Development and implementation of a scalable and versatile test for COVID-19 diagnostics in rural communities.

Rapid and widespread testing of severe acute respiratory coronavirus 2 (SARS-CoV-2) is essential for an effective public health response aimed at containing and mitigating the coronavirus disease 2019 (COVID-19) pandemic. Successful health policy implementation relies on early identification of infected individuals and extensive contact tracing. However, rural communities, where resources for testing are sparse or simply absent, face distinctive challenges to achieving this success. Accordingly, we report the development of an academic, public land grant University laboratory-based detection assay for the identification of SARS-CoV-2 in samples from various clinical specimens that can be readily deployed in areas where access to testing is limited. The test, which is a quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based procedure, was validated on samples provided by the state laboratory and submitted for FDA Emergency Use Authorization. Our test exhibits comparable sensitivity and exceeds specificity and inclusivity values compared to other molecular assays. Additionally, this test can be re-configured to meet supply chain shortages, modified for scale up demands, and is amenable to several clinical specimens. Test development also involved 3D engineering critical supplies and formulating a stable collection media that allowed samples to be transported for hours over a dispersed rural region without the need for a cold-chain. These two elements that were critical when shortages impacted testing and when personnel needed to reach areas that were geographically isolated from the testing center. Overall, using a robust, easy-to-adapt methodology, we show that an academic laboratory can supplement COVID-19 testing needs and help local health departments assess and manage outbreaks. This additional testing capacity is particularly germane for smaller cities and rural regions that would otherwise be unable to meet the testing demand.

Clinical Neuroscience in Practice: An Experiential Learning Course for Undergraduates Offered by Neurosurgeons and Neuroscientists.

Many pre-health students pursue extracurricular shadowing opportunities to gain clinical experience. The Virginia Tech School of Neuroscience introduced a formal course that provides a clinical experience superior to that received by many medical students. This course is composed of weekly 75-minute seminars that cover diseases affecting the nervous system, their diagnosis and treatment, complemented by weekly half-day intensive clinical experiences with unprecedented access to a team of neurosurgeons (in hospital operating rooms, Intensive Care Units, emergency room, angiographic suites, and wards). In the operating rooms, students routinely "scrub-in" for complex surgeries. On hospital rounds, students experience direct patient care and receive in-depth exposure to modern nervous system imaging. Students participate in two 24-hour "on-call" experiences with team providers. After call, students participate in cognitive and psychological studies to assess physiological and psychological effects of call-related sleep deprivation. Students prepare weekly essays on challenging socioeconomic and ethical questions, exploring subjects such as the cost of medicine and inequalities in access to health care. Towards the end of the course, students meet with the admission dean of the Virginia Tech Carilion medical school; they prepare a personal statement for medical school/graduate school applications, and attend a half-day block of mock medical school/graduate school interviews delivered by experienced clinicians. In lieu of a final exam, each student presents to the entire neurosurgery department, an in-depth clinical analysis of a case in which they participated. We provide details on implementation, challenges and outcomes based on experiences from three semesters with a total enrollment of approximately 60 students.

Water permeability through aquaporin-4 is regulated by protein kinase C and becomes rate-limiting for glioma invasion.

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K(+)-Cl(-) cotransporter 1 (KCC1), believed to provide the pathways for Cl(-) and K(+) secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process.

Inhibition of transient receptor potential canonical channels impairs cytokinesis in human malignant gliomas.

OBJECTIVES: Glial-derived primary brain tumours, gliomas, are among the fastest growing malignancies and present a huge clinical challenge. Research suggests an important, yet poorly understood, role of ion channels in growth control of normal and malignant cells. In this study, we sought to functionally characterize Transient Receptor Potential Canoncial (TRPC) channels in glioma cell proliferation. TRPC channels form non-selective cation channels that have been suggested to represent a Ca(2+) influx pathway impacting cellular growth. MATERIALS AND METHODS: Employing a combination of molecular, biochemical and biophysical techniques, we characterized TRPC channels in glioma cells. RESULTS: We showed consistent expression of four channel family members (TRPC-1, -3, -5, -6) in glioma cell lines and acute patient-derived tissues. These channels gave rise to small, non-voltage-dependent cation currents that were blocked by the TRPC inhibitors GdCl(3), 2-APB, or SKF96365. Importantly, TRPC channels contributed to the resting conductance of glioma cells and their acute pharmacological inhibition caused an approximately 10 mV hyperpolarization of the cells' resting potential. Additionally, chronic application of the TRPC inhibitor SKF96365 caused near complete growth arrest. A detailed analysis, by fluorescence-activated cell sorting and time-lapse microscopy, showed that growth inhibition occurred at the G(2)+ M phase of the cell cycle with cytokinesis defects. Cells underwent incomplete cell divisions and became multinucleate, enlarged cells. CONCLUSIONS: Nuclear atypia and enlarged cells are histopathological hallmarks for glioblastoma multiforme, the highest grade glioma, suggesting that a defect in TRPC channel function may contribute to cellular abnormalities in these tumours.

