Focal aggregation of voltage-gated, Kv2.1 subunit-containing, potassium channels at synaptic sites in rat spinal motoneurones

Abstract

Delayed rectifier K+ currents are involved in the control of alpha-motoneurone excitability, but the precise spatial distribution and organization of the membrane ion channels that contribute to these currents have not been defined. Voltage-activated Kv2.1 channels have properties commensurate with a contribution to delayed rectifier currents and are expressed in neurones throughout the mammalian central nervous system. A specific antibody against Kv2.1 channel subunits was used to determine the surface distribution and clustering of Kv2.1 subunit-containing channels in the cell membrane of alpha-motoneurones and other spinal cord neurones. In alpha-motoneurones, Kv2.1 immunoreactivity (-IR) was abundant in the surface membrane of the soma and large proximal dendrites, and was present also in smaller diameter distal dendrites. Plasma membrane-associated Kv2.1-IR in alpha-motoneurones was distributed in a mosaic of small irregularly shaped, and large disc-like, clusters. However, only small to medium clusters of Kv2.1-IR were observed in spinal interneurones and projection neurones, and some interneurones, including Renshaw cells, lacked demonstrable Kv2.1-IR. In alpha-motoneurones, dual immunostaining procedures revealed that the prominent disc-like domains of Kv2.1-IR are invariably apposed to presynaptic cholinergic C-terminals. Further, Kv2.1-IR colocalizes with immunoreactivity against postsynaptic muscarinic (m2) receptors at these locations. Ultrastructural examination confirmed the postsynaptic localization of Kv2.1-IR at C-terminal synapses, and revealed clusters of Kv2.1-IR at a majority of S-type, presumed excitatory, synapses. Kv2.1-IR in alpha-motoneurones is not directly associated with presumed inhibitory (F-type) synapses, nor is it present in presynaptic structures apposed to the motoneurone. Occasionally, small patches of extrasynaptic Kv2.1-IR labelling were observed in surface membrane apposed by glial processes. Voltage-gated potassium channels responsible for the delayed rectifier current, including Kv2.1, are usually assigned roles in the repolarization of the action potential. However, the strategic localization of Kv2.1 subunit-containing channels at specific postsynaptic sites suggests that this family of voltage-activated K+ channels may have additional roles and/or regulatory components.

Figure 1

The mosaic of Kv2.1 subunit-containing channel clusters in the plasma membrane of motoneurones. A, projected confocal image of Kv2.1-IR (red) in two adjacent motoneurones, demonstrating the range in size and complexity of Kv2.1 clusters. The image is a stack of 24 optical sections sampled at 0.5 μm intervals and superimposed to reveal the ‘en face’ architecture of a ‘hemi-neurone’. The inset at upper left is a single optical section to demonstrate Kv2.1-IR (red) surrounding motoneurones identified by ChAT-IR (green). The inset at upper right shows a higher magnification image of a region of surface membrane depicted (arrow) in the main part of the figure. The inset displays the fine structural detail of small and large disc-like clusters and demonstrates that they are formed by aggregates of small punctae. B and C, measurement of cluster area. B shows a screen image of a single optical section capturing an ‘en face’ region of surface membrane, highly magnified (pixel size 0.16 μm). Kv2.1 immunofluorescence intensity is defined by grey scale in C (lighter shades more intense fluorescence). with highlighting (red) of the areas selected by the thresholding paradigm for measurement. Note again the complex structure and shape of the clusters. D, a low power image showing cross-sectioned motoneurones (Nissl; green) and Kv2.1-IR (red), demonstrating the extent of immunofluorescence on the dendrites, as well as the ‘edge-on’ visualization of large discs along the surface membrane.

Figure 2

Quantitative analysis of Kv2.1 subunit-containing channel clusters. The distribution of cluster areas (n = 2673) is highly skewed. Small clusters (area less than 2 μm²) comprise 87% of the population.

Figure 3

A, Renshaw cell interneurones do not expresss Kv2.1. In A1, a Renshaw cell identified by calbindin expression (green cell at lower edge of image) is devoid of plasma membrane Kv2.1-IR, whereas the surface of a nearby motoneurone (seen at upper edge of image) displays typical intense Kv2.1-IR (red). In A2, a calbindin-IR interneurone outside the Renshaw cell area (located in dorso-medial lamina VII) expresses intense Kv2.1-IR. B, surface membrane Kv2.1-IR in motoneurones is apposed to presynaptic boutons labelled by synaptophysin. The inset shows a large synaptophysin-labelled terminal (red) very closely apposed to a large patch of surface membrane Kv2.1-IR (green). C, large discs of Kv2.1-IR (red) are not associated with 5-HT terminals (green) around the soma or proximal dendrites. D, some glutamatergic synaptic terminals are associated with postsynaptic Kv2.1-IR. VGluT1 terminals (green) are apposed to small patches of Kv2.1-IR (red; see inset for higher magnification)

Figure 4

Kv2.1 is specifically associated with large cholinergic terminals. A, a single optical section reveals precise spatial correspondence between VAChT-IR terminals (red) and surface membrane Kv2.1-IR (green). Every large VAChT-IR terminal is associated with a cluster of Kv2.1-IR. At low power, there appears to be some overlap (yellow) between the two channels, but at high power (inset) the red and green fluorescence signals are seen to be adjacent with minimal overlap. B, postsynaptic m2 muscarinic receptor (red) is colocalized with Kv2.1-IR (green). At low and high power (inset), the two channels of fluorescence signal overlap almost completely at the locations of large clusters of Kv2.1 (yellow, inset).

Figure 5

Kv2.1-IR is restricted to the postsynaptic membrane and is clustered at specific synaptic sites on the membrane. Pre-embedding immunohistochemistry with silver intensification reveals Kv2.1-IR sites. A and B, small (arrows) and large (between curved arrows) regions of distinct membrane labelling are apposed to C-terminals (*) on the motoneurone soma. The labelling is clearly postsynaptic, with rare particles of silver-intensified DAB reaction product apparently located (presumably because of diffusion of reaction product) in the synaptic cleft, and is associated with the subsurface cistern (C). D, occasional patches of extrasynaptic Kv2.1-IR are present in the plasma membrane apposed to finger-like astrocytic processes (As). Some cytoplasmic labelling is also visible in B and D (arrowheads), close to the regions of membrane labelling. E, intense postsynaptic Kv2.1-IR (arrow) at a synapse formed by an S-type terminal (*); note that the membrane apposed to the adjacent terminal at left is unlabelled.