Infection promotes Ser-214 phosphorylation important for generation of cytotoxic tau variants.

Patients who recover from hospital-acquired pneumonia exhibit a high incidence of end-organ dysfunction following hospital discharge, including cognitive deficits. We have previously demonstrated that pneumonia induces the production and release of cytotoxic oligomeric tau from pulmonary endothelial cells, and these tau oligomers can enter the circulation and may be a cause of long-term morbidities. Endothelial-derived oligomeric tau is hyperphosphorylated during infection. The purpose of these studies was to determine whether Ser-214 phosphorylation of tau is a necessary stimulus for generation of cytotoxic tau variants. The results of these studies demonstrate that Ser-214 phosphorylation is critical for the cytotoxic properties of infection-induced oligomeric tau. In the lung, Ser-214 phosphorylated tau contributes to disruption of the alveolar-capillary barrier, resulting in increased permeability. However, in the brain, both the Ser-214 phosphorylated tau and the mutant Ser-214-Ala tau, which cannot be phosphorylated, disrupted hippocampal long-term potentiation suggesting that inhibition of long-term potentiation was relatively insensitive to the phosphorylation status of Ser-214. Nonetheless, phosphorylation of tau is essential to its cytotoxicity since global dephosphorylation of the infection-induced cytotoxic tau variants rescued long-term potentiation. Collectively, these data demonstrate that multiple forms of oligomeric tau are generated during infectious pneumonia, with different forms of oligomeric tau being responsible for dysfunction of distinct end-organs during pneumonia.

Modulation of BK channel calcium affinity by differential phosphorylation in developing ovine basilar artery myocytes.

Large-conductance Ca2+-sensitive K+ (BK) channel activity is greater in basilar artery smooth muscle cells (SMCs) of the fetus than the adult, and this increased activity is associated with a lower BK channel Ca2+ set point (Ca0). Associated PKG activity is three times greater in BK channels from fetal than adult myocytes, whereas associated PKA activity is three times greater in channels from adult than fetal myocytes. We hypothesized that the change in Ca0 during development results from different levels of channel phosphorylation. In inside-out membrane patch preparations of basilar artery SMCs from adult and fetal sheep, we measured BK channel activity in four states of phosphorylation: native, dephosphorylated, PKA phosphorylated, and PKG phosphorylated. BK channels from adult and fetus exhibited similar voltage-activation curves, Ca0 values, and Ca2+ dissociation constants (Kd) for the dephosphorylated, PKA phosphorylated, and PKG phosphorylated states. However, voltage-activation curves of native fetal BK channels shifted significantly to the left of those of the adult, with Ca0 and Kd values half those of the adult. For the two age groups at each of the phosphorylation states, Ca0 and Kd produced linear relations when plotted against voltage at half-maximal channel activation. We conclude that the Ca0 and Kd values of the BK channel can be modulated by differential channel phosphorylation. Lower Ca0 and Kd values in BK channels of fetal myocytes can be explained by a greater extent of channel phosphorylation of fetal than adult myocytes.

Ca2+-activated K+ channel-associated phosphatase and kinase activities during development.

In ovine basilar arterial smooth muscle cells (SMCs), the fetal "big" Ca2+-activated K+ (BK) channel activity is significantly greater and has a lower Ca2+ setpoint than BK channels from adult cells. In the present study, we tested the hypothesis that these differences result from developmentally regulated phosphorylation of these channels. Using the patch-clamp technique and a novel in situ enzymological approach, we measured the rates and extents of changes in BK channel voltage activation from SMC inside-out patch preparations in response to selective activation and inhibition of channel-associated protein phosphatases and kinases (CAPAKs). We show that BK channel activity is modulated during development by differential phosphorylation and that the activities of CAPAKs change substantially during development. In particular, excised membrane patches from adult SMCs exhibited greater protein kinase A activity than those from a fetus. In contrast, fetal SMCs exhibited greater protein kinase G activity and phosphatase activity than adult SMCs. These findings extend our previous observation that the BK channel Ca2+ setpoint differs significantly in adult and fetal cerebrovascular myocytes and suggest a biochemical mechanism for this difference. In addition, these findings suggest that the functional stoichiometry of CAPAKs varies significantly during development and that such variation may be a hitherto unrecognized mechanism of ion channel regulation.

Developmental differences in Ca2+-activated K+ channel activity in ovine basilar artery.

A primary determinant of vascular smooth muscle (VSM) tone and contractility is the resting membrane potential, which, in turn, is influenced heavily by K+ channel activity. Previous studies from our laboratory and others have demonstrated differences in the contractility of cerebral arteries from near-term fetal and adult animals. To test the hypothesis that these contractility differences result from maturational changes in voltage-gated K+ channel function, we compared this function in VSM myocytes from adult and fetal sheep cerebral arteries. The primary current-carrying, voltage-gated K+ channels in VSM myocytes are the large conductance Ca2+-activated K+ channels (BKCa) and voltage-activated K+ (KV) channels. We observed that at voltage-clamped membrane potentials of +60 mV in perforated whole cell studies, the normalized outward current densities in fetal myocytes were >30% higher than in those of the adult (P < 0.05) and that these were predominantly due to iberiotoxin-sensitive currents from BKCa channels. Excised, insideout membrane patches revealed nearly identical unitary conductances and Hill coefficients for BKCa channels. The plot of log intracellular [Ca2+] ([Ca2+]i) versus voltage for half-maximal activation (V(1/2)) yielded linear and parallel relationships, and the change in V(1/2) for a 10-fold change in [Ca2+] was also similar. Channel activity increased e-fold for a 19 +/- 2-mV depolarization for adult myocytes and for an 18 +/- 1-mV depolarization for fetal myocytes (P > 0.05). However, the relationship between BKCa open probability and membrane potential had a relative leftward shift for the fetal compared with adult myocytes at different [Ca2+]i. The [Ca2+] for half-maximal activation (i.e., the calcium set points) at 0 mV were 8.8 and 4.7 microM for adult and fetal myocytes, respectively. Thus the increased BKCa current density in fetal myocytes appears to result from a lower calcium set point.