Synaptotagmin 9 Modulates Spontaneous Neurotransmitter Release in Striatal Neurons by Regulating Substance P Secretion.

Synaptotagmin 9 (SYT9) is a tandem C2 domain Ca2+ sensor for exocytosis in neuroendocrine cells; its function in neurons remains unclear. Here, we show that, in mixed-sex cultures, SYT9 does not trigger rapid synaptic vesicle exocytosis in mouse cortical, hippocampal, or striatal neurons, unless it is massively overexpressed. In striatal neurons, loss of SYT9 reduced the frequency of spontaneous neurotransmitter release events (minis). We delved into the underlying mechanism and discovered that SYT9 was localized to dense-core vesicles that contain substance P (SP). Loss of SYT9 impaired SP release, causing the observed decrease in mini frequency. This model is further supported by loss of function mutants. Namely, Ca2+ binding to the C2A domain of SYT9 triggered membrane fusion in vitro, and mutations that disrupted this activity abolished the ability of SYT9 to regulate both SP release and mini frequency. We conclude that SYT9 indirectly regulates synaptic transmission in striatal neurons by controlling SP release.SIGNIFICANCE STATEMENT Synaptotagmin 9 (SYT9) has been described as a Ca2+ sensor for dense-core vesicle (DCV) exocytosis in neuroendocrine cells, but its role in neurons remains unclear, despite widespread expression in the brain. This article examines the role of SYT9 in synaptic transmission across cultured cortical, hippocampal, and striatal neuronal preparations. We found that SYT9 regulates spontaneous neurotransmitter release in striatal neurons by serving as a Ca2+ sensor for the release of the neuromodulator substance P from DCVs. This demonstrates a novel role for SYT9 in neurons and uncovers a new field of study into neuromodulation by SYT9, a protein that is widely expressed in the brain.

External Ion Access in the Na/K Pump: Kinetics of Na+, K+, and Quaternary Amine Interaction.

Na/K pumps build essential ion gradients across the plasmalemma of animal cells by coupling the extrusion of three Na+, with the import of two K+ and the hydrolysis of one ATP molecule. The mechanisms of selectivity and competition between Na+, K+, and inhibitory amines remain unclear. We measured the effects of external tetrapropylammonium (TPA+) and ethylenediamine (EDA2+) on three different Na/K pump transport modes in voltage-clamped Xenopus oocytes: 1) outward pump current (IP), 2) passive inward H+ current at negative voltages without Na+ or K+ (IH), and 3) transient charge movement reporting the voltage-dependent extracellular binding/release of Na+ (QNa). Both amines competed with K+ to inhibit IP. TPA+ inhibited IH without competing with H+, whereas EDA2+ did not alter IH at pH 7.6. TPA+ competed with Na+ in QNa measurements, reducing Na+-apparent affinity, evidenced by a ∼-75 mV shift in the charge-voltage curve (at 20 mM TPA+) without reduction of the total charge moved (Qtot). In contrast, EDA2+ and K+ did not compete with Na+ to inhibit QNa; both reduced Qtot without decreasing Na+-apparent affinity. EDA2+ (15 mM) right-shifted the charge-voltage curve by ∼+50 mV. Simultaneous occlusion of EDA2+ and Na+ by an E2P conformation unable to reach E1P was demonstrated by voltage-clamp fluorometry. Trypsinolysis experiments showed that EDA2+-bound pumps are much more proteolysis-resistant than Na+-, K+-, or TPA+-bound pumps, therefore uncovering unique EDA2+-bound conformations. K+ effects on QNa and IH were also evaluated in pumps inhibited with beryllium fluoride, a phosphate mimic. K+ reduced Qtot without shifting the charge-voltage curve, indicating noncompetitive effects, and partially inhibited IH to the same extent as TPA+ in non-beryllium-fluorinated pumps. These results demonstrate that K+ interacts with beryllium-fluorinated pumps inducing conformational changes that alter QNa and IH, suggesting that there are two external access pathways for proton transport by IH.

