Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in neurons. The human genome includes ten human VGSC α-subunit genes, SCN(X)A, encoding Nav1.1-1.9 plus Nax. To understand the unique role that each VGSC plays in normal and pathophysiological function in neural networks, compounds with high affinity and selectivity for specific VGSC subtypes are required. Toward that goal, a structural analog of the VGSC pore blocker tetrodotoxin, 4,9-anhydrotetrodotoxin (4,9-ah-TTX), has been reported to be more selective in blocking Na+ current mediated by Nav1.6 than other TTX-sensitive VGSCs, including Nav1.2, Nav1.3, Nav1.4, and Nav1.7. While SCN1A, encoding Nav1.1, has been implicated in several neurological diseases, the effects of 4,9-ah-TTX on Nav1.1-mediated Na+ current have not been tested. Here, we compared the binding of 4,9-ah-TTX for human and mouse brain preparations, and the effects of 4,9-ah-TTX on human Nav1.1-, Nav1.3- and Nav1.6-mediated Na+ currents using the whole-cell patch clamp technique in heterologous cells. We show that, while 4,9-ah-TTX administration results in significant blockade of Nav1.6-mediated Na+ current in the nanomolar range, it also has significant effects on Nav1.1-mediated Na+ current. Thus, 4,9-ah-TTX is not a useful tool in identifying Nav1.6-specific effects in human brain networks.

Human variants in voltage-gated sodium channel (VGSC) α and β subunit genes are linked to developmental and epileptic encephalopathies (DEEs). Inherited, biallelic, loss-of-function variants in SCN1B, encoding the β1/β1B subunits, are linked to early infantile DEE (EIEE52). De novo, monoallelic variants in SCN1A (Nav1.1), SCN2A (Nav1.2), SCN3A (Nav1.3), and SCN8A (Nav1.6) are also linked to DEEs. While these VGSC-linked DEEs have similar presentations, they have diverse mechanisms of altered neuronal excitability. Mouse models have suggested that Scn2a-, Scn3a-, and Scn8a-linked DEE variants are, in general, gain of function, resulting in increased persistent or resurgent sodium current (INa ) and pyramidal neuron hyperexcitability. In contrast, Scn1a-linked DEE variants, in general, are loss-of-function, resulting in decreased INa and hypoexcitability of fast-spiking interneurons. VGSC β1 subunits associate with Nav1.1, Nav1.2, Nav1.3, and Nav1.6 and are expressed throughout the brain, raising the possibility that insults to both pyramidal and interneuron excitability may drive EIEE52 pathophysiology.