Modulation of Oxytocin Release by Internal Calcium Stores

This chapter elucidates the role of depolarization-induced oxytocin (OT) vs. arginine vasopressin (AVP) secretion in the absence of external calcium, and calcium release from ryanodine-sensitive internal stores as a significant physiological contributor to neuropeptide secretion from hypothalamic neurohypophysial system (HNS) terminals. This has important therapeutic implications, given that exogenous administration of OT to children with autism spectrum disorders (ASD) has shown some success in improving social behavior and lowering anxiety. However, this nonspecific treatment has side effects, including seizures, increased heart rate variability, and psychotic symptoms. Alternatively, facilitating the physiological neuronal release of OT but not AVP from the HNS via modulation of ryanodine vs. inositol triphosphate receptor (IP3R) calcium stores would specifically facilitate central vs. peripheral OT release in ASD patients

RICORS2040 : The need for collaborative research in chronic kidney disease

Chronic kidney disease (CKD) is a silent and poorly known killer. The current concept of CKD is relatively young and uptake by the public, physicians and health authorities is not widespread. Physicians still confuse CKD with chronic kidney insufficiency or failure. For the wider public and health authorities, CKD evokes kidney replacement therapy (KRT). In Spain, the prevalence of KRT is 0.13%. Thus health authorities may consider CKD a non-issue: very few persons eventually need KRT and, for those in whom kidneys fail, the problem is 'solved' by dialysis or kidney transplantation. However, KRT is the tip of the iceberg in the burden of CKD. The main burden of CKD is accelerated ageing and premature death. The cut-off points for kidney function and kidney damage indexes that define CKD also mark an increased risk for all-cause premature death. CKD is the most prevalent risk factor for lethal coronavirus disease 2019 (COVID-19) and the factor that most increases the risk of death in COVID-19, after old age. Men and women undergoing KRT still have an annual mortality that is 10- to 100-fold higher than similar-age peers, and life expectancy is shortened by ~40 years for young persons on dialysis and by 15 years for young persons with a functioning kidney graft. CKD is expected to become the fifth greatest global cause of death by 2040 and the second greatest cause of death in Spain before the end of the century, a time when one in four Spaniards will have CKD. However, by 2022, CKD will become the only top-15 global predicted cause of death that is not supported by a dedicated well-funded Centres for Biomedical Research (CIBER) network structure in Spain. Realizing the underestimation of the CKD burden of disease by health authorities, the Decade of the Kidney initiative for 2020-2030 was launched by the American Association of Kidney Patients and the European Kidney Health Alliance. Leading Spanish kidney researchers grouped in the kidney collaborative research network Red de Investigación Renal have now applied for the Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS) call for collaborative research in Spain with the support of the Spanish Society of Nephrology, Federación Nacional de Asociaciones para la Lucha Contra las Enfermedades del Riñón and ONT: RICORS2040 aims to prevent the dire predictions for the global 2040 burden of CKD from becoming true

Lipids modulate the increase of BK channel calcium sensitivity by the β1 subunit.

Co-expression of the auxiliary β1 subunit with the pore forming α subunit of BK dramatically alters apparent calcium sensitivity. Investigation of the mechanism underlying the increase in calcium sensitivity of BK in smooth muscle has concentrated on the energetic effect of β1's interaction with α. We take a novel approach, exploring whether β1 modification of calcium sensitivity reflects altered interaction between the channel protein and surrounding lipids. We reconstituted hSlo BK α and BK α+β1 channels into two sets of bilayers. One set contained POPE with POPS, POPG, POPA and POPC, where the length of acyl chains is constant, but surface charge differs. The second set is a series of neutral bilayers formed from DOPE with phosphatidylcholines (PCs) of varying acyl chain lengths: C (14:1), C (18:1), C (22:1) and C (24:1), and with brain sphingomyelin (SPM), in which surface charge is constant, but bilayer thickness varies. The increase in calcium sensitivity caused by the β1 subunit was preserved in negatively charged lipid bilayers but not in neutral bilayers, indicating that modification of apparent Ca(2+) sensitivity by β1 is modulated by membrane lipids, requiring negatively charged lipids in the membrane. Moreover, the presence of β1 reduces BK activity in thin bilayers of PC 14:1 and thick bilayers containing SPM, but has no significant effect on activity of BK in PC 18:1, PC 22:1 and PC 24:1 bilayers. These data suggest that auxiliary β1 subunits fine-tune channel gating not only through direct subunit-subunit interactions but also by modulating lipid-protein interactions

BK ZERO isoform HEK293 stably transfected cell lines differing 3'UTRs to assess miR-9 regulation.

