determining-membrane-protein-topology-using-fluorescence-protease

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Chicago Medical School

Analysis of CFTR endocytosis by cell surface biotinylation.

Demostramos que la base de la terminal C del hIK1 resulta en una p&eacute;rdida de canales funcionales. Esto puede ser causado por cualquiera de los dos (i) una falla en el canal de tr&aacute;fico a la membrana plasm&aacute;tica o (ii) la expresi&oacute;n de canales no funcionales. Delinear entre estas posibilidades, un epitopo de la hemaglutinina fue insertado en el lazo extracelular entre dominios transmembrana S3 y S4. Expresi&oacute;n superficial y funci&oacute;n del canal fueron medidos por inmunofluorescencia, inmunoprecipitaci&oacute;n de superficie celular y t&eacute;cnicas de abrazadera de parche de c&eacute;lulas completas. Aunque la eliminaci&oacute;n de los &uacute;ltimos 14 amino&aacute;cidos de hIK1 (L414STOP) no tuvo ning&uacute;n efecto sobre la expresi&oacute;n de la membrana plasm&aacute;tica y funci&oacute;n, eliminaci&oacute;n de los &uacute;ltimos 26 amino&aacute;cidos (K402STOP) dio lugar a una p&eacute;rdida completa de expresi&oacute;n de la membrana. Mutaci&oacute;n de la Heptada leucina repetir terminando en Leu(406) (L399A/L406A) totalmente abrogado la localizaci&oacute;n de la membrana. Mutaciones adicionales dentro de la repetici&oacute;n de la Heptada (L385A/L392A, L392A/L406A) o de la a posiciones (I396A/L403A) resultaron en una p&eacute;rdida casi total del canal de membrana localizada. Por el contrario, mutando leucines individuales no comprometer canal tr&aacute;fico o funci&oacute;n. Tanto la localizaci&oacute;n de la membrana y la funci&oacute;n de L399A/L406A podr&iacute;an ser parcialmente restaurados por incubaci&oacute;n a 27 grados C. Co-inmunoprecipitaci&oacute;n estudios demostraron que las mutaciones de cremallera de leucina hacen no formaci&oacute;n de multimer del compromiso. Por el contrario, hemos demostrado que la regi&oacute;n de cremallera de leucina de hIK1 es capaz de co-assembly y que esto depende de una cremallera de leucina intacto. Finalmente, en otro miembro de la familia del gene, SK3 se conserva esta cremallera de leucina. Sin embargo, la mutaci&oacute;n de la cremallera de leucina en SK3 no ten&iacute;a ning&uacute;n efecto sobre la localizaci&oacute;n de la membrana plasm&aacute;tica o funci&oacute;n. En conclusi&oacute;n, demostramos que la cremallera de leucina c-terminal es esencial para facilitar el correcto plegamiento y la membrana plasm&aacute;tica trata de hIK1, mientras que esta funci&oacute;n no se conserva en otros miembros de la familia de gene.

Tr&aacute;fico del Ca2 +-K + canal activado, hIK1, depende de una cremallera de leucina c-terminal.

