Key points• The recently identified TMEM16/anoctamin protein family includes Ca 2+ -activated Cl − channels (TMEM16A and TMEM16B), a Ca 2+ -activated non-selective cation channel (TMEM16F) and proteins for which the function remains unclear.• TMEM16 channel proteins consist of eight putative transmembrane domains (TMs) with the 5th and 6th TMs flanking a loop predicted to protrude deep into the membrane. Recent studies suggest that this re-entrant loop may compose part of the pore of TMEM16A channels while also containing residues involved in Ca 2+ binding.• Here, we investigate the functional role of the putative pore-loop by examining the electrophysiological properties of chimeras produced by transplanting this region between TMEM16 family members with different conduction properties and Ca 2+ sensitivities.• We revealed that the putative pore-loop of TMEM16 channels has an unexpected role in controlling the whole-cell Ca 2+ -activated Cl − conductance by regulating the number of functional channels present on the plasma membrane. AbstractThe recently identified TMEM16/anoctamin protein family includes Ca 2+ -activated anion channels (TMEM16A, TMEM16B), a cation channel (TMEM16F) and proteins with unclear function. TMEM16 channels consist of eight putative transmembrane domains (TMs) with TM5-TM6 flanking a re-entrant loop thought to form the pore. In TMEM16A this region has also been suggested to contain residues involved in Ca 2+ binding. The role of the putative pore-loop of TMEM16 channels was investigated using a chimeric approach. Heterologous expression of either TMEM16A or TMEM16B resulted in whole-cell anion currents with very similar conduction properties but distinct kinetics and degrees of sensitivity to Ca 2+ . Furthermore, whole-cell currents mediated by TMEM16A channels were ∼six times larger than TMEM16B-mediated currents. Replacement of the putative pore-loop of TMEM16A with that of TMEM16B (TMEM16A-B channels) reduced the currents by ∼six-fold, while the opposite modification (TMEM16B-A channels) produced a ∼six-fold increase in the currents. Unexpectedly, these changes were not secondary to variations in channel gating by Ca 2+ or voltage, nor were they due to changes in single-channel conductance. Instead, they depended on the number of functional channels present on the plasma membrane. Generation of additional, smaller chimeras within the putative pore-loop of TMEM16A and TMEM16B led to the identification of a region containing a non-canonical trafficking motif. Chimeras composed of the putative pore-loop of TMEM16F transplanted into the TMEM16A protein scaffold did not conduct anions or cations. These data suggest that the putative pore-loop does not form a complete, transferable pore domain. Furthermore, our data reveal an unexpected role for

Background and PurposeCalcium‐activated chloride channels (CaCCs) play varied physiological roles and constitute potential therapeutic targets for conditions such as asthma and hypertension. TMEM16A encodes a CaCC. CaCC pharmacology is restricted to compounds with relatively low potency and poorly defined selectivity. Anthracene‐9‐carboxylic acid (A9C), an inhibitor of various chloride channel types, exhibits complex effects on native CaCCs and cloned TMEM16A channels providing both activation and inhibition. The mechanisms underlying these effects are not fully defined.Experimental ApproachPatch‐clamp electrophysiology in conjunction with concentration jump experiments was employed to define the mode of interaction of A9C with TMEM16A channels.Key ResultsIn the presence of high intracellular Ca2+, A9C inhibited TMEM16A currents in a voltage‐dependent manner by entering the channel from the outside. A9C activation, revealed in the presence of submaximal intracellular Ca2+ concentrations, was also voltage‐dependent. The electric distance of A9C inhibiting and activating binding site was ~0.6 in each case. Inhibition occurred according to an open‐channel block mechanism. Activation was due to a dramatic leftward shift in the steady‐state activation curve and slowed deactivation kinetics. Extracellular A9C competed with extracellular Cl−, suggesting that A9C binds deep in the channel's pore to exert both inhibiting and activating effects.Conclusions and ImplicationsA9C is an open TMEM16A channel blocker and gating modifier. These effects require A9C to bind to a region within the pore that is accessible from the extracellular side of the membrane. These data will aid the future drug design of compounds that selectively activate or inhibit TMEM16A channels.

