MOLECULAR GENETIC DISSECTION OF THE VDAC ION CHANNEL

The goal of this proposal is to use the voltage dependent anion channel
(VDAC) found in the outer mitochondrial membrane as a model system in which
to study the general phenomena of voltage regulation of ion channel
function. It takes advantage of the uniqueness of VDAC as an experimental
system in which to analyze voltage-sensitive ion channel function and of
the elegance of molecular genetic analysis as is possible in yeast. VDAC
has been isolated from the outer mitochondrial membrane of yeast and
analyzed biochemically and biophysically (see Section II and accompanying
manuscripts). Antibodies to this protein have been raised in rabbits,
affinity purified and used to demonstrate antigenic conservation among
VDACs from a wide range of organisms. The antibodies have been used as
well to screen yeast genomic expression libraries. The expressed sequences
in positive clones have then been used to isolate homologous sequences from
both yeast genomic and cDNA libraries. Clones (cDNA) containing VDAC
sequences have authenticated using hybrid select translation and
immunoprecipitation. In addition, the amino acid sequence derived by DNA
sequencing of cDNA clones aligns with the partial amino acid sequence of a
CNBr fragment obtained from the purified protein. From the primary amino
acid sequence, a tentative structural model of the VDAC protein can be
constructed based on computer predictions of secondary structure. From
these cloned genes, we will attempt to construct a system in which
sufficient VDAC for biochemical and physiological analysis can be produced
completely in vitro. The cloned genes will be manipulated in vitro, first
by the introduction of random mutations to define domains of the protein
involved in voltage-sensitivity followed by site-directed mutagenesis to
investigate the role of specific or a series of specific amino acids.
These mutant proteins, produced in vivo or in vitro, will then be
physiologically examined in artificial planar bilayers.