Differential distribution of Kir4.1 in spinal cord astrocytes suggests regional differences in K+ homeostasis.

Neuronal activity in the spinal cord results in extracellular potassium accumulation that is significantly higher in the dorsal horn than in the ventral horn. This is suggestive of differences in K(+) clearance, widely thought to involve diffusional K(+) uptake by astrocytes. We previously identified the inward rectifying K(+) channel Kir4.1 as the major K(+) conductance in spinal cord astrocytes in situ and hence hypothesized that different expression levels of Kir4.1 may account for the observed differences in potassium dynamics in spinal cord. Our results with immunohistochemical staining demonstrated highest Kir4.1 channel expression in the ventral horn and very low levels of Kir4.1 in the apex of the dorsal horn. Western blots from tissue of these two regions similarly confirmed much lower levels of Kir4.1 in the apex of the dorsal horn. Whole cell patch-clamp recordings from astrocytes in rat spinal cord slices also showed a difference in inwardly rectifying currents in these two regions. However, no statistical difference in either fast-inactivating (Ka) or delayed rectifying potassium currents (Kd) was observed, suggesting these differences were specific to Kir currents. Importantly, when astrocytes in each region were challenged with high [K(+)](o), astrocytes from the dorsal horn showed significantly smaller (60%) K(+) uptake currents than astrocytes from the ventral horn. Taken together, these data support the conclusion that regional differences in astrocytic expression of Kir4.1 channels result in marked changes in potassium clearance rates in these two regions of the spinal cord.

Functional expression of Kir4.1 channels in spinal cord astrocytes.

Spinal cord astrocytes (SCA) have a high permeability to K+ and hence have hyperpolarized resting membrane potentials. The underlying K+ channels are believed to participate in the uptake of neuronally released K+. These K+ channels have been studied extensively with regard to their biophysics and pharmacology, but their molecular identity in spinal cord is currently unknown. Using a combination of approaches, we demonstrate that channels composed of the Kir4.1 subunit are responsible for mediating the resting K+ conductance in SCA. Biophysical analysis demonstrates astrocytic Kir currents as weakly rectifying, potentiated by increasing [K+]o, and inhibited by micromolar concentrations of Ba2+. These currents were insensitive to tolbutemide, a selective blocker of Kir6.x channels, and to tertiapin, a blocker for Kir1.1 and Kir3.1/3.4 channels. PCR and Western blot analysis show prominent expression of Kir4.1 in SCA, and immunocytochemistry shows localization Kir4.1 channels to the plasma membrane. Kir4.1 protein levels show a developmental upregulation in vivo that parallels an increase in currents recorded over the same time period. Kir4.1 is highly expressed throughout most areas of the gray matter in spinal cord in vivo and recordings from spinal cord slices show prominent Kir currents. Electrophysiological recordings comparing SCA of wild-type mice with those of homozygote Kir4.1 knockout mice confirm a complete and selective absence of Kir channels in the knockout mice, suggesting that Kir4.1 is the principle channel mediating the resting K+ conductance in SCA in vitro and in situ.

Modulation of glioma BK channels via erbB2.

