Structure-activity relationships for the interaction of bovine pancreatic trypsin inhibitor with an intracellular site on a large conductance Ca(2+)-activated K(+) channel
Large conductance Ca(2+)-activated K(+) channels (BK(Ca)) contain an intracellular binding site for bovine pancreatic trypsin inhibitor (BPTI), a well-known inhibitor of various serine proteinase (SerP) enzymes. To investigate the structural basis of this interaction, we examined the activity of 11...
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| Veröffentlicht in: | Biochemistry (Easton) 2000-02, Vol.39 (8), p.2001 |
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| creator | Favre, I Moss, G W Goldenberg, D P Otlewski, J Moczydlowski, E |
| description | Large conductance Ca(2+)-activated K(+) channels (BK(Ca)) contain an intracellular binding site for bovine pancreatic trypsin inhibitor (BPTI), a well-known inhibitor of various serine proteinase (SerP) enzymes. To investigate the structural basis of this interaction, we examined the activity of 11 BPTI mutants using single BK(Ca) channels from rat skeletal muscle incorporated into planar lipid bilayers. All of the mutants induced discrete substate events at the single-channel level. The dwell time of the substate, which is inversely related to the dissociation rate constant of BPTI, exhibited relatively small changes ( |
| doi_str_mv | 10.1021/bi992140v |
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To investigate the structural basis of this interaction, we examined the activity of 11 BPTI mutants using single BK(Ca) channels from rat skeletal muscle incorporated into planar lipid bilayers. All of the mutants induced discrete substate events at the single-channel level. The dwell time of the substate, which is inversely related to the dissociation rate constant of BPTI, exhibited relatively small changes (<9-fold) for the various mutants. However, the apparent association rate constant varied up to 190-fold and exhibited a positive correlation with the net charge of the molecule, suggesting the presence of a negative electrostatic surface potential in the vicinity of the binding site. The substate current level was unaffected by most of the mutations except for substitutions of Lys15. Different residues at this position were found to modulate the apparent conductance of the BPTI-induced substate to 0% (K15G), 10% (K15F), 30% (K15 wild-type), and 55% (K15V) of the open state at +20 mV. Lys15 is located on a loop of BPTI that forms the primary contact region for binding to many SerPs such as trypsin, chymotrypsin, and elastase. The finding that Lys15 is a determinant of the conductance behavior of the BK(Ca) channel when BPTI is bound implies that the same inhibitory loop that contacts SerP's is located close to the protein interface in the BK(Ca) channel complex. This supports the hypothesis that the C-terminal region of the BK(Ca) channel protein contains a domain homologous to SerP's. We propose a domain interaction model for the mechanism of substate production by Kunitz inhibitors based on current ideas for allosteric activation of BK(Ca) channels by voltage and Ca(2+).</description><identifier>ISSN: 0006-2960</identifier><identifier>DOI: 10.1021/bi992140v</identifier><identifier>PMID: 10684650</identifier><language>eng</language><publisher>United States</publisher><subject>Amino Acid Sequence ; Animals ; Aprotinin - chemistry ; Binding Sites ; Cattle ; Electrophysiology ; Ion Channel Gating ; Kinetics ; Large-Conductance Calcium-Activated Potassium Channels ; Models, Molecular ; Molecular Sequence Data ; Muscle, Skeletal - chemistry ; Mutagenesis ; Potassium Channels - chemistry ; Potassium Channels, Calcium-Activated ; Protein Structure, Tertiary ; Rats ; Recombinant Proteins - chemistry ; Static Electricity ; Structure-Activity Relationship</subject><ispartof>Biochemistry (Easton), 2000-02, Vol.39 (8), p.2001</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,782,786,27933,27934</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10684650$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Favre, I</creatorcontrib><creatorcontrib>Moss, G W</creatorcontrib><creatorcontrib>Goldenberg, D