Nuclear Patch Clamping for Determining Ion Channel Activities of Bc1 Apoptosis Proteins in Endoplasmic Reticulum and Nuclear Envelope Intracellular Membrane

摘要

Apoptosis plays a critical role in growth and development of the mammary gland in normal and pathologic states. An important regulator of apoptosis is the bcl-2 oncogene, whose expression prevents apoptosis and is associated with poor responses to cancer therapies. Other bcl-2-related genes have been identified, defining a gene family with anti- and pro-apoptotic members. The molecular mechanisms which link bcl proteins to apoptosis are unclear. bcl-X(sub L) forms ion channels in artificial membranes. To determine whether these proteins form or regulate ion channels in the endoplasmic reticulum in vivo, we have employed a novel Xenopus oocyte nuclear envelope patch-clamp technique. During the second funding period, we have developed a novel mammalian expression system for patch clamp electrophysiology of recombinant endoplasmic reticulum localized membrane proteins. The system has been validated, and it will now be possible to use it for expression of bcl- reated proteins. We discovered that caspase 3, a key intermediate in apoptosis pathways, cleaves the inositol trisphosphate receptor calcium channel in the endoplasmic reticulum, and causes it to become spontaneously activated, leaking calcium into the cytoplasm. This observation may provide a molecular insight into disruption of calcium homeostasis observed in apoptosis. We will determine whether other capsases have similar effects. In addition, we will examine the cellular consequqnces of this effect, and whether its modulation affects the time-course or extent of apoptosis.