Glioma cells show up-regulation and constitutive activation of erbB2, and its expression correlates positively with increased malignancy. A similar correlation has been demonstrated for the expression of gBK, a calcium-sensitive, large-conductance K(+) channel. We show here that glioma BK channels are a downstream target of erbB2/neuregulin signaling. Tyrphostin AG825 was able to disrupt the constituitive erbB2 activation in a dose-dependent manner, causing a 30-mV positive shift in gBK channel activation in cell-attached patches. Conversely, maximal stimulation of erbB2 with a recombinant neuregulin (NRG-1beta) caused a 12-mV shift in the opposite direction. RT-PCR studies reveal no change in the BK splice variants expressed in treated glioma cells. Furthermore, isolation of surface proteins through biotinylation did not show a change in gBK channel expression, and probing with phospho-specific antibodies showed no alteration in channel phosphorylation. However, fura-II Ca(2+) fluorescence imaging revealed a 35% decrease in the free intracellular Ca(2+) concentration after erbB2 inhibition and an increase in NRG-1beta-treated cells, suggesting that the observed changes most likely were due to alterations in [Ca(2+)](i). Consistent with this conclusion, neither tyrphostin AG825 nor NRG-1beta was able to modulate gBK channels under inside-out or whole-cell recording conditions when intracellular Ca(2+) was fixed. Thus, gBK channels are a downstream target for the abundantly expressed neuregulin-1 receptor erbB2 in glioma cells. However, unlike the case in other systems, this modulation appears to occur via changes in [Ca(2+)](i) without changes in channel expression or phosphorylation. The enhanced sensitivity of gBK channels in glioma cells to small, physiological Ca(2+) changes appears to be a prerequisite for this modulation.

Mislocalization of Kir channels in malignant glia.

Inwardly rectifying potassium (K(ir)) channels are a prominent feature of mature, postmitotic astrocytes. These channels are believed to set the resting membrane potential near the potassium equilibrium potential (E(K)) and are implicated in potassium buffering. A number of previous studies suggest that K(ir) channel expression is indicative of cell differentiation. We therefore set out to examine K(ir) channel expression in malignant glia, which are incapable of differentiation. We used two established and widely used glioma cell lines, D54MG (a WHO grade 4 glioma) and STTG-1 (a WHO grade 3 glioma), and compared them to immature and differentiated astrocytes. Both glioma cell lines were characterized by large outward K(+) currents, depolarized resting membrane potentials (V(m)) (-38.5 +/- 4.2 mV, D54 and -28.1 +/- 3.5 mV, STTG1), and relatively high input resistances (R(m)) (260.6 +/- 64.7 MOmega, D54 and 687.2 +/- 160.3 MOmega, STTG1). These features were reminiscent of immature astrocytes, which also displayed large outward K(+) currents, had a mean V(m) of -51.1 +/- 3.7 and a mean R(m) value of 627.5 +/- 164 MOmega. In contrast, mature astrocytes had a significantly more negative resting membrane potential (-75.2 +/- 0.56 mV), and a mean R(m) of 25.4 +/- 7.4 MOmega. Barium (Ba(2+)) sensitive K(ir) currents were >20-fold larger in mature astrocytes (4.06 +/- 1.1 nS/pF) than in glioma cells (0.169 +/- 0.033 nS/pF D54, 0.244 +/- 0.04 nS/pF STTG1), which had current densities closer to those of dividing, immature astrocytes (0.474 +/- 0.12 nS/pF). Surprisingly, Western blot analysis shows expression of several K(ir) channel subunits in glioma cells (K(ir)2.3, 3.1, and 4.1). However, while in astrocytes these channels localize diffusely throughout the cell, in glioma cells they are found almost exclusively in either the cell nucleus (K(ir)2.3 and 4.1) or ER/Golgi (3.1). These data suggest that mislocalization of K(ir) channel proteins to intracellular compartments is responsible for a lack of appreciable K(ir) currents in glioma cells.

Current transients associated with BK channels in human glioma cells.

