Involvement of Kv1.3 and p38 MAPK signaling in HIV-1 glycoprotein 120-induced microglia neurotoxicity

Inflammatory responses mediated by activated microglia play a pivotal role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorders. Studies on identification of specific targets to control microglia activation and resultant neurotoxic activity are imperative. Increasing evidence indicate that voltage-gated K⁺ (K_v) channels are involved in the regulation of microglia functionality. In this study, we investigated K_v1.3 channels in the regulation of neurotoxic activity mediated by HIV-1 glycoprotein 120 (gp120)-stimulated rat microglia. Our results showed treatment of microglia with gp120 increased the expression levels of K_v1.3 mRNA and protein. In parallel, whole-cell patch-clamp studies revealed that gp120 enhanced microglia K_v1.3 current, which was blocked by margatoxin, a K_v1.3 blocker. The association of gp120 enhancement of K_v1.3 current with microglia neurotoxicity was demonstrated by experimental results that blocking microglia K_v1.3 attenuated gp120-associated microglia production of neurotoxins and neurotoxicity. Knockdown of K_v1.3 gene by transfection of microglia with K_v1.3-siRNA abrogated gp120-associated microglia neurotoxic activity. Further investigation unraveled an involvement of p38 MAPK in gp120 enhancement of microglia K_v1.3 expression and resultant neurotoxic activity. These results suggest not only a role K_v1.3 may have in gp120-associated microglia neurotoxic activity, but also a potential target for the development of therapeutic strategies.     

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Anti-allodynic effect of 2-(aminomethyl)adamantane-1-carboxylic acid in a rat model of neuropathic pain: A mechanism dependent on CaV2.2 channel inhibition

Neuropathic pain is a serious physical disabling condition resulting from lesion or dysfunction of the peripheral sensory nervous system. Despite the fact that the mechanisms underlying neuropathic pain are poorly understood, the involvement of voltage-gated calcium (Ca_V) channels in its pathophysiology has justified the use of drugs that bind the Ca_V channel α₂δ auxiliary subunit, such as gabapentin (GBP), to attain analgesic and anti-allodynic effects in models involving neuronal sensitization and nerve injury. GBP binding to α₂δ inhibits nerve injury-induced trafficking of the α₁ pore forming subunits of Ca_V channels, particularly of the N-type, from the cytoplasm to the plasma membrane of pre-synaptic terminals in dorsal root ganglion neurons and dorsal horn spinal neurons. In the search for alternative forms of treatment, in this study we describe the synthesis and pharmacological profile of a GABA derivative, 2-aminoadamantane-1-carboxylic acid (GZ4), which displays a close structure–activity relationship with GBP. Behavioral assessment using von Frey filament stimuli showed that GZ4 treatment reverted mechanical allodynia/hyperalgesia in an animal model of spinal nerve ligation-induced neuropathic pain. In addition, using the patch clamp technique we show that GZ4 treatment significantly decreased whole-cell currents through N-type Ca_V channels heterologously expressed in HEK-293 cells. Interestingly, the behavioral and electrophysiological time course of GZ4 actions reflects that its mechanism of action is similar but not identical to that of GBP. While GBP actions require at least 24 h and imply uptake of the drug, which suggests that the drug acts mainly intracellularly affecting channels trafficking to the plasma membrane, the faster time course (1–3 h) of GZ4 effects suggests also a direct inhibition of Ca²⁺ currents acting on cell surface channels.     

The Alpha and Omega of Galactosylceramides in T Cell Immune Function

Glycosphingolipids are a subgroup of glycolipids that contain an amino alcohol sphingoid base linked to sugars. They are found in the membranes of cells ranging from bacteria to vertebrates. This group of lipids is known to stimulate the immune system through activation of a type of white blood cell known as natural killer T cell (NKT cell). Here we summarize the extensive research that has been done to identify the structures of natural glycolipids that stimulate NKT cells and to determine how these antigens are recognized. We also review studies designed to understand how glycolipid variants, both natural and synthetic, can alter the responses of NKT cells, leading to dramatic changes in the global immune response.     

The Effects of Aging on Information-Processing Channels in the Sense of Touch: I. Absolute Sensitivity

Thresholds for detecting vibrotactile signals of variable frequency applied to the thenar eminence of the hand by small and large contactors were measured in subjects ranging in age from 10 to 89 years. Thresholds were found to increase as a function of age, but the rate of increase was greater after than before the age of 65 years. The rate of loss of vibrotactile sensitivity was substantially greater in the P channel (mediated by Pacinian corpuscles) than in the NP I channel (mediated by rapidly adapting fibers), the NP II channel (mediated by slowly adapting type II fibers), or the NP HI channel (mediated by slowly adapting type I fibers). Women were frequently found to have greater sensitivity than men.     

Correlation of open cell-attached and excised patch clamp techniques

The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na⁺ channel, located on the apical surface of A₆ cells, to determine whether the nystatininduced open cell-attached patch could serve as an alternative configuration.Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and E _rev was zero in symmetrical Na⁺ solutions, indicating similar Na⁺ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3–4pS) and E _rev (75 mV), when the bath was perfused with a high K:low Na solution (E _Na=80 mV), were nearly equal in both patch configurations.Our results therefore indicate that the nystatininduced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.We thank Dr. Olaf S. Andersen for his suggestions in the development of the open cell-attached recording technique. This work was supported by a National Institutes of Health grant (DK-18061)     

The Ca2+ channel β2 subunit is selectively targeted to the axon terminals of supraoptic neurons

The assembly of high voltage-activated Ca²⁺ channels with different β subunits influences channel properties and possibly subcellular targeting. We studied β subunit expression in the somata and axon terminals of the magnocellular neurosecretory cells, which are located in the supraoptic nucleus (SON) and neurohypophysis, respectively. Antibodies directed against the 4 Ca_Vβ subunits (Ca_Vβ₁-Ca_Vβ₄) were used for immunoblots and for immunostaining of slices of these two tissues. We found that all 4 β subunits are expressed in both locations, but that Ca_Vβ₂ had the highest relative expression in the neurohypophysis. These data suggest that the Ca_Vβ₂ subunit is selectively targeted to axon terminals and may play a role in targeting and/or regulating the properties of Ca²⁺ channels.     

Spontaneous opening at zero membrane potential of sodium channels from eel electroplax reconstituted into lipid vesicles

Summary The voltage-dependent sodium channel from the eel electroplax was purified and reconstituted into vesicles of varying lipid composition. Isotopic sodium uptake experiments were conducted with vesicles at zero membrane potential, using veratridine to activate channels and tetrodotoxin to block them. Under these conditions, channel-dependent uptake of isotopic sodium by the vesicles was observed, demonstrating that a certain fraction of the reconstituted protein was capable of mediating ion fluxes. In addition, vesicles untreated with veratridine showed significant background uptake of sodium; a considerable proportion of this flux was blocked by tetrodotoxin. Thus these measurements showed that a significant subpopulation of channels was present that could mediate ionic fluxes in the absence of activating toxins. The proportion of channels exhibiting this behavior was dependent on the lipid composition of the vesicles and the temperature at which the uptake was measured; furthermore, the effect of temperature was reversible. However, the phenomenon was not affected by the degree of purification of the protein used for reconstitution, and channels in resealed electroplax membrane fragments or reconstituted, solely into native eel lipids did not show this behavior. The kinetics of vesicular uptake through these spontaneously-opening channels was slow, and we attribute this behavior to a modification of sodium channel inactivation.