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

Inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) are intracellu lar Ca2+ channels gated by the second messenger InsP(3). Here we describe a novel approach for recording single-channel currents through recombinant I nsP(3)Rs in mammalian cells that applies patch-clamp electrophysiology to n uclei isolated from COS-7 cells transiently transfected with the neuronal ( SII(+)) and peripheral (SII(-)) alternatively-spliced variants of the rat t ype 1 InsP(3)R. Single channels that were activated by InsP(3) and inhibite d by heparin were observed in 45% of patches from nuclei prepared from tran sfected cells overexpressing recombinant InsP(3)Rs. In contrast, nuclei fro m cells transfected with the vector alone had InsP(3)-dependent channel act ivity in only 1.5% of patches. With K+ (140 mM) as the permeant ion, recomb inant SII(+) and SII(-) channels had slope conductances of 370 pS and 390 p S, respectively. The recombinant channels were 4-fold more selective for Ca 2+ over K+, and their open probabilities were biphasically regulated by cyt oplasmic [Ca2+]. This approach provides a powerful new methodology to study the permeation and gating properties of recombinant mammalian InsP(3)Rs in a native mammalian membrane environment at the single-channel level.