We have previously demonstrated the expression of BK channels in human glioma cells. There was a curious feature to the whole-cell currents of glioma cells seen during whole-cell patch-clamp: large, outward current transients accompanied repolarization of the cell membrane following an activating voltage step. This transient current, Itransient, activated and inactivated rapidly (approximately 1 ms). The I-V relationship of Itransient had features that were inconsistent with simple ionic current through open ion channels: (i) Itransient amplitude peaked with a -80 mV voltage change and was invariant over a 200 mV range, and (ii) Itransient remained large and outward at -140 mV. We provide evidence for a direct relationship of Itransient to glioma BK currents. They had an identical time course of activation, identical pharmacology, identical voltage-dependence, and small, random variations in the amplitude of the steady-state BK current and Itransient seen over time were often perfectly in phase. Substituting intracellular K+ with Cs+, Li+, or Na+ ions reversibly reduced Itransient and BK currents. Itransient was not observed in recordings of other BK currents (hbr5 expressed in HEK cells and BK currents in rat neurons), suggesting Itransient is unique to BK currents in human glioma cells. We conclude that Itransient is generated by a mechanism related to the deactivation, and level of prior activation, of glioma BK channels. To account for these findings we propose that K+ ions are "trapped" within glioma BK channels during deactivation and are forced to exit to the extracellular side in a manner independent of membrane potential.

Expression of voltage-gated chloride channels in human glioma cells.

Voltage-gated chloride channels have recently been implicated as being important for cell proliferation and invasive cell migration of primary brain tumors cells. In the present study we provide several lines of evidence that glioma Cl- currents are primarily mediated by ClC-2 and ClC-3, two genes that belong to the ClC superfamily. Transcripts for ClC-2 thru ClC-7 were detected in a human glioma cell line by PCR, whereas only ClC-2, ClC-3, and ClC-5 protein could be identified by Western blot. Prominent ClC-2, -3, and -5 channel expression was also detected in acute patient biopsies from low- and high-grade malignant gliomas. Immunogold electron microscopic studies as well as digital confocal imaging localized a portion of these ClC channels to the plasma membrane. Whole-cell patch-clamp recordings show the presence of two pharmacologically and biophysically distinct Cl- currents that could be specifically reduced by 48 hr exposure of cells to channel-specific antisense oligonucleotides. ClC-3 antisense selectively and significantly reduced the expression of outwardly rectifying current with pronounced voltage-dependent inactivation. Such currents were sensitive to DIDS (200-500 microm) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (165 microm). ClC-2 antisense significantly reduced expression of inwardly rectifying currents, which were potentiated by hyperpolarizing prepulses and inhibited by Cd2+ (200-500 microm). Currents that were mediated by ClC-5 could not be demonstrated. We suggest that ClC-2 and ClC-3 channels are specifically upregulated in glioma membranes and endow glioma cells with an enhanced ability to transport Cl-. This may in turn facilitate rapid changes in cell size and shape as cells divide or invade through tortuous extracellular brain spaces.

(1R,3S)-1-Aminocyclopentane-1,3-dicarboxylic acid (RS-ACPD) reduces intracellular glutamate levels in astrocytes.

(+/-)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) is an equimolar mixture of two enantiomers: (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (SR-ACPD) and 1R,3S-1-Aminocyclopentane-1,3-dicarboxylic acid (RS-ACPD). t-ACPD and SR-ACPD have been commonly used as agonists for metabotropic glutamate receptors (mGluR). Here we demonstrated that RS-ACPD, but not SR-ACPD, is transported into astrocytes with a K(m) of 6.51 +/- 2.38 mM and V(max) of 22.8 +/- 3.4 nmol/mg/min. This low-affinity transport is Na(+)-dependent and is competitively blocked by glutamate or other substrates for the glutamate transporter. RS-ACPD therefore is probably taken up by the glutamate transporter. Prolonged incubation with high levels of RS-ACPD (> 500 microM) induced significant swelling of astrocytes. At lower concentrations (100 microM), RS-ACPD reduced intracellular glutamate content ([Glu](i)) by > 50% without obvious morphological changes. The reduction in [Glu](i) was accompanied by an increase in [glutamine](i). The RS-ACPD's effect on [Glu](i) required glutamine and high levels of phosphate, suggesting that RS-ACPD inhibited phosphate-activated glutaminase (PAG). These data suggest that astrocytic PAG is actively involved in determining the equilibrium between intracellular glutamate and glutamine. By reducing [Glu](i), RS-ACPD reduces the amount of glutamate available for release.

Volume-activated chloride currents contribute to the resting conductance and invasive migration of human glioma cells.