Intracellular Requirements for Passive Proton Transport through the Na+,K+-ATPase.

The Na+,K+-ATPase (NKA or Na/K pump) hydrolyzes one ATP to exchange three intracellular Na+ (Na+i) for two extracellular K+ (K+o) across the plasma membrane by cycling through a set of reversible transitions between phosphorylated and dephosphorylated conformations, alternately opening ion-binding sites externally (E2) or internally (E1). With subsaturating [Na+]o and [K+]o, the phosphorylated E2P conformation passively imports protons generating an inward current (IH), which may be exacerbated in NKA-subunit mutations associated with human disease. To elucidate the mechanisms of IH, we studied the effects of intracellular ligands (transported ions, nucleotides, and beryllium fluoride) on IH and, for comparison, on transient currents measured at normal Na+o (QNa). Utilizing inside-out patches from Xenopus oocytes heterologously expressing NKA, we observed that 1) in the presence of Na+i, IH and QNa were both activated by ATP, but not ADP; 2) the [Na+]i dependence of IH in saturating ATP showed K0.5,Na = 1.8 ± 0.2 mM and the [ATP] dependence at saturating [Na+]i yielded K0.5,ATP = 48 ± 11 µM (in comparison, Na+i-dependent QNa yields K0.5,Na = 0.8 ± 0.2 mM and K0.5,ATP = 0.43 ± 0.03 µM; 3) ATP activated IH in the presence of K+i (∼15% of the IH observed in Na+i) only when Mg2+i was also present; and 4) beryllium fluoride induced maximal IH even in the absence of nucleotide. These data indicate that IH occurs when NKA is in an externally open E2P state with nucleotide bound, a conformation that can be reached through forward Na/K pump phosphorylation of E1, with Na+i and ATP, or by backward binding of K+i to E1, which drives the pump to the occluded E2(2K), where free Pi (at the micromolar levels found in millimolar ATP solutions) promotes external release of occluded K+ by backdoor NKA phosphorylation. Maximal IH through beryllium-fluorinated NKA indicates that this complex mimics ATP-bound E2P states.

Retrospective Review of Critically Ill Patients Experiencing Alcohol Withdrawal: Dexmedetomidine Versus Propofol and/or Lorazepam Continuous Infusions.

BACKGROUND: Alcohol withdrawal symptoms can be difficult to manage and may lead to an intensive care unit (ICU) admission. Patients experiencing severe alcohol withdrawal often require high doses of sedatives, which can lead to respiratory depression and the need for endotracheal intubation. Dexmedetomidine, an alpha-2 adrenoreceptor agonist, provides adequate sedation with little effect on respiratory function when compared to other sedatives. OBJECTIVE: To evaluate sedation with a continuous infusion of dexmedetomidine versus propofol and/or lorazepam in critically ill patients experiencing alcohol withdrawal. METHODS: A retrospective chart review was conducted on ICU admissions between March 2002 and April 2009 for alcohol withdrawal patients who necessitated treatment with a continuous infusion of dexmedetomidine, propofol, and/or lorazepam. Primary outcomes included the incidence of mechanical ventilation, length of mechanical ventilation (if applicable), and ICU and hospital length of stay. RESULTS: Fifteen patients were treated with a continuous infusion of dexmedetomidine, and 17 were treated with an infusion of propofol and/or lorazepam. Two patients (13.3%) required intubation and mechanical ventilation in the dexmedetomidine group versus 10 (58.8%) in the propofol and/or lorazepam group (P = .006). Length of stay in the ICU was 53 hours for patients treated with dexmedetomidine versus 114.9 hours in the propofol and/or lorazepam group (P = .016). Hospital length of stay was less for the dexmedetomidine group, 135.8 hours versus 241.1 hours in the propofol and/or lorazepam group (P = .008). CONCLUSIONS: Dexmedetomidine use was associated with a decrease in the incidence of endotracheal intubation when used to sedate patients experiencing alcohol withdrawal. Patients transferred to a lower level of care faster and were discharged from the hospital sooner when treated with dexmedetomidine.