Key points• Spinal cord α-motoneurons display strong membrane immunoreactivity (IR) against small-conductance calcium-activated potassium channel (SK) isoform SK2, and a specific subpopulation of motoneurons also express SK3-IR.• Rat α-motoneurons expressing SK3-IR are significantly smaller, have significantly longer after-hyperpolarization half-decay time, significantly larger after-hyperpolarization amplitude and significantly slower axon conduction velocity than α-motoneurons that lack SK3-IR.• Motoneuron pools innervating slow-twitch muscles have a higher percentage of SK3-IR α-motoneurons than those innervating fast-twitch muscles.• Expression of SK3 may contribute to variability in after-hyperpolarization duration and amplitude across different types of rat α-motoneurons and may be a molecular factor differentiating between slow-and fast-type motoneurons.• In the soma and proximal dendrites of α-motoneurons, large clusters of SK2 and SK3 channel subunits appose cholinergic C-boutons and colocalize with muscarinic type 2 receptors and Kv2.1 channels, which suggests a novel cellular mechanism for state-dependent regulation of neuronal excitability.Abstract Small-conductance calcium-activated potassium (SK) channels mediate medium after-hyperpolarization (AHP) conductances in neurons throughout the central nervous system. However, the expression profile and subcellular localization of different SK channel isoforms in lumbar spinal α-motoneurons (α-MNs) is unknown. Using immunohistochemical labelling of rat, mouse and cat spinal cord, we reveal a differential and overlapping expression of SK2 and SK3 isoforms across specific types of α-MNs. In rodents, SK2 is expressed in all α-MNs, whereas SK3 is expressed preferentially in small-diameter α-MNs; in cats, SK3 is expressed in all α-MNs. Function-specific expression of SK3 was explored using post hoc immunostaining of electrophysiologically characterized rat α-MNs in vivo. These studies revealed strong relationships between SK3 expression and medium AHP properties. Motoneurons with SK3-immunoreactivity exhibit significantly longer AHP half-decay times (24.67 vs. 11.02 ms) and greater AHP amplitudes (3.27 vs. 1.56 mV) than MNs lacking SK3-immunoreactivity. We conclude that the differential expression of SK isoforms in rat and mouse spinal cord may contribute to the range of medium AHP durations across specific MN functional types and may be a molecular factor distinguishing between slow-and fast-type α-MNs in rodents. Furthermore, our results show that SK2-and SK3-immunoreactivity is enriched in distinct postsynaptic domains that contain Kv2.1 channel clusters associated with cholinergic C-boutons on the soma and proximal dendrites of α-MNs. We suggest that this remarkably specific subcellular membrane localization of SK channels is likely to represent the basis for a cholinergic mechanism for effective regulation of channel function and cell excitability.

C-boutons are important cholinergic modulatory loci for state-dependent alterations in motoneuron firing rate. m2 receptors are concentrated postsynaptic to C-boutons, and m2 receptor activation increases motoneuron excitability by reducing the action potential afterhyperpolarization. Here, using an intensive review of the current literature as well as data from our laboratory, we illustrate that C-bouton postsynaptic sites comprise a unique structural/functional domain containing appropriate cellular machinery (a “signaling ensemble”) for cholinergic regulation of outward K+ currents. Moreover, synaptic reorganization at these critical sites has been observed in a variety of pathologic states. Yet despite recent advances, there are still great challenges for understanding the role of C-bouton regulation and dysregulation in human health and disease. The development of new therapeutic interventions for devastating neurological conditions will rely on a complete understanding of the molecular mechanisms that underlie these complex synapses. Therefore, to close this review, we propose a comprehensive hypothetical mechanism for the cholinergic modification of α-MN excitability at C-bouton synapses, based on findings in several well-characterized neuronal systems.

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Pathophysiological responses to peripheral nerve injury include alterations in the activity, intrinsic membrane properties and excitability of spinal neurons. The intrinsic excitability of α-motoneurons is controlled in part by the expression, regulation, and distribution of membrane-bound ion channels. Ion channels, such as Kv2.1 and SK, which underlie delayed rectifier potassium currents and afterhyperpolarization respectively, are localized in high-density clusters at specific postsynaptic sites (Deardorff et al., 2013; Muennich and Fyffe, 2004). Previous work has indicated that Kv2.1 channel clustering and kinetics are regulated by a variety of stimuli including ischemia, hypoxia, neuromodulator action and increased activity. Regulation occurs via channel dephosphorylation leading to both declustering and alterations in channel kinetics, thus normalizing activity (Misonou et al., 2004; Misonou et al., 2005; Misonou et al., 2008; Mohapatra et al., 2009; Park et al., 2006). Here we demonstrate using immunohistochemistry that peripheral nerve injury is also sufficient to alter the surface distribution of Kv2.1 channels on motoneurons. The dynamic changes in channel localization include a rapid progressive decline in cluster size, beginning immediately after axotomy, reaching maximum within one week. With reinnervation, the organization and size of Kv2.1 clusters do not fully recover. However, in the absence of reinnervation Kv2.1 cluster sizes fully recover. Moreover, unilateral peripheral nerve injury evokes parallel, but smaller, effects bilaterally. These results suggest that homeostatic regulation of motoneuron Kv2.1 membrane distribution after axon injury is largely independent of axon reinnervation.

Key points Kv2 currents maintain and regulate motoneuron (MN) repetitive firing properties. Kv2.1 channel clustering properties are dynamic and respond to both high and low activity conditions. The enzyme calcineurin regulates Kv2.1 ion channel declustering. In patholophysiological conditions of high activity, Kv2.1 channels homeostatically reduce MN repetitive firing. Modulation of Kv2.1 channel kinetics and clustering allows these channels to act in a variable way across a spectrum of MN activity states. Abstract Kv2.1 channels are widely expressed in the central nervous system, including in spinal motoneurons (MNs) where they aggregate as distinct membrane clusters associated with highly regulated signalling ensembles at specific postsynaptic sites. Multiple roles for Kv2 channels have been proposed but the physiological role of Kv2.1 ion channels in mammalian spinal MNs is unknown. To determine the contribution of Kv2.1 channels to rat α‐motoneuron activity, the Kv2 inhibitor stromatoxin was used to block Kv2 currents in whole‐cell current clamp electrophysiological recordings in rat lumbar MNs. The results indicate that Kv2 currents permit shorter interspike intervals and higher repetitive firing rates, possibly by relieving Na+ channel inactivation, and thus contribute to maintenance of repetitive firing properties. We also demonstrate that Kv2.1 clustering properties in motoneurons are dynamic and respond to both high and low activity conditions. Furthermore, we show that the enzyme calcineurin regulates Kv2.1 ion channel clustering status. Finally, in a high activity state, Kv2.1 channels homeostatically reduce motoneuron repetitive firing. These results suggest that the activity‐dependent regulation of Kv2.1 channel kinetics allows these channels to modulate repetitive firing properties across a spectrum of motoneuron activity states.