We used an in vitro model for glioma cell invasion (transwell migration assay) and patch-clamp techniques to investigate the role of volume-activated Cl(-) currents (I(Cl,Vol)) in glioma cell invasion. Hypotonic solutions ( approximately 230 mOsm) activated outwardly rectifying currents that reversed near the equilibrium potential for Cl(-) ions (E(Cl)). These currents (I(Cl,Vol)) were sensitive to several known Cl(-) channel inhibitors, including DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The IC(50) for NPPB inhibition of I(Cl,Vol) was 21 microm. Under isotonic conditions, NPPB (165 microm) blocked inward currents (at -40 mV) and increased input resistance in both standard whole-cell recordings and amphotericin perforated-patch recordings. Reducing [Cl(-)](o) under isotonic conditions positively shifted the reversal potential of whole-cell currents. These findings suggest a significant resting Cl(-) conductance in glioma cells. Under isotonic and hypotonic conditions, Cl(-) channels displayed voltage- and time-dependent inactivation and had an I(-) > Cl(-) permeability. To assess the potential role of these channels in cell migration, we studied the chemotactic migration of glioma cells toward laminin or vitronectin in a Boyden chamber containing transwell filters with 8 microm pores. Inhibition of I(Cl,Vol) with NPPB reduced chemotactic migration in a dose-dependent fashion with an IC(50) of 27 microm. Time-lapse video microscopy during patch-clamp recordings revealed visible changes in cell shape and/or movement that accompanied spontaneous activation of I(Cl,Vol), suggesting that I(Cl,Vol) is activated during cell movement, consistent with the effects of NPPB in migration assays. We propose that I(Cl,Vol) contributes to cell shape and volume changes required for glioma cell migration through brain tissue.

Inhibition of glial Na+ and K+ currents by tamoxifen.

Tamoxifen (tmx) is a non-steroidal triphenylethylene derivative that is predominantly known as a competitive antagonist at the estrogen receptor and is used in the treatment of breast cancer. Recent studies suggest that tamoxifen is also beneficial in the treatment of brain metastases and primary brain tumors. Tmx accumulates in brain and its concentration can be up to 46-fold higher than in serum. Therefore, astrocytes may be exposed to tmx in vivo. We use the whole-cell patch-clamp technique to examine the effects of tmx on voltage-dependent cation currents in rat cortical cultures. Using biophysical and pharmacological methods, we isolate sustained and transient outward potassium currents (I(KS) and I(KT), respectively), inwardly rectifying potassium currents (I(KIR)), and transient inward sodium currents (I(Na)). We show that that TTX-sensitive I(Na) is completely inhibited by 10 microm tmx within 5 min. Similarly, tmx blocks I(KS), but does not inhibit I(KT) or I(KIR) at these concentrations. Tmx effects are irreversible with 10 min wash. Interestingly, the currents sensitive to tmx are important in growth control of glial cells (MacFarlane & Sontheimer, 1997). Therefore, we examine cytotoxic and proliferative effects of tmx. Tmx (10 microm) is not cytotoxic as judged by trypan blue exclusion. However, incubation with tmx significantly reduces cell proliferation as examined by 3[H]-thymidine uptake.

Electrophysiological characteristics of reactive astrocytes in experimental cortical dysplasia.

Neocortical freeze lesions have been widely used to study neuronal mechanisms underlying hyperexcitability in dysplastic cortex. Comparatively little attention has been given to biophysical changes in the surrounding astrocytes that show profound morphological and biochemical alterations, often referred to as reactive gliosis. Astrocytes are thought to aid normal neuronal function by buffering extracellular K(+). Compromised astrocytic K(+) buffering has been proposed to contribute to neuronal dysfunction. Astrocytic K(+) buffering is mediated, partially, by the activity of inwardly rectifying K(+) channels (K(IR)) and may involve intracellular redistribution of K(+) through gap-junctions. We characterized K(+) channel expression and gap-junction coupling between astrocytes in freeze-lesion-induced dysplastic neocortex. Whole cell patch-clamp recordings were obtained from astrocytes in slices from postnatal day (P) 16--P24 rats that had received a freeze-lesion on P1. A marked increase in glial fibrillary acidic protein immunoreactivity was observed along the entire length of the freeze lesion. Clusters of proliferative (bromo-deoxyuridine nuclear staining, BrdU+) astrocytes were seen near the depth of the microsulcus. Astrocytes in cortical layer I surrounding the lesion were characterized by a significant reduction in K(IR). BrdU-positive astrocytes near the depth of the microsulcus showed essentially no expression of K(IR) channels but markedly enhanced expression of delayed rectifier K(+) (K(DR)) channels. These proliferative cells showed virtually no dye coupling, whereas astrocytes in the hyperexcitable zone adjacent to the microsulcus displayed prominent dye-coupling as well as large K(IR) and outward K(+) currents. These findings suggest that reactive gliosis is accompanied by a loss of K(IR) currents and reduced gap junction coupling, which in turn suggests a compromised K(+) buffering capacity.

