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

Biophys J. 2001 Jul;81(1):117-24. doi: 10.1016/s0006-3495(01)75685-8.

Abstract

Inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) are intracellular Ca(2+) channels gated by the second messenger InsP(3). Here we describe a novel approach for recording single-channel currents through recombinant InsP(3)Rs 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 InsP(3)R. Single channels that were activated by InsP(3) and inhibited by heparin were observed in 45% of patches from nuclei prepared from transfected cells overexpressing recombinant InsP(3)Rs. In contrast, nuclei from cells transfected with the vector alone had InsP(3)-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 Ca(2+) over K(+), and their open probabilities were biphasically regulated by cytoplasmic [Ca(2+)]. 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alternative Splicing / genetics
  • Animals
  • COS Cells
  • Calcium / metabolism
  • Calcium / pharmacology
  • Calcium Channels / chemistry
  • Calcium Channels / genetics
  • Calcium Channels / metabolism*
  • Cell Line
  • Electrophysiology
  • Inositol 1,4,5-Trisphosphate Receptors
  • Ion Channel Gating / drug effects
  • Nuclear Envelope / metabolism*
  • Patch-Clamp Techniques
  • Probability
  • Rats
  • Receptors, Cytoplasmic and Nuclear / chemistry
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism

Substances

  • Calcium Channels
  • Inositol 1,4,5-Trisphosphate Receptors
  • Receptors, Cytoplasmic and Nuclear
  • Recombinant Proteins
  • Calcium