Activation of ferret erythrocyte Na+–K+–2Cl− cotransport by deoxygenation

Deoxygenation of ferret erythrocytes stimulates Na⁺–K⁺–2Cl⁻ cotransport by 111% ( s.d. , 46) compared to controls in air. Half-maximal activation occurs at a P _O2 of 24 mmHg ( s.d. , 2) indicating that physiological changes in oxygen tension can influence cotransport function. Approximately 25–35% of this stimulation can be attributed to the rise of intracellular free magnesium concentration that occurs on deoxygenation (from 0.82 ( s.d. , 0.07) to 1.40 m m ( s.d. , 0.17)). Most of the stimulation is probably caused by activation of a kinase which can be prevented or reversed by treating cells with the kinase inhibitors PP1 or staurosporine, or by reducing cell magnesium content to submicromolar levels. Stimulation by deoxygenation is comparable with that caused by calyculin A or sodium arsenite, compounds that cause a 2- to 3-fold increase in threonine phosphorylation of the cotransporter which can be detected with phospho-specific antibodies. However, the same approach failed to detect significant changes in threonine phosphorylation following deoxygenation. The results suggest that deoxygenation causes activation of a kinase that either phosphorylates the transporter, but probably not on threonine, or phosphorylates another protein that in turn influences cotransporter behaviour. They also indicate that more than one kinase and phosphatase are involved in cotransporter phosphorylation.     

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.     

Increased Ca2+ influx through Na+/Ca2+ exchanger during long-term facilitation at crayfish neuromuscular junctions

Intense motor neuron activity induces a long-term facilitation (LTF) of synaptic transmission at crayfish neuromuscular junctions (NMJs) that is accompanied by an increase in the accumulation of presynaptic Ca²⁺ ions during a test train of action potentials. It is natural to assume that the increased Ca²⁺ influx during action potentials is directly responsible for the increased transmitter release in LTF, especially as the magnitudes of LTF and increased Ca²⁺ influx are positively correlated. However, our results indicate that the elevated Ca²⁺ entry occurs through the reverse mode operation of presynaptic Na⁺/Ca²⁺ exchangers that are activated by an LTF-inducing tetanus. Inhibition of Na⁺/Ca²⁺ exchange blocks this additional Ca²⁺ influx without affecting LTF, showing that LTF is not a consequence of the regulation of these transporters and is not directly related to the increase in [Ca²⁺]_i reached during a train of action potentials. Their correlation is probably due to both being induced independently by the strong [Ca²⁺]_i elevation accompanying LTF-inducing stimuli. Our results reveal a new form of regulation of neuronal Na⁺/Ca²⁺ exchange that does not directly alter the strength of synaptic transmission.