Reduced expression of connexin-43 and functional gap junction coupling in human gliomas.

Gap junctions are an important means for intercellular communication during development, processes of tissue differentiation, and in maintenance of adult tissue homeostasis. We investigated the expression levels and distribution of connexin-43 (Cx-43), the most abundant astrocytic gap junction protein, in acutely isolated astrocytes and glioma cells from biopsy tissue obtained from patients diagnosed with glioblastoma multiforme (GBM), low-grade astrocytomas (LGAs), or mesial temporal lobe epilepsy. Western blot and immunohistochemical analyses indicated an inverse correlation between the amount of Cx-43 protein and tumor malignancy grade, as assessed by calculating tissue mitotic indexes (MI) obtained using anti-Ki-67 nuclear antigen staining. Samples from epilepsy patients had a low MI and were intensely positive for Cx-43 staining, while LGA tissue samples exhibited moderate staining for Cx-43 and average MI, and GBM biopsies showed significantly lower levels of Cx-43 and high MI. Functional coupling was assayed using fluorescence recovery after photobleach (FRAP). We found that cells from glioma cell lines and primary cultures of human astrocytes from GBM tissues displayed significantly lower degrees of gap junction intercellular communication (GJIC) as indicated by longer and less complete recovery from photobleaching. Mean recovery values were GBM 23.8% +/- 11.4%, LGA 49.4% +/- 47%, and nontumor astrocytes 67.2% +/- 8.4%. Western blot analysis of several human glioma cell lines and tissue biopsies showed variable expression levels of Cx-43, which correlated negatively with the extent of recovery in the same samples. Taken together, our findings suggest that high-grade brain tumors show reduced intercellular communication and a decrease in connexin-43 protein levels.

BK channels in human glioma cells.

Ion channels in inexcitable cells are involved in proliferation and volume regulation. Glioma cells robustly proliferate and undergo shape and volume changes during invasive migration. We investigated ion channel expression in two human glioma cell lines (D54MG and STTG-1). With low [Ca2+]i, both cell types displayed voltage-dependent currents that activated at positive voltages (more than +50 mV). Current density was sensitive to intracellular cation replacement with the following rank order; K+ > Cs+ approximately = Li+ > Na+. Currents were >80% inhibited by iberiotoxin (33 nM), charybdotoxin (50 nM), quinine (1 mM), tetrandrine (30 microM), and tetraethylammonium ion (TEA; 1 mM). Extracellular phloretin (100 microM), an activator of BK(Ca2+) channels, and elevated intracellular Ca2+ negatively shifted the I-V curve of whole cell currents. With 0, 0.1, and 1 microM [Ca2+]i, the half-maximal voltages, V(0.5), for whole cell current activation were +150, +65, and +12 mV, respectively. Elevating [K+]o potentiated whole cell currents in a fashion proportional to the square-root of [K+]o. Recording from cell-attached patches revealed large conductance channels (150-200 pS) with similar voltage dependence and activation kinetics as whole cell currents. These data indicate that human glioma cells express large-conductance, Ca2+ activated K+ (BK) channels. In amphotericin-perforated patches bradykinin (1 microM) activated TEA-sensitive currents that were abolished by preincubation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). The BK channels described here may influence the responses of glioma cells to stimuli that increase [Ca2+]i.

Reactive astrocytes show enhanced inwardly rectifying K+ currents in situ.

