LS-14-P-3241 Participation of calcium-activated potassium channels in the formation of plasmerosomes in neurons

The endoplasmic reticulum (ER) forms a continuous network of tubules and cisterns in neurons. However, recent evidence suggests that neuronal ER does not represent a uniform Ca²⁺ pool but rather a spatially heterogeneous system organized into subcompartments. These ER domains, or calciosomes, are usually enriched in certain isoforms of Ca²⁺ pumps, Ca²⁺ binding proteins and Ca²⁺ permeant channels, and are supposed to unload and refill Ca²⁺ independently [1]. Ca²⁺ release from the ER is mediated by two families of Ca²⁺ permeable channels, namely the ryanodine receptors (RyRs) and the inositol 1,4,5-trisphosphate receptors (IP3-Rs), each with three major isoforms. Areas of the plasma membrane overlying calciosomes also form specialized microdomains that contain unique sets of ion channels. These plasma membrane domains together with the underlying calciosomes are proposed to build functional units, termed plasmerosomes [2].

This study was undertaken (i) to unravel exact morphological parameters of subsurface cisterns (SSCs), representing particular types of calciosomes in cerebellar Purkinje cells (PCs), and (ii) to analyze the molecular composition of SSCs as well as overlying plasma membrane domains with respect to Ca²⁺ release channels (IP3-Rs, RyRs), voltage-gated Ca²⁺ channels and Ca²⁺ sensors such as large-conductance Ca²⁺ activated K⁺ (BKCa) channels. The morphological parameters of SSCs are established by 3D-reconstruction plane-by-plane from series of ultrathin sections by using the software CAR (Contour Alignment Reconstruction). The molecular composition is studied by means of pre- and post-embedding immunogold electron microscopy and SDS-digested freeze-fracture replica immunolabeling.

SSCs are discoid flattened cisterns, 0.4-1.5 µm wide, with a luminal depth of 4-5 nm (widening at their lateral edges), situated beneath the inner leaflet of the plasma membrane at a regular distance of 10-15 nm. IP3-Rs and RyRs are both localized to SSCs indicating that these Ca²⁺ release channels share a common Ca²⁺ pool and dispose SSCs to the generation of both Ca²⁺ puffs and sparks. Clustered BKCa channels are always associated with plasma membrane domains overlying SSCs and likely facilitate the generation of small transient outward currents. These findings indicate that functional units exist in cerebellar PCs resembling plasmerosomes in myocytes [3], and these units may contribute significantly to spatial signalling in central principal neurons.

[1] Verkhratsky A (2005) Physiol Rev 85:201-79. [2] Blaustein MP and Golovina VA (2001) Trends Neurosci 24:602-8. [3] Wellman GC and Nelson MT (2003) Cell Calcium 34(3):211-29.