P</creatorcontrib><creatorcontrib>Otlewski, J</creatorcontrib><creatorcontrib>Moczydlowski, E</creatorcontrib><title>Structure-activity relationships for the interaction of bovine pancreatic trypsin inhibitor with an intracellular site on a large conductance Ca(2+)-activated K(+) channel</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Large conductance Ca(2+)-activated K(+) channels (BK(Ca)) contain an intracellular binding site for bovine pancreatic trypsin inhibitor (BPTI), a well-known inhibitor of various serine proteinase (SerP) enzymes. To investigate the structural basis of this interaction, we examined the activity of 11 BPTI mutants using single BK(Ca) channels from rat skeletal muscle incorporated into planar lipid bilayers. All of the mutants induced discrete substate events at the single-channel level. The dwell time of the substate, which is inversely related to the dissociation rate constant of BPTI, exhibited relatively small changes (<9-fold) for the various mutants. However, the apparent association rate constant varied up to 190-fold and exhibited a positive correlation with the net charge of the molecule, suggesting the presence of a negative electrostatic surface potential in the vicinity of the binding site. The substate current level was unaffected by most of the mutations except for substitutions of Lys15. Different residues at this position were found to modulate the apparent conductance of the BPTI-induced substate to 0% (K15G), 10% (K15F), 30% (K15 wild-type), and 55% (K15V) of the open state at +20 mV. Lys15 is located on a loop of BPTI that forms the primary contact region for binding to many SerPs such as trypsin, chymotrypsin, and elastase. The finding that Lys15 is a determinant of the conductance behavior of the BK(Ca) channel when BPTI is bound implies that the same inhibitory loop that contacts SerP's is located close to the protein interface in the BK(Ca) channel complex. This supports the hypothesis that the C-terminal region of the BK(Ca) channel protein contains a domain homologous to SerP's. We propose a domain interaction model for the mechanism of substate production by Kunitz inhibitors based on current ideas for allosteric activation of BK(Ca) channels by voltage and Ca(2+).</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Aprotinin - chemistry</subject><subject>Binding Sites</subject><subject>Cattle</subject><subject>Electrophysiology</subject><subject>Ion Channel Gating</subject><subject>Kinetics</subject><subject>Large-Conductance Calcium-Activated Potassium Channels</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Muscle, Skeletal - chemistry</subject><subject>Mutagenesis</subject><subject>Potassium Channels - chemistry</subject><subject>Potassium Channels, Calcium-Activated</subject><subject>Protein Structure, Tertiary</subject><subject>Rats</subject><subject>Recombinant Proteins - chemistry</subject><subject>Static Electricity</subject><subject>Structure-Activity Relationship</subject><issn>0006-2960</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo1UMtOwzAQ9AFES-HADyAfW1WB9SNuc0QVL1GJA3CubGdDjFIncpyifhM_iavCZVczOzMaLSFXDG4YcHZrXFFwJmF3QsYAoDJeKBiR877_SlDCQp6REQO1lCqHMfl5i2GwcQiYaRvdzsU9Ddjo6Frf167radUGGmukzkcMB03raVtR0-6cR9ppbwMmuaUx7Lve-SSsnXEx2b5drKk-MDE5sWmGRgfau4g0hWia0CdS2_oyVUhBSFd6yuezYxUdsaQv0_mM2lp7j80FOa100-Pl356Qj4f799VTtn59fF7drbOOcR6zSuZQALe5qcAytcyVKMFKKzBnXGhMYwEqnYQucrBCFGKhCmm04QaVkGJCro-53WC2WG664LY67Df_XxO_5V5utw</recordid><startdate>20000229</startdate><enddate>20000229</enddate><creator>Favre, I</creator><creator>Moss, G W</creator><creator>Goldenberg, D P</creator><creator>Otlewski, J</creator><creator>Moczydlowski, E</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope></search><sort><creationdate>20000229</creationdate><title>Structure-activity relationships for the interaction of bovine pancreatic trypsin inhibitor with an intracellular site on a large conductance Ca(2+)-activated K(+) channel</title><author>Favre, I ; Moss, G W ; Goldenberg, D P ; Otlewski, J ; Moczydlowski, E</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p122t-f450902c5bf0c168563d0c4c3e5123ae1237060c13a950c33937694bab2be6343</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Aprotinin - chemistry</topic><topic>Binding Sites</topic><topic>Cattle</topic><topic>Electrophysiology</topic><topic>Ion Channel Gating</topic><topic>Kinetics</topic><topic>Large-Conductance Calcium-Activated Potassium Channels</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Muscle, Skeletal - chemistry</topic><topic>Mutagenesis</topic><topic>Potassium Channels - chemistry</topic><topic>Potassium Channels, Calcium-Activated</topic><topic>Protein Structure, Tertiary</topic><topic>Rats</topic><topic>Recombinant Proteins - chemistry</topic><topic>Static Electricity</topic><topic>Structure-Activity Relationship</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Favre, I</creatorcontrib><creatorcontrib>Moss, G W</creatorcontrib><creatorcontrib>Goldenberg, D P</creatorcontrib><creatorcontrib>Otlewski, J</creatorcontrib><creatorcontrib>Moczydlowski, E</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Favre, I</au><au>Moss, G W</au><au>Goldenberg, D P</au><au>Otlewski, J</au><au>Moczydlowski, E</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Structure-activity relationships for the interaction of bovine pancreatic trypsin inhibitor with an intracellular site on a large conductance Ca(2+)-activated K(+) channel</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2000-02-29</date><risdate>2000</risdate><volume>39</volume><issue>8</issue><spage>2001</spage><pages>2001-</pages><issn>0006-2960</issn><abstract>Large conductance Ca(2+)-activated K(+) channels (BK(Ca)) contain an intracellular binding site for bovine pancreatic trypsin inhibitor (BPTI), a well-known inhibitor of various serine proteinase (SerP) enzymes. To investigate the structural basis of this interaction, we examined the activity of 11 BPTI mutants using single BK(Ca) channels from rat skeletal muscle incorporated into planar lipid bilayers. All of the mutants induced discrete substate events at the single-channel level. The dwell time of the substate, which is inversely related to the dissociation rate constant of BPTI, exhibited relatively small changes (<9-fold) for the various mutants. However, the apparent association rate constant varied up to 190-fold and exhibited a positive correlation with the net charge of the molecule, suggesting the presence of a negative electrostatic surface potential in the vicinity of the binding site. The substate current level was unaffected by most of the mutations except for substitutions of Lys15. Different residues at this position were found to modulate the apparent conductance of the BPTI-induced substate to 0% (K15G), 10% (K15F), 30% (K15 wild-type), and 55% (K15V) of the open state at +20 mV. Lys15 is located on a loop of BPTI that forms the primary contact region for binding to many SerPs such as trypsin, chymotrypsin, and elastase. The finding that Lys15 is a determinant of the conductance behavior of the BK(Ca) channel when BPTI is bound implies that the same inhibitory loop that contacts SerP's is located close to the protein interface in the BK(Ca) channel complex. This supports the hypothesis that the C-terminal region of the BK(Ca) channel protein contains a domain homologous to SerP's. We propose a domain interaction model for the mechanism of substate production by Kunitz inhibitors based on current ideas for allosteric activation of BK(Ca) channels by voltage and Ca(2+).</abstract><cop>United States</cop><pmid>10684650</pmid><doi>10.1021/bi992140v</doi></addata></record> |
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| subjects | Amino Acid Sequence Animals Aprotinin - chemistry Binding Sites Cattle Electrophysiology Ion Channel Gating Kinetics Large-Conductance Calcium-Activated Potassium Channels Models, Molecular Molecular Sequence Data Muscle, Skeletal - chemistry Mutagenesis Potassium Channels - chemistry Potassium Channels, Calcium-Activated Protein Structure, Tertiary Rats Recombinant Proteins - chemistry Static Electricity Structure-Activity Relationship |
| title | Structure-activity relationships for the interaction of bovine pancreatic trypsin inhibitor with an intracellular site on a large conductance Ca(2+)-activated K(+) channel |
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