP-activated protein kinase (AMPK), p38 MAPK (p38) and sarcolemmal ATP-sensitive potassium (sarcK(ATP)) channels in phenylephrine preconditioning using isolated rat ventricular myocytes. Preconditioning of ventricular myocytes with phenylephrine increased the recovery of contractile activity following metabolic inhibition and re-energisation from 30.1±1.9% to 66.5±5.2% (P<0.01) and increased the peak sarcK(ATP) current activated during metabolic inhibition from 32.1±1.8 pA/pF to 46.0±5.0 pA/pF (P<0.05), which was required for protection. Phenylephrine preconditioning resulted in a sustained activation of PKCε and PKCδ, and transient activation of AMPK, which was dependent upon activation of PKCδ but not PKCε. P38 was also activated by phenylephrine preconditioning and this was blocked by inhibitors of PKCε, PKCδ or AMPK. Inhibition of PKCδ, AMPK or p38 was sufficient to prevent the increase in current, suggesting that these kinases are involved in modulation of sarcK(ATP) channel current by phenylephrine preconditioning. However, whilst inhibition of AMPK and p38 prevented the protection from phenylephrine preconditioning, PKCδ inhibition paradoxically had no effect. The increase in sarcK(ATP) current induced by phenylephrine preconditioning requires PKCδ, AMPK and p38 and may contribute to the observed improvement in contractile recovery.

Assessing the effect of empathy-enhancing interventions in health education and training: a systematic review of randomised controlled trials.

OBJECTIVE: To estimate the effect of empathy interventions in health education and training from randomised controlled trials (RCTs). METHODS: MEDLINE, PsycINFO, EMBASE, CINAHL and Cochrane databases were searched from inception to June 2019 for RCTs investigating the effect of empathy-enhancing interventions in medical and healthcare students and professionals. Studies measuring any aspect of 'clinical empathy' as a primary or secondary outcome were included. Two reviewers extracted data and assessed the risk of bias of eligible studies using the Cochrane Risk of Bias Tool. Random effects meta-analyses of the impact of empathy training on participants' empathy levels were performed. RESULTS: Twenty-six trials were included, with 22 providing adequate data for meta-analysis. An overall moderate effect on participant empathy postintervention (standardised mean difference 0.52, 95% CI 0.36 to 0.67) was found. Heterogeneity across trial results was substantial (I2=63%). Data on sustainability of effect was provided by 11 trials and found a moderate effect size for improved empathy up until 12 weeks (0.69, 95% CI 0.23 to 1.15), and a small but statistically significant effect size for sustainability at 12 weeks and beyond (standardised mean difference 0.34, 95% CI 0.11 to 0.57). In total, 15 studies were considered to be either unclear or high risk of bias. The quality of evidence of included studies was low. CONCLUSION: Findings suggest that empathy-enhancing interventions can be effective at cultivating and sustaining empathy with intervention specifics contributing to effectiveness. This review focuses on an important, growing area of medical education and provides guidance to those looking to develop effective interventions to enhance empathy in the healthcare setting. Further high-quality trials are needed that include patient-led outcome assessments and further evaluate the long-term sustainability of empathy training. PROTOCOL REGISTRATION NUMBER: PROSPERO (CRD42019126843).

ATP-sensitive K(+) channels regulate the concentrative adenosine transporter CNT2 following activation by A(1) adenosine receptors.

This study describes a novel mechanism of regulation of the high-affinity Na(+)-dependent adenosine transporter (CNT2) via the activation of A(1) adenosine receptors (A(1)R). This regulation is mediated by the activation of ATP-sensitive K(+) (K(ATP)) channels. The high-affinity Na(+)-dependent adenosine transporter CNT2 and A(1)R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (-)-N(6)-(2-phenylisopropyl)adenosine is fully inhibited by K(ATP) channel blockers and mimicked by a K(ATP) channel opener. A(1)R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A(1)R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A(1)R, CNT2, and K(ATP) channels. These results also suggest that the activation of K(ATP) channels under metabolic stress can be mediated by the activation of A(1)R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for K(ATP) channels.

Proximal C-terminal domain of sulphonylurea receptor 2A interacts with pore-forming Kir6 subunits in KATP channels.

Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein-SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254-1545), rSUR2A-CTB (residues 1254-1403) and rSUR2A-CTC (residues 1294-1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358-1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294-1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294-1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels.

Nuclear immunostaining in rat neuronal cells using two anti-Kir2.2 ion channel polyclonal antibodies.

The inwardly rectifying potassium ion channel Kir2.2 has recently been demonstrated to have nuclear and plasma membrane subcellular localization. Nuclear expression of Kir2.2 is controversial, as a functional role for Kir2.0 potassium channels in the nucleus has not been investigated. However, in this report we have demonstrated Kir2.2 nuclear localization in sections of rat hindbrain and dorsal root ganglia tissue, using two anti- Kir2.2 polyclonal antisera with different epitope specificities. These data confirm nuclear localization and are suggestive of new functions of Kir2.0 potassium ion channels in the nucleus.

Endothelin-I and angiotensin II inhibit arterial voltage-gated K+ channels through different protein kinase C isoenzymes.

AIMS: Voltage-gated K+ (Kv) channels of arterial smooth muscle (ASM) modulate arterial tone and are inhibited by vasoconstrictors through protein kinase C (PKC). We aimed to determine whether endothelin-1 (ET-1) and angiotensin II (AngII), which cause similar inhibition of Kv, use the same signalling pathway and PKC isoenzyme to exert their effects on Kv and to compare the involvement of PKC isoenzymes in contractile responses to these agents. METHODS AND RESULTS: Kv currents recorded using the patch clamp technique with freshly isolated rat mesenteric ASM cells were inhibited by ET-1 or AngII. Inclusion of a PKCepsilon inhibitor peptide in the intracellular solution substantially reduced inhibition by AngII, but did not affect that by ET-1. Kv inhibition by ET-1 was reduced by the conventional PKC inhibitor Gö 6976 but not by the PKCbeta inhibitor LY333531. Selective peptide inhibitors of PKCalpha and PKCepsilon were linked to a Tat carrier peptide to make them membrane permeable and used to show that inhibition of PKCalpha prevented ET-1 inhibition of Kv current, but did not affect that by AngII. In contrast, inhibition of PKCepsilon prevented Kv inhibition by AngII but not by ET-1. The Tat-linked inhibitor peptides were also used to investigate the involvement of PKCalpha and PKCepsilon in the contractile responses of mesenteric arterial rings, showing that ET-1 contractions were substantially reduced by inhibition of PKCalpha, but unaffected by inhibition of PKCepsilon. AngII contractions were unaffected by inhibition of PKCalpha but substantially reduced by inhibition of PKCepsilon. CONCLUSION: ET-1 inhibits Kv channels of mesenteric ASM through activation of PKCalpha, while AngII does so through PKCepsilon. This implies that ET-1 and AngII target Kv channels of ASM through different pathways of PKC-interacting proteins, so each vasoconstrictor enables its distinct PKC isoenzyme to interact functionally with the Kv channel.

Syntaxin-1A actions on sulfonylurea receptor 2A can block acidic pH-induced cardiac K(ATP) channel activation.

During cardiac ischemia, ATP stores are depleted, and cardiomyocyte intracellular pH lowers to <7.0. The acidic pH acts on the Kir6.2 subunit of K(ATP) channels to reduce its sensitivity to ATP, causing channel opening. We recently reported that syntaxin-1A (Syn-1A) binds nucleotide binding folds (NBF)-1 and NBF2 of sulfonylurea receptor 2A (SUR2A) to inhibit channel activity (Kang, Y., Leung, Y. M., Manning-Fox, J. E., Xia, F., Xie, H., Sheu, L., Tsushima, R. G., Light, P. E., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 47125-47131). Here, we examined Syn-1A actions on SUR2A to influence the pH regulation of cardiac K(ATP) channels. K(ATP) channel currents from inside-out patches excised from Kir6.2/SUR2A expressing HEK293 cells and freshly isolated cardiac myocytes were increased by reducing intracellular pH from 7.4 to 6.8, which could be blocked by increasing concentrations of Syn-1A added to the cytoplasmic surface. Syn-1A had no effect on C-terminal truncated Kir6.2 (Kir6.2-deltaC26) channels expressed in TSA cells without the SUR subunit. In vitro binding and co-immunoprecipitation studies show that Syn-1A binding to SUR2A or its NBF-1 and NBF-2 domain proteins increased progressively as pH was reduced from 7.4 to 6.0. The enhancement of Syn-1A binding to SUR2A by acidic pH was further regulated by Mg2+ and ATP. Therefore, pH regulates Kir.6.2/SUR2A channels not only by its direct actions on the Kir6.2 subunit but also by modulation of Syn-1A binding to SUR2A. The increased Syn-1A binding to the SUR2A at acidic pH would assert some inhibition of the K(ATP) channels, which may serve as a "brake" to temper the fluctuation of low pH-induced K(ATP) channel opening that could induce fatal reentrant arrhythmias.

