Nuclear Patch Clamping for Determining Ion Channel Activities of Bc1 Apoptosis Proteins in Endoplasmic Reticulum and Nuclear Envelope Intracellular Membranes
Annual rept. 1 Jul 1998-30 Jun 1999
PENNSYLVANIA UNIV PHILADELPHIA
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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 to the 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 nuclear envelope patch-clamp technique. During the first funding period, we have cloned bcl-X to the L and bax into vectors for transient expression in Xenopus oocytes. We injected in vitro-transcribed mRNA into oocytes, and have achieved expression as evidenced by Western blotting. Bax expression caused oocyte cell death, as expected based on it pro-apoptotic activity, indicating functional activity. A series of patch clamp of nuclei from injected oocytes has been initiated, although novel ion channel activity has not been detected to date. These results suggest that we have not yet identified the proper conditions for observing channel activity, and that further studies as well as initiation of the other specific aims are necessary.
- Anatomy and Physiology
- Medicine and Medical Research