&#x6211;&#x4EEC;&#x8868;&#x73B0;&#x51FA;&#x7684; hIK1 C &#x7EC8;&#x7AEF;&#x622A;&#x65AD;&#x7ED3;&#x679C;&#x529F;&#x80FD;&#x6E20;&#x9053;&#x7684;&#x635F;&#x5931;&#x3002;&#x8FD9;&#x53EF;&#x80FD;&#x662F;&#x7531; (i) &#x5931;&#x8D25; &#xFF1B; &#x6216;&#x8005;&#x5C06;&#x901A;&#x4FE1;&#x5230;&#x7EC6;&#x80DE;&#x8D28;&#x819C;&#x7684;&#x901A;&#x9053;&#x6216; (ii) &#x975E;&#x529F;&#x80FD;&#x6027;&#x7684;&#x6E20;&#x9053;&#x8868;&#x8FBE;&#x5F15;&#x8D77;&#x7684;&#x3002;&#x6765;&#x63CF;&#x7ED8;&#x8FD9;&#x4E9B;&#x53EF;&#x80FD;&#x6027;&#x4E4B;&#x95F4;&#xFF0C;&#x8840;&#x51DD;&#x7D20;&#x6297;&#x539F;&#x8868;&#x4F4D;&#x63D2;&#x5165;&#x4E86;&#x8DE8;&#x819C;&#x57DF; S3 &#x548C; S4 &#x4E4B;&#x95F4;&#x7684;&#x80DE;&#x5916;&#x5FAA;&#x73AF;&#x3002;&#x901A;&#x8FC7;&#x514D;&#x75AB;&#x8367;&#x5149;&#x6CD5;&#x3001; &#x7EC6;&#x80DE;&#x8868;&#x9762;&#x6C89;&#x6DC0;&#x548C;&#x5168;&#x7EC6;&#x80DE;&#x4FEE;&#x8865;&#x94B3;&#x6280;&#x672F;&#x6D4B;&#x91CF;&#x4E86;&#x8868;&#x9762;&#x8868;&#x8FBE;&#x548C;&#x901A;&#x9053;&#x529F;&#x80FD;&#x3002;&#x867D;&#x7136;&#x5220;&#x9664;&#x4E0A;&#x6B21; 14 &#x6C28;&#x57FA;&#x9178;&#x7684; hIK1 (L414STOP) &#x66FE;&#x5BF9;&#x7EC6;&#x80DE;&#x819C;&#x8868;&#x8FBE;&#x53CA;&#x529F;&#x80FD;&#x6CA1;&#x6709;&#x5F71;&#x54CD;&#xFF0C;&#x6700;&#x540E; 26 &#x6C28;&#x57FA;&#x9178; &#xFF08;K402STOP&#xFF09; &#x5220;&#x9664;&#x5BFC;&#x81F4;&#x819C;&#x8868;&#x8FBE;&#x5B8C;&#x5168;&#x4E27;&#x5931;&#x3002;&#x7A81;&#x53D8;&#x7684;&#x4EAE;&#x6C28;&#x9178;&#x80BD;&#x91CD;&#x590D;&#x7ED3;&#x675F; Leu(406) &#xFF08;L399A/L406A&#xFF09; &#x5B8C;&#x5168;&#x5E9F;&#x9664;&#x819C;&#x672C;&#x5730;&#x5316;&#x3002;&#x989D;&#x5916;&#x7A81;&#x53D8;&#x6216;&#x5185;&#x4E03;&#x80BD;&#x91CD;&#x590D; &#xFF08;L385A/L392A&#x3001; L392A/L406A&#xFF09; &#x7684;&#x804C;&#x4F4D; &#xFF08;I396A/L403A&#xFF09; &#x9020;&#x6210;&#x7684;&#x819C;&#x672C;&#x5730;&#x5316;&#x901A;&#x9053;&#x51E0;&#x4E4E;&#x5B8C;&#x5168;&#x635F;&#x5931;&#x3002;&#x76F8;&#x6BD4;&#x4E4B;&#x4E0B;&#xFF0C;&#x53D8;&#x5F02;&#x4E2A;&#x522B; leucines &#x4E5F;&#x4E0D;&#x4F1A;&#x59A5;&#x534F;&#x6E20;&#x9053;&#x8D29;&#x8FD0;&#x6216;&#x51FD;&#x6570;&#x3002;&#x819C;&#x672C;&#x5730;&#x5316;&#x548C; L399A/L406A &#x51FD;&#x6570;&#x53EF;&#x4EE5;&#x901A;&#x8FC7; 27 &#x5EA6; C.Co &#x6C89;&#x6DC0;&#x7684;&#x7814;&#x7A76;&#x8868;&#x660E;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#x7A81;&#x53D8;&#x505A;&#x4E0D;&#x59A5;&#x534F; multimer &#x5F62;&#x6210;&#x5B75;&#x5316;&#x90E8;&#x5206;&#x8FD8;&#x539F;&#x3002;&#x76F8;&#x6BD4;&#x4E4B;&#x4E0B;&#xFF0C;&#x6211;&#x4EEC;&#x5C55;&#x793A;&#x4E86;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#x533A;&#x7684; hIK1 &#x662F; co-assembly &#x7684;&#x80FD;&#x529B;&#xFF0C;&#x8FD9;&#x662F;&#x4F9D;&#x8D56;&#x4E8E;&#x4E0D;&#x53D8;&#x7684;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x5728;&#x53E6;&#x4E00;&#x540D;&#x6210;&#x5458;&#x7684;&#x57FA;&#x56E0;&#x5BB6;&#x65CF;&#xFF0C;SK3 &#x6B64;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#x88AB;&#x5B88;&#x6052;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#x4E2D; SK3 &#x7A81;&#x53D8;&#x4E86;&#x7EC6;&#x80DE;&#x819C;&#x672C;&#x5730;&#x5316;&#x6216;&#x529F;&#x80FD;&#x6CA1;&#x6709;&#x5F71;&#x54CD;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x8BC1;&#x660E; C &#x7EC8;&#x7AEF;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#x662F;&#x5173;&#x952E;&#xFF0C;&#x4EE5;&#x4FBF;&#x6B63;&#x786E;&#x6298;&#x53E0;&#x548C;&#x7EC6;&#x80DE;&#x819C;&#x8D29;&#x8FD0;&#x7684; hIK1&#xFF0C;&#x800C;&#x6B64;&#x529F;&#x80FD;&#x5728;&#x5176;&#x4ED6;&#x57FA;&#x56E0;&#x5BB6;&#x65CF;&#x6210;&#x5458;&#x4E0D;&#x5B88;&#x6052;&#x3002;

