Elucidating the molecular mechanisms controlling synaptic strength in mammalian central nervous system

Brain functions are critical to the human body, from basic activities such as breathing and heart beating to complex activities, such as learning and memorizing. These regulations largely depend on the information flow between neurons. In a brief overview, this process involves neuron structures called the presynaptic and postsynaptic terminals and chemicals released by the neurons as the signal passengers. Chemical release is closely regulated by voltage gated calcium channels (Ca_V) in the presynaptic terminal. Influx of Ca²⁺ through calcium channels triggers neurotransmitter release, which activates the response of other neurons upon neurotransmitter binding to the postsynaptic terminals. Therefore, levels of Ca_V are a critical factor controlling the strength of information flow between neurons. In mammalian brains, there is Ca_V2 family composed of Ca_V2.1, Ca_V2.2 and Ca_V2.3 subtypes. The level of Ca_V2 isoforms changes along with nervous system maturation to accommodate the needs of highly efficient communication between neurons. Ca_V2.1 is dominant in channel levels and activate earlier compared to Ca_V2.2 and Ca_V2.3, the level of Ca_V2.1 increase accompanied by the reduction of Ca_V2.2 and Ca_V2.3 levels during development, which makes Ca_V2.1 the most efficient and preferable subtype in mediating neurotransmitter release. Moreover, mutations in Ca_V2.1 result in neurological disorders, such as epilepsy and ataxia, which affect 3 billion people worldwide. However, little is known about the mechanisms controlling Ca_V2 isoform levels and preference. The Ca_V2 channels are composed of three substructures, α₁, β, and α₂δ subunit. The α₁ subunit forms the channel pore and has been indicated to control the level of calcium channels due to multiple functional motifs within the α₁ subunit. Motifs in the α₁ subunit loop I-II, loop II-III and C-terminus are heavily discussed in the previous research. To determine how the α₁ subunit regulates channel levels, I made Ca_V2.1/2.3 chimeric channels by replacing these regions in Ca_V2.1 with that in Ca_V2.2 or Ca_V2.3. We also generated mice in which the majority of the original Ca_V2 channels can be genetically deleted to provide a null background. By expressing the chimeric channels in the gene manipulated mice, we expect to investigate the level of Ca²⁺ currents, which indicates the level of the chimeric channels in the presynaptic terminal. Moreover, we also investigated the sensitivity of the Ca²⁺ currents to Ca_V2.1 specific blockers, which indicates whether the chimeric channels are preferably used by the presynaptic terminals over the native Ca_V2.3 channels. In the results, we observed that the Ca_V2.1 chimeric channel with the loop II-III or the C-terminus being swapped yielded significantly lower Ca²⁺ currents. Interestingly, the remaining currents mediated by Ca_V2.1 chimeric channels are largely sensitive to Ca_V2.1 blocks, which indicates they compete away the native Ca_V2.3 and hence, being preferably used by the presynaptic terminal. In this research, we delineated the molecular mechanisms controlling Ca_V2 subtype levels and preference. For the first time, we demonstrated that Ca_V2 subtype levels and preference are controlled by distinct mechanisms. Our research findings are critical to understanding the molecular mechanisms controlling synaptic function that ensures high fidelity synaptic transmission during development of mammalian central nervous system.