Spontaneously active and InsP₃-activated ion channels in cell nuclei from rat cerebellar Purkinje and granule neurones

Abstract

Increases in Ca²⁺ concentration in the nucleus of neurones modulate gene transcription and may be involved in activity-dependent long-term plasticity, apoptosis, and neurotoxicity. Little is currently known about the regulation of Ca²⁺ in the nuclei of neurones. Investigation of neuronal nuclei is hampered by the cellular heterogeneity of the brain where neurones comprise no more than 10% of the cells. The situation is further complicated by large differences in properties of different neurones. Here we report a method for isolating nuclei from identified central neurones. We employed this technique to study nuclei from rat cerebellar Purkinje and granule neurones. Patch-clamp recording from the nuclear membrane of Purkinje neurones revealed numerous large-conductance channels selective for monovalent cations. The nuclear membrane of Purkinje neurones also contained multiple InsP₃- activated ion channels localized exclusively in the inner nuclear membrane with their receptor loci facing the nucleoplasm. In contrast, the nuclear membrane of granule neurones contained only a small number of mainly anion channels. Nuclear InsP₃ receptors (InsP₃Rs) were activated by InsP₃ with EC₅₀ = 0.67 μm and a Hill coefficient of 2.5. Ca²⁺ exhibited a biphasic effect on the receptors elevating its activity at low concentrations and inhibiting it at micromolar concentrations. InsP₃ in saturating concentrations did not prevent the inhibitory effect of Ca²⁺, but strongly increased InsP₃R activity at resting Ca²⁺ concentrations. These data are the first evidence for the presence of intranuclear sources of Ca²⁺ in neurones. Ca²⁺ release from the nuclear envelope may amplify Ca²⁺ transients penetrating the nucleus from the cytoplasm or generate Ca²⁺ transients in the nucleus independently of the cytoplasm.