Activation of ryanodine receptors in the nuclear envelope alters the conformation of the nuclear pore complex

Abstract

Nuclear pore complexes (NPCs) are supramolecular protein pores that traverse the nuclear envelope and form the only known direct route of transport between the cytoplasmic and nuclear spaces. Detailed studies have identified both active and passive mechanisms of transport through the NPC and structural studies have revealed its three-dimensional architecture. Under certain conditions, structural studies have found evidence for a mass in the central pore of the NPC whose identity remains unclear. Some studies suggest this mass represents cargo caught in transit, while others suggest it is an integral component of the NPC, the position of which is sensitive to sample conditions. Regardless of its identity, previous studies have shown that the central mass location within the NPC pore is influenced by the presence of calcium in the cisternal spaces of the nuclear membrane. Specific depletion of these calcium stores through inositol 1,4,5-trisphosphate (IP(3)) receptor activation leads to the apparent displacement of the central mass towards both the cytoplasmic and nucleoplasmic sides of the NPC. Whether the central mass is cargo or a NPC component, these observations may offer interesting insights linking transport and calcium signaling pathways. Here, we show that ryanodine (Ry) receptors are also present in the nuclear envelope of Xenopus laevis oocytes, and their specific activation can affect the conformational state of the NPC. Although previously undetected, Western blot analysis of isolated oocyte nuclei reveals the presence of Ry receptors in the nuclear envelope, albeit in low abundance. Extensive atomic force microscopy (AFM) studies at the single pore level of isolated, fixed nuclei reveal changes in the NPC conformational state following treatments that stimulate Ry receptor activity. At resting calcium levels ( approximately 200 nM Ca(2+)), the central mass within the lumen of the NPC is recessed 5.3 nm below the cytoplasmic rim of the NPC. Following treatment with 10 nM ryanodine, the central mass displaces towards the cytoplasmic face occupying a new position only 2.9 nm below the cytoplasmic rim. Interestingly, at high ryanodine concentrations (20 microM), which are reported to deactivate Ry receptors, the central mass is observed to return to the recessed position, 5.4 nm below the cytoplasmic rim. Treatments with caffeine also lead to large changes in the NPC conformation, confirming the link to specific activation of Ry receptors. These observations are consistent with a new mechanism of NPC regulation in which specific activation of Ry receptors located in the nuclear envelope can modulate cisternal calcium levels, leading to changes in the NPC conformation. Together with previous studies, it now appears that both IP(3) and Ry receptors are present in the nuclear envelope of Xenopus oocytes and are capable, through activation, of indirectly influencing the conformational state of the NPC.