&#x8D29;&#x8FD0;&#x7684; Ca2 +-K + &#x901A;&#x9053;&#x6FC0;&#x6D3B;&#xFF0C;hIK1&#xFF0C;&#x662F;&#x53D6;&#x51B3;&#x4E8E; C &#x7EC8;&#x7AEF;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#x3002;

We demonstrate that the C-terminal truncation of hIK1 results in a loss of functional channels. This could be caused by either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of non-functional channels. To delineate among these possibilities, a hemagglutinin epitope was inserted into the extracellular loop between transmembrane domains S3 and S4. Surface expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp techniques. Although deletion of the last 14 amino acids of hIK1 (L414STOP) had no effect on plasma membrane expression and function, deletion of the last 26 amino acids (K402STOP) resulted in a complete loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu(406) (L399A/L406A) completely abrogated membrane localization. Additional mutations within the heptad repeat (L385A/L392A, L392A/L406A) or of the a positions (I396A/L403A) resulted in a near-complete loss of membrane-localized channel. In contrast, mutating individual leucines did not compromise channel trafficking or function. Both membrane localization and function of L399A/L406A could be partially restored by incubation at 27 degrees C. Co-immunoprecipitation studies demonstrated that leucine zipper mutations do not compromise multimer formation. In contrast, we demonstrated that the leucine zipper region of hIK1 is capable of co-assembly and that this is dependent upon an intact leucine zipper. Finally, this leucine zipper is conserved in another member of the gene family, SK3. However, mutation of the leucine zipper in SK3 had no effect on plasma membrane localization or function. In conclusion, we demonstrate that the C-terminal leucine zipper is critical to facilitate correct folding and plasma membrane trafficking of hIK1, whereas this function is not conserved in other gene family members.

Trafficking of the Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal leucine zipper.

Nous d&eacute;montrons que la troncation de C-terminal de hIK1 provoque une perte de canaux fonctionnels. Cela peut &ecirc;tre d&ucirc; &agrave; soit (i) une d&eacute;faillance de la cha&icirc;ne du trafic vers la membrane plasmique ou (ii) l&#39;expression des canaux non fonctionnel. Pour d&eacute;limiter parmi ces possibilit&eacute;s, un &eacute;pitope h&eacute;magglutinine a &eacute;t&eacute; ins&eacute;r&eacute; dans la boucle extracellulaire entre domaines transmembranaires S3 et S4. Expression en surface et la fonction des canaux ont &eacute;t&eacute; mesur&eacute;s par immunofluorescence, immunopr&eacute;cipitation de surface cellulaire et techniques de cellules enti&egrave;res de patch clamp. Bien que la suppression des derni&egrave;re 14 acides amin&eacute;s de hIK1 (L414STOP) n&#39;a aucun effet sur l&#39;expression de la membrane plasmique et de la fonction, la suppression des derniers 26 acides amin&eacute;s (K402STOP) a entra&icirc;n&eacute; une perte compl&egrave;te de l&#39;expression de la membrane. Mutation de l&#39;Heptade leucine r&eacute;p&eacute;ter se terminant &agrave; Leu(406) (L399A/L406A) compl&egrave;tement abrog&eacute;e localisation de membrane. Autres mutations au sein de la r&eacute;p&eacute;tition Heptade (L385A/L392A, L392A/L406A) ou de l&#39;une position (I396A/L403A) a entra&icirc;n&eacute; une perte presque compl&egrave;te des canaux membranaires localis&eacute;e. En revanche, leucines individuels de mutation n&#39;a pas compromis traite de canal ou de la fonction. Localisation de membrane et fonction de L399A/L406A pourraient &ecirc;tre partiellement restaur&eacute;s par incubation &agrave; 27 degr&eacute;s C. Co-immunopr&eacute;cipitation des &eacute;tudes ont d&eacute;montr&eacute; que les mutations de tirette de leucine ne pas compromis multim&egrave;res de formation. En revanche, nous avons d&eacute;montr&eacute; que la r&eacute;gion de tirette de leucine de hIK1 est capable de co-assembly et que cela d&eacute;pend une tirette de leucine intact. Enfin, cette tirette de leucine est conserv&eacute;e chez un autre membre de la famille de g&egrave;nes, SK3. Cependant, la mutation de la tirette de leucine dans SK3 n&#39;a aucun effet sur la localisation de la membrane plasmique ou fonction. En conclusion, nous d&eacute;montrons que la tirette de leucine de C-terminal est essentielle pour faciliter le pliage correcte et la membrane plasmique traite des hIK1, alors que cette fonction n&#39;est pas conserv&eacute;e chez les autres membres de famille de g&egrave;nes.