Background and PurposeOptogenetic control of electromechanical coupling in vascular smooth muscle cells (VSMCs) is emerging as a powerful research tool with potential applications in drug discovery and therapeutics. However, the precise ionic mechanisms involved in this control remain unclear.Experimental ApproachCell imaging, patch‐clamp electrophysiology and muscle tension recordings were used to define these mechanisms over a wide range of light stimulations.Key ResultsTransgenic mice expressing a channelrhodopsin‐2 variant [ChR2(H134R)] selectively in VSMCs were generated. Isolated VSMCs obtained from these mice demonstrated blue light‐induced depolarizing whole‐cell currents. Fine control of artery tone was attained by varying the intensity of the light stimulus. This arterial response was sufficient to overcome the endogenous, melanopsin‐mediated, light‐evoked, arterial relaxation observed in the presence of contractile agonists. Ca2+ entry through voltage‐gated Ca2+ channels, and opening of plasmalemmal depolarizing channels (TMEM16A and TRPM) and intracellular IP3 receptors were involved in the ChR2(H134R)‐dependent arterial response to blue light at intensities lower than ~0.1 mW·mm−2. Light stimuli of greater intensity evoked a significant Ca2+ influx directly through ChR2(H134R) and produced marked intracellular alkalinization of VSMCs.Conclusions and ImplicationsWe identified the range of light intensity allowing optical control of arterial tone, primarily by means of endogenous channels and without substantial alteration to intracellular pH. Within this range, mice expressing ChR2(H134R) in VSMCs are a powerful experimental model for achieving accurate and tuneable optical voltage‐clamp of VSMCs and finely graded control of arterial tone, offering new approaches to the discovery of vasorelaxant drugs.

1: Left ventricular twist mechanics in hypertensive patients with preserved left ventricular ejection fraction and its relation to left atrial phasic functionFigure 4 Chart depicting the numbers of patients assigned into each category of mitral stenosis by each of the methods of mitral valve orifice area assessment. PHT, pressure half time; 2D, 2D planimetry; 3D, 3D planimetry. https://erp.bioscientifica.com Abstract 9: 2-Year event rates of patients discharged from rapid access chest pain clinic after normal stress echocardiogram a Disease classification not available for one patient. b Patients were categorised with the highest disease type. c Disease types were similar on each side. https://erp.bioscientifica.com

IntroductionEthnicity is associated with certain HBV phenotypes which in turn affect prognosis.MethodsWe performed a search for patients with HBV DNA measured at GSTT(March 2007 to March 2015). sAg+ve patients were analysed for:Ethnic group,HIV,Hep C and D co-infection,eAg status, ALT and viral load. Phenotypes were assigned to patients at diagnosis and at last f/up.ALT cut offs were >30 U/L for men and >20 U/L for females.The American Association for the Study of Liver Disease guidelines divide HBV phenotype into 4 groups although a recent study defined an “Indeterminant” group for patients who do not fit into any phenotypeResultsAbstract PTH-094 Table 1PhenotypeAll n = 1799Diagnosis:Last F/upAfrican/Afro-Caribbean n = 872 (48%)Caucasian n = 254 (14%)Chinese n = 186 (10%)Immunotolerant (eAg+ve, normal ALT,viral load > 105)39 (2%):256(<1%):53(<1%):218 (10%):8Chronic Hepatitis B (eAg-ve with a viral load > 104 AND high ALT487 (27%):338188 (22%):15684 (33%):4264 (34%):37Inactive(eAg –ve with a viral load of <104)621 (35%):590357 (41%):33270 (28%):7154 (29%):45Indeterminant565 (31%):398290 (33%):20276 (30%):5340 (22%):35Cirrhosis62 (3%):7923 (3%):2915 (6%):186 (3%):6Acute Hepatitis B25 (1%)8(<1%)6 (2%)4 (2%)On treatment but not cirrhotic0:2830:1100:510:47On treatment AND cirrhotic0:220:90:10:4Seroconversion0:640:290:160:41799 patients were sAg+ve,mean f/up duration was 39.2 months(SD 41.2).45% of the cohort were lost to f/up.47% were male, mean age at diagnosis was 36(No difference between ethnicities).82% were eAg-ve,1.5% Hepatitis D+ve,10% HIV+ve,2% hepatitis C+ve.49% of the cohort were African/Afro-Caribbean,14% Caucasian,10% Chinese,1.5% South Asian, 2.6% South East Asian(other than Chinese),0.3% Arab,3.4% Mixed,2.3% unknown,6.6% “other”.The table shows HBV phenotype at diagnosis compared to last f/up in all patients and then according to the 3 main ethnic groups.Rates of seroconversion and treatment are also shown. 90.5% of African/Afro-Caribbeans were eAg-ve compared to 71% of Caucasians and 63.9% of Chinese(P < 0.01).There was an association between the risk of cirrhosis and older age at diagnosis,HIV or Hep C co-infection(P < 0.01).Comparing the 3 largest ethnic groups only,in 2007,70% were African/Afro-Caribbean,19% Caucasian,11% Chinese compared to 51%,36% and 13% respectively in 2014.ConclusionEthnicity is significantly associated with HBV phenotypes with a higher percentage of immunotolerants being Chinese and a higher percentage of African/Afro-Caribbeans being inactive.Compared to 2007,a higher percentage of HBV patients in 2014 were Caucasian reflecting changes in immigration.This may affect HBV outcomes and demand on Hepatology services.Disclosure of InterestNone Declared