De novo variants in CAMK2A and CAMK2B cause neurodevelopmental disorders.

Objective: α (CAMK2A) and ß (CAMK2B) isoforms of Calcium/calmodulin-dependent protein kinase II (CaMKII) play a pivotal role in neuronal plasticity and in learning and memory processes in the brain. Here, we explore the possible involvement of α- and ß-CaMKII variants in neurodevelopmental disorders. Methods: Whole-exome sequencing was performed for 976 individuals with intellectual disability, developmental delay, and epilepsy. The effect of CAMK2A and CAMK2B variants on CaMKII structure and firing of neurons was evaluated by computational structural analysis, immunoblotting, and electrophysiological analysis. Results: We identified a total of five de novo CAMK2A and CAMK2B variants in three and two individuals, respectively. Seizures were common to three individuals with CAMK2A variants. Using a minigene splicing assay, we demonstrated that a splice site variant caused skipping of exon 11 leading to an in-frame deletion of the regulatory segment of CaMKII α. By structural analysis, four missense variants are predicted to impair the interaction between the kinase domain and the regulatory segment responsible for the autoinhibition of its kinase activity. The Thr286/Thr287 phosphorylation as a result of release from autoinhibition was increased in three mutants when the mutants were stably expressed in Neuro-2a neuroblastoma cells. Expression of a CaMKII α mutant in primary hippocampal neurons significantly increased A-type K+ currents, which facilitated spike repolarization of single action potentials. Interpretation: Our data highlight the importance of CaMKII α and CaMKII ß and their autoinhibitory regulation in human brain function, and suggest the enhancement of A-type K+ currents as a possible pathophysiological basis.

Ca2+ nanodomain-mediated component of swelling-induced volume-sensitive outwardly rectifying anion current triggered by autocrine action of ATP in mouse astrocytes.

The volume-sensitive outwardly rectifying (VSOR) anion channel provides a major pathway for anion transport during cell volume regulation. It is typically activated in response to cell swelling, but how the channel senses the swelling remains unclear. Meanwhile, we recently found that in mouse astrocytes the channel is activated by an inflammatory chemical mediator, bradykinin, without cell swelling and that the activation is regulated via high concentration regions of intracellular Ca(2+) ([Ca(2+)](i)) in the immediate vicinity of open Ca(2+)-permeable channels, so-called Ca(2+) nanodomains. Here we investigated whether a similar mechanism is involved in the swelling-induced VSOR channel activation in the astrocytes. A hypotonic stimulus (25% reduction in osmolality) caused the [Ca(2+)](i) rises in the astrocytes, and the rises were abolished in the presence of an ATP-degrading enzyme, apyrase (10 U/ml). Application of ATP (100 µM) under isotonic conditions generated the current through VSOR channels via Ca(2+) nanodomains, as bradykinin does. The current induced by the hypotonic stimulus was suppressed by ~40% in the Ca(2+)-depleted condition where the ATP-induced VSOR current was totally prevented. Thus the swelling-induced VSOR channel activation in mouse astrocytes is partly regulated via Ca(2+) nanodomains, whose generation is triggered by an autocrine action of ATP.

Overexpression of human CD38/ADP-ribosyl cyclase enhances acetylcholine-induced Ca2+ signalling in rodent NG108-15 neuroblastoma cells.

The role of cyclic ADP-ribose (cADPR) and its synthetic enzyme, CD38, as a downstream signal of muscarinic acetylcholine receptors (mAChRs) was examined in neuroblastoma cells expressing M1 mAChRs (NGM1). NGM1 cells were further transformed with both wild-type and mutant (C119K/C201E) human CD38. The dual transformed cells exhibited higher cADPR formation than ADPR production and elevated intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in response to ACh. These phenotypes were analyzed in detail in a representative CD38 clone. The intracellular cADPR concentration by ACh application was significantly increased by CD38 overexpression. Digital image analysis by a confocal microscopy revealed that topographical distribution of the sites of Ca(2+) release was unchanged between control and overexpressed cells. These results indicate that cADPR is an intracellular messenger of Ca(2+) signalling, suggesting that CD38 can contribute to mAChR-cADPR signalling.

Adenosine depresses a Ca(2+)-independent step in transmitter exocytosis at frog motor nerve terminals.

The depressant action of adenosine on acetylcholine release at frog motor nerve terminals was studied by intracellular recording and Ca(2+)-imaging techniques. Adenosine (200 microm) quickly and reversibly decreased the amplitude and quantal content of end-plate potentials (EPPs) with no change in quantal size in a low-Ca(2+), high-Mg(2+) solution, and EPP amplitude in normal Ringer containing d-tubocurarine. Likewise, adenosine (200 microm) reduced miniature EPP (MEPP) frequency, but not amplitude, in a high-K(+) (6 mm) solution. Adenosine (40-200 microm), however, did not affect single or repetitive impulse(s)-induced rises in Ca(2+) in the nerve terminals or its basal level. Adenosine (100-200 microm) reduced the Ca(2+)-independent enhancement of MEPP frequency caused by hypertonicity. EPPs induced by tetanic stimulation (33 Hz) in Ringer with d-tubocurarine initially increased in amplitude within 10 stimuli and then declined to the minimum. Adenosine (200 microm) decreased EPP amplitude in the initial phase of the tetanus, but enhanced it in the middle phase, thus prolonging the decay of EPP amplitude. The total sum of these EPPs, reflecting the readily releasable pool of vesicles and its refilling, however, was not changed. The results suggest that adenosine inhibits a Ca(2+)-independent step of transmitter exocytosis at frog motor nerve terminals.