; R o o t E n t r y F p+- C o m p O b j b W o r d D o c u m e n t ; O b j e c t P o o l >*- >*- 4 @ 5 6 7 8 9 : ; < = > ? F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; 9513439 Kinnally Oxidative phosphorylation is intimately connected to the flux of ions and metabolites across the two membranes enclosing mitochondria; one pathway for ion flux is through ion channels. The regulatory role of the outer membrane and of the contact sites between the inner an outer membranes is just beginning to be recognized and understood. This proposal focusses on the energy-coupling inner membrane and its predominant channel activity, a multiple conductance channel, MCC. Gated channels are adjustable molecular apertures through membranes whose opening is regulated by physiological stimuli such as voltage and the intracellular calcium in the case of MCC. The main goal of this proposal is to determine the physiological and molecular basis of MCC. This knowledge is necessary for understanding factors controlling the inner membrane permeability at the molecular level. Furthermore, this information may provide insight into the mechanisms by which all five inner membrane channels interact with the regulation of energy production in mitochondria under changing conditions, e.g. durin g exercise or ischemia. This proposal has several objectives related to its overall goal, which are to be achieved through a combination of electrophysiological and biophysical techniques. It has been proposed by others that channels are involved in the translocation of mitochondrial proteins synthesized in the cytoplasm and we have evidence MCC may play a role in protein import. Antibodies to and several mutants of the putative inner membrane channel portion of the hetero-oligomeric import apparatus will be examined for effects on MCC activity primarily using patch-clamp techniques. At the same time, MCC activity will be examined in deletion mutants of proteins implicated in MCC activity by other studies, e.g. voltage dependent anion channel and adenine nucleotide translocator. These two lines of investigation will continue until MCC s molecular basis and physiological role are determined. A biophysical characterization of the purified MCC constituent(s) will include electrophysiological comparisons to MCC in mitoplasts and circular dichroism determination of (-helix and (-sheet content. Finally, structure/function studies will be made of MCC and its relationship with contact sites between the inner and outer membranes. *** ; Oh +' 0 $ H l D h R:WWUSERTEMPLATENORMAL.DOT S u m m a r y I n f o r m a t i o n ( 4 9513439 Thomasina M Osborne Thomasina M Osborne @ >*- @ @ >*- @ F # Microsoft Word 6.0 2 ; e = e A ; A j j j j j j j u 1 # 7 W T A u j u j j j j ~ j j j j j 9513439 Kinnally Oxidative phosphorylation is intimately connected to the flux of ions and metabolites across the two membranes enclosing mitochondria; one pathway for ion flux is through ion channels. The regulatory role of the outer membrane and of the contact sites between the inner an outer membranes is just beginning to be recognized and understood. This proposal focusses on the energy-coupling inner membrane and its predominant channel activity, a multiple conductance channel, MCC. Gated channels are adjustable molecular apertures through membranes whose op