Direct inhibition of rat detrusor muscle contraction by erythromycin.

PURPOSE: Detrusor instability is a common problem in the elderly, which is usually treated with anti-cholinergic medication. This study investigates the effect of erythromycin on rat detrusor muscle contractile response to characterise its potential as an alternative inhibitor of bladder muscle contraction. MATERIALS AND METHODS: Strips of rat detrusor muscle were suspended in a perfusion organ bath. The contractile response to direct muscle stimulation, electrical field stimulation (EFS, 0.5-60 Hz), carbachol (10(-5) M), and potassium (10-80 x 10(-3) M) were determined before and after the addition of erythromycin (10(-4)-10(-3) M). The contractile response to carbachol (10(-5) M) in the presence of nifedipine (10(-8) or 10(-6) M) or in calcium-free Kreb's solution was also determined in the absence and presence of erythromycin. RESULTS: Erythromycin 5 x 10(-4) M inhibited the maximum contractile response to EFS, carbachol, and potassium by 38% (P < 0.01), 62% (P < 0.001), and 17% (P < 0.05), respectively, but did not significantly reduce the response to direct muscle stimulation. The atropine-resistant component of EFS-evoked contraction was inhibited by 19.5% (P < 0.01) in the presence of erythromycin. In calcium-free Krebs solution, the maximum contractile response to carbachol was reduced by 42% of control (P < 0.0001) and nifedipine 10(-8) M had no additional effect. When erythromycin 5 x 10(-4) M was added together with nifedipine 10(-8) M, the response to carbachol was inhibited by a further 25% (P < 0.005). CONCLUSIONS: Erythromycin inhibits rat detrusor muscle contraction through the inhibition of calcium influx and the modulation of intracellular calcium movement.

Pore loop-mutated rat KIR6.1 and KIR6.2 suppress KATP current in rat cardiomyocytes.

Cardiomyocytes express mRNA for all major subunits of ATP-sensitive potassium (K(ATP)) channels: KIR6.1, KIR6.2, SUR1A, SUR2A, and SUR2B. It has remained controversial as to whether KIR6.1 may associate with KIR6.2 to form the tetrameric pore of K(ATP) channels in cardiomyocytes. To explore this possibility, cultured rat cardiomyocytes were examined for an inhibition of K(ATP) current by overexpression of pore loop-mutated (inactive) KIR6.x. Bicistronic plasmids were constructed encoding loop-mutated (AFA or SFG for GFG) rat KIR6.x followed by EGFP. In ventricular myocytes, the overexpression of KIR6.1SFG-pIRES(2)-EGFP or KIR6.2AFA-pIRES(2)-EGFP DNA caused, after 72 h, a major decrease of K(ATP) current density of 85.8% and 82.7%, respectively (P < 0.01), relative to EGFP controls (59 +/- 9 pA/pF). In atrial myocytes, overexpression of these pore-mutated KIR6.x by 6.0-fold and 10.6-fold, as assessed by quantitative immunohistochemistry, caused a decrease of K(ATP) current density of 73.7% and 58.5%, respectively (P < 0.01). Expression of wild-type rat KIR6.2 increased the ventricular and atrial K(ATP) current density by 58.3% and 42.9%, respectively (P < 0.01), relative to corresponding EGFP controls, indicating a reserve of SUR to accommodate increased KIR6.x trafficking to the sarcolemma. The results favor the view that KIR6.1 may associate with KIR6.2 to form heterotetrameric pores of native K(ATP) channels in cardiomyocytes.