The long-term objectives are two-fold: (1) to obtain a detailed understanding of the mechanism(s) by which channels are opened and closed (i.e., gated) by voltage; (2) to determine if the translocation of polypeptide chains across membranes can occur through wide channels that function as """"""""tunnels"""""""" for these chains. The methodology to be employed is the study of the size, ion-selectivity, and particularly the voltage-dependent properties of channels incorporated into planar phospholipid bilayer membranes. These channels include both those inserted into membranes by bacterial proteins, such as diptheria toxin, tetanus toxin, botulinum toxin, and colicins of the E1 class (E1, Ib, A and K), and those normally present in plasma membranes, such as the voltage-dependent calcium, sodium, and potassium channels. With respect to the first objective: the genes for fice of the channel-forming proteins mentioned above (diptheria toxin, colicins E1, Ib, and A, and the sodium channel) have been cloned and sequences, so that detailed models of channel structure and gating can now be developed. Moreover, proposed models can be stringently tested by comparing properties of specicvically modified channels (formed by site-mutated proteins) with their predicted behavior; this will be done mainly with colicin E1. Mutants of the calcium channel in Paramecia and of the sodium channel in Drosophila also provide a handle on channel structure and gating mechanisms, and these will also be studied. With respect to the second objective: all of the above-mentioned bacterial toxins are single polypeptides with at least three domains, only one of which is necessary for channel formation. In diptheria toxin, and probably also in tetanus and botulinum toxin, one of the other domains is an enzyme that must cross a vesicular membrane to enter the cytosol and thereby cause cell intoxication. Whether these or other domains of the toxins cross planar bilayers in conjunction with the opening and closing of the wide channels formed by their channel-forming domains will be investigated to determine if these wide channels provide a pathway for peptide chain transport across membranes. Voltage regulation of channels is a ubiquitous phenomenon found in almost all cells and tissues of the body, and it is particularly crucial for the functioning of the nervous system and the heart. An understanding of the structure of these channels and the mechanism(s) of their voltage control can lead to a more rational understanding and treatment of the myriad of neurological and cardiac maladies that aflict mankind.

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National Institute of General Medical Sciences (NIGMS)
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Physiology Study Section (PHY)
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Albert Einstein College of Medicine
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Kienker, Paul K; Wu, Zhengyan; Finkelstein, Alan (2016) Topography of the TH5 Segment in the Diphtheria Toxin T-Domain Channel. J Membr Biol 249:181-96
Schiffmiller, Aviva; Anderson, Damon; Finkelstein, Alan (2015) Ion selectivity of the anthrax toxin channel and its effect on protein translocation. J Gen Physiol 146:183-92
Thomson, Russell; Finkelstein, Alan (2015) Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: relevance to trypanosome lysis. Proc Natl Acad Sci U S A 112:2894-9
Schiffmiller, Aviva; Finkelstein, Alan (2015) Ion conductance of the stem of the anthrax toxin channel during lethal factor translocation. J Mol Biol 427:1211-23
Kienker, Paul K; Wu, Zhengyan; Finkelstein, Alan (2015) Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel. J Gen Physiol 145:107-25
Udho, Eshwar; Jakes, Karen S; Finkelstein, Alan (2012) TonB-dependent transporter FhuA in planar lipid bilayers: partial exit of its plug from the barrel. Biochemistry 51:6753-9
Basilio, Daniel; Kienker, Paul K; Briggs, Stephen W et al. (2011) A kinetic analysis of protein transport through the anthrax toxin channel. J Gen Physiol 137:521-31
Rodnin, Mykola V; Kyrychenko, Alexander; Kienker, Paul et al. (2011) Replacement of C-terminal histidines uncouples membrane insertion and translocation in diphtheria toxin T-domain. Biophys J 101:L41-3
Basilio, Daniel; Jennings-Antipov, Laura D; Jakes, Karen S et al. (2011) Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins. J Gen Physiol 137:343-56
Jakes, Karen S; Finkelstein, Alan (2010) The colicin Ia receptor, Cir, is also the translocator for colicin Ia. Mol Microbiol 75:567-78

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