Injury and diseases of the nervous system can induce astrocytes to form tenacious glial scars. We induced focal cortical freeze-lesions in neonatal rats and examined scars histologically and electrophysiologically in tissue slices isolated 2-3 weeks after lesioning. Lesions displayed marked gliosis, characterized by upregulation of GFAP labeling. Reactive astrocytes surrounding the scar showed marked hypertrophy, enlarged cell bodies and extended processes frequently terminating with endfeet-like structures on blood vessels. These reactive astrocytes showed enhanced expression of inwardly rectifying K+ (K(IR)) channels, widely believed to be an important pathway for astrocytic K+ buffering. These results suggest that a subpopulation of reactive astrocytes along a glial scar might be instrumental in buffering K+ away from the lesion.

Modulation of Kv1.5 currents by Src tyrosine phosphorylation: potential role in the differentiation of astrocytes.

Using biophysical techniques, we previously have implicated outwardly rectifying potassium currents in the proliferation of cultured spinal cord astrocytes and have demonstrated that delayed rectifier potassium currents (I(Kd)), in particular, are upregulated on entry into the cell cycle and downregulated with cell cycle exit and differentiation. In the present study, using specific antibodies and antisense oligodeoxynucleotides, we show that this proliferation-dependent potassium current is mediated by the Shaker potassium channel Kv1.5. Downregulation of Kv1.5 protein by antisense oligodeoxynucleotides reduces astrocyte proliferation by approximately 50%, although no observed changes occur in Kv1.5 protein expression during spontaneous differentiation in culture. Tyrosine phosphorylation of Kv1.5, however, is downregulated markedly in differentiated cells but unaltered on cell cycle arrest. Using immunoprecipitation, we show that Kv1.5 is associated with Src family protein tyrosine kinases and that this association does not change with cell differentiation. Inhibition of kinase activity with the Src-specific inhibitor PP2 decreases Kv1.5 phosphorylation, reduces I(Kd), and inhibits astrocyte proliferation, specifically in the G(0)/G(1) phase of cell cycle. Conversely, I(Kd) are potentiated when active Src is present in the pipette. Transfection of quiescent astrocytes with constitutively active Src (Src Y529F) causes marked upregulation of astrocyte proliferation. These data suggest that Kv1. 5 is phosphorylated constitutively by Src kinases during growth and that downregulation of Src activity may underlie both astrocyte differentiation and the accompanying changes in delayed rectifier potassium channel activity.

Muscarinic activation of BK channels induces membrane oscillations in glioma cells and leads to inhibition of cell migration.

Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca2+]i) associated with periodic Ca2+ oscillations during prolonged ACh applications. The early transient rise in [Ca2+]i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca2+ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca2+]i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca2+-sensitive K+ currents. These K+ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca2+ from intracellular stores which in turn activate Ca2+-dependent (BK-type) K+ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration.

Ion channel expression by astrocytes in situ: comparison of different CNS regions.

Patch-clamp recordings were obtained in brain slices from 283 rat astrocytes. The expression of voltage-activated whole-cell currents was compared in four different CNS regions (hippocampus, cerebral cortex, spinal cord, and cerebellum). Our data show that CNS astrocytes do not show significant regional differences in their ion channel complement. With the exception of cerebellar Bergmann glial cells, essentially all astrocytes express a combination of delayed rectifying outward K(+) currents, transient A-type K(+) currents, and small Na(+) currents. Developmentally, an increasing percentage of astrocytes and Bergmann glial cells express inwardly rectifying K(+) currents. We did not observe cells that were passive, i.e., lacking voltage-activated currents. A few cells that appeared "passive" in initial recordings showed voltage-activated K(+) currents after off-line leak subtraction. The heterogeneity observed in the ion channel complement was found to be identical when cell-to-cell variations observed within a given CNS region and between various CNS regions were compared, suggesting a common and fairly stereotypical complement of ion channels in CNS astrocytes. Ion channel expression in Bergmann glial cells differed from that of all other CNS regions studied. These cells typically showed very low input resistances attributable to a significant time- and voltage-independent resting K(+) conductance. However, as with electrophysiologically "passive"-appearing astrocytes, Bergmann glial cells showed expression of delayed rectifying K(+) currents after off-line leak subtraction. Inwardly rectifying K(+) currents were observed in Bergmann glial cells after postnatal day 17. Collectively, our data suggest that all astrocytes contain voltage-gated ion channels that display a common pattern of expression during development.