Sodium Channel β2 Subunits Prevent Action Potential Propagation Failures at Axonal Branch Points

Neurotransmitter release depends on voltage-gated Na⁺ channels (Na_vs) to propagate an action potential (AP) successfully from the axon hillock to a synaptic terminal. Unmyelinated sections of axon are very diverse structures encompassing branch points and numerous presynaptic terminals with undefined molecular partners of Na⁺ channels. Using optical recordings of Ca²⁺ and membrane voltage, we demonstrate here that Na⁺ channel β2 subunits (Na_vβ2s) are required to prevent AP propagation failures across the axonal arborization of cultured rat hippocampal neurons (mixed male and female). When Na_vβ2 expression was reduced, we identified two specific phenotypes: (1) membrane excitability and AP-evoked Ca²⁺ entry were impaired at synapses and (2) AP propagation was severely compromised with >40% of axonal branches no longer responding to AP-stimulation. We went on to show that a great deal of electrical signaling heterogeneity exists in AP waveforms across the axonal arborization independent of axon morphology. Therefore, Na_vβ2 is a critical regulator of axonal excitability and synaptic function in unmyelinated axons.SIGNIFICANCE STATEMENT Voltage-gated Ca²⁺ channels are fulcrums of neurotransmission that convert electrical inputs into chemical outputs in the form of vesicle fusion at synaptic terminals. However, the role of the electrical signal, the presynaptic action potential (AP), in modulating synaptic transmission is less clear. What is the fidelity of a propagating AP waveform in the axon and what molecules shape it throughout the axonal arborization? Our work identifies several new features of AP propagation in unmyelinated axons: (1) branches of a single axonal arborization have variable AP waveforms independent of morphology, (2) Na⁺ channel β2 subunits modulate AP-evoked Ca²⁺-influx, and (3) β2 subunits maintain successful AP propagation across the axonal arbor. These findings are relevant to understanding the flow of excitation in the brain.