Kv2/Kv6.4 heteromeric potassium channels are expressed in spinal motor neurons and localized at C-bouton synapses

channels of the Kv2 family co-assemble with electrically silent KvS subunits in specific subpopulations of brain neurons, forming heteromeric Kv2/KvS channels with distinct functional properties. Little is known about the composition and function of Kv2 channels in spinal cord neurons, however. Here, we show that while Kv2.1 is broadly expressed in multiple classes of spinal cord neurons, the Kv6.4 "electrically-silent" subunit is specifically expressed in motor neurons. In motor neurons, we find that Kv6.4 protein is co-clustered with Kv2.1 and Kv2.2 subunits at endoplasmic reticulum-plasma membrane (ER-PM) junctions beneath C-bouton synapses. In Kv2.1 S590A mutant mice, in which Kv2.1 is unable to bind ER VAP proteins, Kv2.1 and Kv6.4 clustering at ER-PM junctions is severely reduced suggesting Kv2 channels are localized at ER-PM junctions by the same molecular mechanism in motor neurons and brain neurons. Moreover, clustering of Kv6.4, as well as the AMIGO-1 auxiliary subunit, are severely reduced in Kv2.1 knockout mice and moderately reduced in Kv2.2 knockout mice. Thus, expression and localization of Kv6.4 subunits is dependent on Kv2 subunits, likely through their co-assembly into heteromeric channels. Finally, we find that presynaptic C-boutons and postsynaptic clusters of the ER-resident sigma1-receptor are preserved in motor neurons of Kv2 knockout mice. Together, these findings identify a specific Kv2/KvS channel subtype expressed in motor neurons that localizes to C-bouton junctions where it could regulate neuronal excitability and signaling at ER-PM junctions.