Dual Ca2+ modulation of glycinergic synaptic currents in rodent hypoglossal motoneurones

Glycinergic synapses are implicated in the coordination of reflex responses, sensory signal processing and pain sensation. Their activity is pre- and postsynaptically regulated, although mechanisms are poorly understood. Using patch-clamp recording and Ca²⁺ imaging in hypoglossal motoneurones from rat and mouse brainstem slices, we address here the role of cytoplasmic Ca²⁺ (Ca_i) in glycinergic synapse modulation. Ca²⁺ influx through voltage-gated or NMDA receptor channels caused powerful transient inhibition of glycinergic IPSCs. This effect was accompanied by an increase in both the failure rate and paired-pulse ratio, as well as a decrease in the frequency of mIPSCs, suggesting a presynaptic mechanism of depression. Inhibition was reduced by the cannabinoid receptor antagonist SR141716A and occluded by the agonist WIN55,212-2, indicating involvement of endocannabinoid retrograde signalling. Conversely, in the presence of SR141716A, glycinergic IPSCs were potentiated postsynaptically by glutamate or NMDA, displaying a Ca²⁺-dependent increase in amplitude and decay prolongation. Both presynaptic inhibition and postsynaptic potentiation were completely prevented by strong Ca_i buffering (20 m m BAPTA). Our findings demonstrate two independent mechanisms by which Ca²⁺ modulates glycinergic synaptic transmission: (i) presynaptic inhibition of glycine release and (ii) postsynaptic potentiation of GlyR-mediated responses. This dual Ca²⁺-induced regulation might be important for feedback control of neurotransmission in a variety of glycinergic networks in mammalian nervous systems.     

TRPC3 properties of a native constitutively active Ca2+-permeable cation channel in rabbit ear artery myocytes

Previously we have described a constitutively active, Ca²⁺-permeable, non-selective cation channel in freshly dispersed rabbit ear artery myocytes which has similar properties to some of the canonical transient receptor potential (TRPC) channel proteins. In the present work we have compared the properties of constitutive channel activity with known properties of TRPC proteins by investigating the effect of selective anti-TRPC antibodies and pharmacological agents on whole-cell and single cation channel activity. Bath application of anti-TRPC3 antibodies markedly reduced channel activity in inside-out patches and also produced a pronounced reduction of both current amplitude and variance of constitutively active whole-cell cation currents whereas anti-TRPC1/4/5/6/7 antibodies had no effect on channel activity. In the presence of antigenic peptide, anti-TRPC3 antibodies had no effect on whole-cell or single cation channel activity. Bath application of flufenamic acid, Gd³⁺, La³⁺ and Ca²⁺ inhibited spontaneous channel activity in outside-out patches with IC₅₀ values of 6.8 μ m , 25 n m , 1.5 μ m and 0.124 m m , respectively, which are similar values to those against TRPC3 proteins. Immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 proteins in ear artery myocytes, and these were predominately distributed at, or close to, the plasma membrane. These data provide strong evidence that native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 channel proteins and indicate that these proteins may have an important role in mediating this conductance.     

Modulation of Ca2+ release and Ca2+ oscillations in HeLa cells and fibroblasts by mitochondrial Ca2+ uniporter stimulation

The recent availability of activators of the mitochondrial Ca²⁺ uniporter allows direct testing of the influence of mitochondrial Ca²⁺ uptake on the overall Ca²⁺ homeostasis of the cell. We show here that activation of mitochondrial Ca²⁺ uptake by 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or kaempferol stimulates histamine-induced Ca²⁺ release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na⁺–Ca²⁺ exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca²⁺ uptake and release from mitochondria control the ability of local Ca²⁺ microdomains to produce feedback inhibition of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). In addition, the ability of mitochondria to control Ca²⁺ release from the ER allows them to modulate cytosolic Ca²⁺ oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca²⁺ oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca²⁺ oscillations are exquisitely sensitive to the rates of mitochondrial Ca²⁺ uptake and release, which precisely control the size of the local Ca²⁺ microdomains around InsP₃Rs and thus the ability to produce feedback activation or inhibition of Ca²⁺ release.     

Epithelial Ca2+ entry channels: transcellular Ca2+ transport and beyond

The recently discovered apical calcium channels CaT1 (TRPV6) and ECaC (TRPV5) belong to a family of six members called the 'TRPV family'. Unlike the other four members which are nonselective cation channels functioning as heat or osmolarity sensors in the body, CaT1 and ECaC are remarkably calcium-selective channels which serve as apical calcium entry mechanisms in absorptive and secretory tissues. CaT1 is highly expressed in the proximal intestine, placenta and exocrine tissues, whereas ECaC expression is most prominent in the distal convoluted and connecting tubules of the kidney. CaT1 in the intestine is highly responsive to 1,25-dihydroxyvitamin D₃ and shows both fast and slow calcium-dependent feedback inhibition to prevent calcium overload. In contrast, ECaC only shows slow inactivation kinetics and appears to be mostly regulated by the calcium load in the kidney. Outside the calcium-transporting epithelia, CaT1 is highly expressed in exocrine tissues such as pancreas, prostate and salivary gland. In these tissues it probably mediates re-uptake of calcium following its release by secretory vesicles. CaT1 also contributes to store-operated calcium entry in Jurkat T-lymphocytes and prostate cancer LNCaP cells, possibly in conjunction with other cellular components which link CaT1 activity to the filling state of the calcium stores. Finally, CaT1 expression is upregulated in prostate cancer and other cancers of epithelial origin, highlighting its potential as a target for cancer therapy.     

Ca2+ imaging of mouse neocortical interneurone dendrites: Contribution of Ca2+-permeable AMPA and NMDA receptors to subthreshold Ca2+dynamics

In this second study, we have combined two-photon calcium imaging with whole-cell recording and anatomic reconstructions to directly characterize synaptically evoked calcium signals in three types of mouse V1 supragranular interneurones: parvalbumin-positive fast spikers (FS), calretinin-positive irregular spikers (IS), and adapting cells (AD). We observed that subthreshold synaptic activation evoked calcium signals locally restricted to individual dendritic compartments. These signals were mediated by NMDA receptors (NMDARs) in AD and IS cells, whereas in FS cells, calcium-permeable AMPA receptors (CP-AMPARs) provided an additional and kinetically distinct influx. Furthermore, even a single, subthreshold synaptic activation evoked a larger dendritic calcium influx than backpropagating action potentials. Our results demonstrate that NMDARs dominate subthreshold calcium dynamics in interneurones and reveal the functional contribution of CP-AMPARs to a specific subclass of cortical interneurone. These data highlight different strategies in dendritic signal processing by distinct classes of interneurones.