The functionality of the endoplasmic reticulum (ER) as a Ca²⁺ storage organelle is supported by families of Ca²⁺ pumps, buffers and channels that regulate Ca²⁺ fluxes between the ER lumen and cytosol. Although many studies have identified heterogeneities in Ca²⁺ fluxes throughout the ER, the question of how differential functionality of Ca²⁺ channels is regulated within proximal regions of the same organelle is unresolved. Here, we studied the in vivo dynamics of an ER subdomain known as annulate lamellae (AL), a cytoplasmic nucleoporin containing organelle widely used in vitro to study the mechanics of nuclear envelope breakdown. We show that nuclear pore complexes (NPCs) within AL suppress local Ca²⁺ signalling activity, an inhibitory influence relieved by heterogeneous dissociation of nucleoporins to yield NPC-denuded ER domains competent at Ca²⁺ signalling. Consequently, we propose a novel generalized role for AL – reversible attenuation of resident protein activity – such that regulated AL (dis)assembly via a kinase/phosphatase cycle allows cells to support rapid gain/loss-of-function transitions in cellular physiology.