Research has identified the large conductance voltage- and calcium-activated potassium channel (BK) as a key regulator of neuronal excitability genetically associated to behavioral alcohol tolerance. Sensitivity to ethanol at the molecular level is characterized by acute potentiation of channel activity. BK isoforms show variations in alcohol sensitivity and are differentially distributed on the plasma membrane surface in response to prolonged exposure. MicroRNA (MiRNA) targeting of alcohol-sensitive isoforms coupled with active internalization of BK channels in response to ethanol are believed to be key in establishing homeostatic adaptations that produce persistent changes within the plasma membrane of neurons. In fact, microRNA 9 (miR-9) upregulated expression is a key event in persistent alcohol tolerance mediating acute EtOH desensitization of BK channels. The exact nature of these interactions remains a current topic of discussion. To further study the effects of miR-9 on the expression and distribution of BK channel isoforms we designed an experimental model by transfecting human BK channel isoforms ZERO heterologous constructs in human embryonic kidney cells 293 (HEK293) cells respectively expressing 2.1 (miR-9 responsive), 2.2 (unresponsive) and control (no sequence) 3'untranslated region (3'UTR) miRNA recognition sites. We used imaging techniques to characterize the stably transfected monoclonal cell lines, and electrophysiology to validate channel activity. Finally, we used immunocytochemistry to validate isoform responsiveness to miR-9. Our findings suggest the cell lines were successfully transfected to express either the 2.1 or 2.2 version of ZERO. Patch clamp recordings confirm that these channels retain their functionality and immunohistochemistry shows differential responses to miR-9, making these cells viable for use in future alcohol dependence studies

β1 has little effect on the apparent Ca²⁺ sensitivity of BK in the neutral POPE/POPC bilayer, but dramatically increases the apparent Ca²⁺ in negatively charged lipid bilayers of POPE/POPA, POPE/POPS, and POPE/POPG.

<p>A, Sample currents from a single channel of hSlo α and hSlo αβ1 recorded in POPE/POPC bilayer at low [Ca²⁺] of 6.2 µM with holding potentials from −40 mV to +40 mV. B, Sample currents of a single channel of hSlo α and hSlo αβ1 recorded in POPE/POPC bilayer at high [Ca²⁺] of 22.5 µM with varied holding potentials from −40 mV to +40 mV. C, plots of log Po vs V of BK α (open symbols) and BK αβ1 (filled symbols) recorded at three different [Ca²⁺] concentrations of 6.2 µM (triangles), 15.6 µM (squares) and 22.5 µM (circles) in POPE/POPC bilayers. These data demonstrate that β1 has little effect on the apparent Ca²⁺ sensitivity of the BK channel in POPE/POPC bilayers. D, Plots of log Po – V of BK α (open symbols) and BK αβ1 (closed symbols) in bilayers of POPE/POPC, POPE/POPG, POPE/POPS and POPE/POPA at [Ca²⁺] = 15.6 µM. Error bars represent SEM, <i>n</i> = 3–7.</p

The open and closed time distribution of BK hSlo α and hSlo αβ1 recorded at free [Ca²⁺] of 6. 2 µM.

<p>Dwell-time data were plotted with a logarithmic time axis along the <i>abscissa</i> and a square-root <i>ordinate</i> exhibiting the number of events in each bin. The bin density is 20 bins per decade. A lower limit of 0.6 ms was set for the dwell time distribution of open and closed intervals, consistent with the time resolution of sampling and filtering. The time constants for each fit of the open and closed time distribution are listed in the figure, and the fractional contribution of each particular component to the composite fit is given in <i>parentheses</i>.</p

Molecular structure of the lipids used in this study.

<p>The left panel shows lipids having identical acyl chains but with different polar headgroups. The right panel shows lipids having identical polar headgroups but different acyl chain length.</p

β1 creates a leftward shift in the G-V relationship of BK channels.

<p>A) The macroscopic currents recorded from HEK 293 cells stably transected with hSlo α with 5 uM Ca²⁺ inside the pipette and in the bath. The holding potential is −60 mV, traces from 0 to 220 mV with delta 20 mV. B) G-V relations determined at 1, 5, and 10 µM Ca²⁺ concentrations. C) The macroscopic currents recorded for hSlo αβ1 at 5 µM Ca²⁺. The holding potential is −60 mV, and traces from −60 to 160 mV with delta 20 mV. D) G-V relations determined at 1, 5, and 10 µM Ca²⁺ concentrations for hSlo αβ1. Each curve in B and D represents the average of between 3 and 7 individual curves. Error bars indicate SEM. E) Plots of half-maximal activation voltage (V_1/2) vs. Ca²⁺ concentration. V1/2 were statistically different between hSlo and hSlo αβ1 for every calcium concentration tested. Error bars represent SEM, <i>n</i>  = 4–7.</p