Single-channel recordings of recombinant inositol trisphosphate receptors in mammalian nuclear envelope

Inositol 1,4,5-trisphosphate (InsP₃) receptors (InsP₃Rs) are intracellular Ca²⁺ channels gated by the second messenger InsP₃. Here we describe a novel approach for recording single-channel currents through recombinant InsP₃Rs in mammalian cells that applies patch-clamp electrophysiology to nuclei isolated from COS-7 cells transiently transfected with the neuronal (SII(+)) and peripheral (SII(-)) alternatively-spliced variants of the rat type 1 InsP₃R. Single channels that were activated by InsP₃ and inhibited by heparin were observed in 45% of patches from nuclei prepared from transfected cells overexpressing recombinant InsP₃Rs. In contrast, nuclei from cells transfected with the vector alone had InsP₃-dependent channel activity in only 1.5% of patches. With K⁺ (140 mM) as the permeant ion, recombinant SII(+) and SII(-) channels had slope conductances of 370 pS and 390 pS, respectively. The recombinant channels were 4-fold more selective for Ca²⁺ over K⁺, and their open probabilities were biphasically regulated by cytoplasmic [Ca²⁺]. This approach provides a powerful new methodology to study the permeation and gating properties of recombinant mammalian InsP₃Rs in a native mammalian membrane environment at the single-channel level.