The tonoplast monosaccharide transporter (TMT) family comprises three isoforms in Arabidopsis thaliana, and TMT–green fluorescent protein fusion proteins are targeted to the vacuolar membrane. TMT promoter–β-glucuronidase plants revealed that the TONOPLAST MONOSACCHARIDE TRANSPORTER1 (TMT1) and TMT2 genes exhibit a tissue- and cell type–specific expression pattern, whereas TMT3 is only weakly expressed. TMT1 and TMT2 expression is induced by drought, salt, and cold treatments and by sugar. During cold adaptation, tmt knockout lines accumulated less glucose and fructose compared with wild-type plants, whereas no differences were observed for sucrose. Cold adaptation of wild-type plants substantially promoted glucose uptake into isolated leaf mesophyll vacuoles. Glucose uptake into isolated vacuoles was inhibited by NH4 +, fructose, and phlorizin, indicating that transport is energy-dependent and that both glucose and fructose were taken up by the same carrier. Glucose import into vacuoles from two cold-induced tmt1 knockout lines or from triple knockout plants was substantially lower than into corresponding wild-type vacuoles. Monosaccharide feeding into leaf discs revealed the strongest response to sugar in tmt1 knockout lines compared with wild-type plants, suggesting that TMT1 is required for cytosolic glucose homeostasis. Our results indicate that TMT1 is involved in vacuolar monosaccharide transport and plays a major role during stress responses.

We studied GABAergic signaling in astrocytes of olfactory bulb slices using confocal Ca ²⁺ imaging and two-photon Na ⁺ imaging. GABA evoked Ca ²⁺ transients in astrocytes that persisted in the presence of GABA _A and GABA _B receptor antagonists, but were suppressed by inhibition of GABA uptake by SNAP 5114. Withdrawal of external Ca ²⁺ blocked GABA-induced Ca ²⁺ transients, and depletion of Ca ²⁺ stores with cyclopiazonic acid reduced Ca ²⁺ transients by approximately 90%. This indicates that the Ca ²⁺ transients depend on external Ca ²⁺ , but are mainly mediated by intracellular Ca ²⁺ release, conforming with Ca ²⁺ -induced Ca ²⁺ release. Inhibition of ryanodine receptors did not affect GABA-induced Ca ²⁺ transients, whereas the InsP ₃ receptor blocker 2-APB inhibited the Ca ²⁺ transients. GABA also induced Na ⁺ increases in astrocytes, potentially reducing Na ⁺ /Ca ²⁺ exchange. To test whether reduction of Na ⁺ /Ca ²⁺ exchange induces Ca ²⁺ signaling, we inhibited Na ⁺ /Ca ²⁺ exchange with KB-R7943, which mimicked GABA-induced Ca ²⁺ transients. Endogenous GABA release from neurons, activated by stimulation of afferent axons or NMDA application, also triggered Ca ²⁺ transients in astrocytes. The significance of GABAergic Ca ²⁺ signaling in astrocytes for control of blood flow is demonstrated by SNAP 5114-sensitive constriction of blood vessels accompanying GABA uptake. The results suggest that GABAergic signaling is composed of GABA uptake-mediated Na ⁺ rises that reduce Na ⁺ /Ca ²⁺ exchange, thereby leading to a Ca ²⁺ increase sufficient to trigger Ca ²⁺ -induced Ca ²⁺ release via InsP ₃ receptors. Hence, GABA transporters not only remove GABA from the extracellular space, but may also contribute to intracellular signaling and astrocyte function, such as control of blood flow.

Within the Ca_v1 family of voltage-gated calcium channels, Ca_v1.2 and Ca_v1.3 channels are the predominant subtypes in the brain. Whereas specific functions for each subtype were described in the adult brain, their role in brain development is poorly understood. Here we assess the role of Ca_v1.3 subunits in the activity-dependent development of the auditory brainstem. We used Ca_v1.3-deficient (Ca_v1.3^−/−) mice because these mice lack cochlea-driven activity that deprives the auditory centers from peripheral input. We found a drastically reduced volume in all auditory brainstem centers (range 25–59%, total 35%), which was manifest before hearing onset. A reduction was not obvious outside the auditory system. The lateral superior olive (LSO) was strikingly malformed in Ca_v1.3^−/−mice and had fewer neurons (1/3 less). The remaining LSO neurons displayed normal dendritic trees and received functional glutamatergic input, yet they fired action potentials predominantly with a multiple pattern upon depolarization, in contrast to the single firing pattern prevalent in controls. The latter finding appears to be due to a reduction of dendrototoxin-sensitive potassium conductances, presumably mediated through the K_v1.2 subtype. Fura2 imaging provided evidence for functional Ca_v1.3 channels in the LSO of wild-type mice. Our results imply that Ca_v1.3 channels are indispensable for the development of the central auditory system. We propose that the unique LSO phenotype in Ca_v1.3^−/−mice, which hitherto was not described in other hereditary deafness models, is caused by the synergistic contribution of two factors: on-site loss of Ca_v1.3 channels in the neurons plus lack of peripheral input.

We have studied store-operated Ca²⁺entry (SOCE) in Bergmann glia and granule cell layer astrocytes in acute brain slices of the rat cerebellum, using the Ca²⁺-sensitive fluorescent dye Fluo-4 and confocal laser scanning microscopy. Astrocytes were identified by their morphology, location, and their Ca²⁺response in K⁺-free solution. Depletion of Ca²⁺stores by cyclopiazonic acid (CPA) (20 μm) induced SOCE in both types of astrocyte. A similar Ca²⁺influx was elicited by the calmodulin antagonist calmidazolium (CMZ) (1 μm). The SOCE channel blocker 2-aminoethoxy-diphenylborate (2-APB) (100 μm) and the Ca²⁺release-activated channel blocker 3,5-bistrifluoromethyl pyrazole derivative (BTP2) (20 μm) suppressed the CPA- and the CMZ-induced Ca²⁺influx. Pretreatment of acute slices with the specific Ca²⁺-independent phospholipase A₂(iPLA₂) inhibitor bromoenol lactone (BEL) (25 μm) blocked the CPA- and the CMZ-induced Ca²⁺influx. The lysophospholipid products of iPLA₂, lysophosphatidylcholine (250 nm) and lysophosphatidylinositol (250 nm), but not lysophosphatidic acid (250 nm), induced a BTP2- and 2-APB-sensitive, but BEL-insensitive, Ca²⁺influx. CPA or CMZ enhanced the BEL-sensitive enzymatic activity of iPLA₂in cerebellar astrocyte culture. Inhibition of iPLA₂expression by specific antisense oligodeoxynucleotide of iPLA₂reduced the SOCE and the Ca²⁺store refilling in cultured astrocytes. Spontaneous Ca²⁺oscillations in astrocytesin situwere reduced after inhibiting SOCE channels or iPLA₂activity. The results suggest that the depletion of Ca²⁺stores activates iPLA₂to open Ca²⁺channels in the plasma membrane by the formation of lysophospholipids in astrocytes, presumably to refill the stores and allow normal Ca²⁺signaling.