Traite de la Ca2 +-canal K + activ&eacute;, hIK1, d&eacute;pend d&#39;une tirette de leucine de C-terminal.

Wir zeigen, dass das C-terminale Abschneiden von hIK1 einen Verlust von funktionalen Kan&auml;le f&uuml;hrt. Dies k&ouml;nnte entweder (i) einen Fehler des Kanals um Besucher auf die Plasmamembran oder (Ii) den Ausdruck nicht funktionierende Kan&auml;le haben. Um unter diesen M&ouml;glichkeiten abzugrenzen, wurde die extrazellul&auml;re Schleife zwischen transmembrane Dom&auml;nen S3 und S4 ein H&auml;magglutinin-Epitop eingef&uuml;gt. Oberfl&auml;che Ausdruck und Kanal Funktion wurden durch Immunfluoreszenz, Zelle Oberfl&auml;che Immunopr&auml;zipitation und ganze Zellen Patch Clamp Techniken gemessen. Obwohl L&ouml;schung der letzten 14 Aminos&auml;uren von hIK1 (L414STOP) hatte keine Auswirkung auf die Plasmamembran Ausdruck und Funktion, L&ouml;schung der letzte 26 Aminos&auml;uren (K402STOP) f&uuml;hrte zu einem v&ouml;lligen Verlust von Membran-Ausdruck. Mutation der Leucin-Heptad wiederholen Sie Membran Lokalisierung endet beim Leu(406) (L399A/L406A) vollst&auml;ndig aufgehoben werden. Weitere Mutationen innerhalb der Heptad Repeat (L385A/L392A, L392A/L406A) oder von der eine Positionen (I396A/L403A) f&uuml;hrte zu einem nahezu vollst&auml;ndigen Verlust der Membran lokalisierte Kanal. Im Gegensatz dazu das mutierende einzelne Leucine nicht Kanal Menschenhandel oder Funktion beeintr&auml;chtigen. Membran-Lokalisierung und Funktion des L399A/L406A konnte durch Inkubation bei 27 Grad C. Co-Immunopr&auml;zipitation Studien nachgewiesen, dass Leucin Rei&szlig;verschluss Mutationen nicht Kompromiss Multimer Bildung tun, teilweise wiederhergestellt werden. Im Gegensatz dazu zeigte wir, dass die Leucin-Rei&szlig;verschluss-Region von hIK1 Co-assembly kann und dass dies eine intakte Leucins abh&auml;ngig. Schlie&szlig;lich ist diese des Leucins in ein weiteres Mitglied der Familie der Gen SK3 konserviert. Mutation des Leucins in SK3 hatte jedoch keine Auswirkungen auf die Plasmamembran Lokalisierung oder Funktion. Abschlie&szlig;end zeigen wir, dass der C-terminalen des Leucins entscheidend f&uuml;r die korrekte Faltung und Plasmamembran Menschenhandel hIK1, zu erleichtern, w&auml;hrend diese Funktion nicht in andere gen-Familienmitglieder erhalten ist.

Handel mit den Ca2 +-aktivierte K + Kanal, hIK1, ist abh&auml;ngig von einer C-terminalen des Leucins.

Dimostriamo che il troncamento del C-terminale di hIK1 si traduce in una perdita di canali funzionali. Questo potrebbe essere causato da o (i) un fallimento del canale per il traffico verso la membrana plasmatica o (ii) l&#39;espressione dei canali non funzionali. Per delineare tra queste possibilit&agrave;, un epitopo emagglutinina &egrave; stato inserito nel ciclo extracellulare tra domini transmembrana S3 e S4. Espressione superficiale e funzione dei canali sono stati misurati da immunofluorescenza, immunoprecipitazione di superficie delle cellule e cellule intere patch clamp tecniche. Sebbene eliminazione degli ultimi 14 amminoacidi di hIK1 (L414STOP) ha avuto alcun effetto sulla membrana plasmatica espressione e funzione, eliminazione degli ultimi 26 amminoacidi (K402STOP) ha provocato una perdita completa di espressione di membrana. Mutazione di heptad la leucina ripetere termina presso Leu(406) (L399A/L406A) completamente abolita la localizzazione di membrana. Ulteriori mutazioni all&#39;interno la ripetizione heptad (L385A/L392A, L392A/L406A) o della a posizioni (I396A/L403A) ha provocato una perdita quasi completa del canale di membrana localizzati. In contrasto, la mutazione di singoli leucines non compromesso canale traffico o funzione. Localizzazione di membrana e la funzione di L399A/L406A potrebbe essere parzialmente restaurate da incubazione a 27 gradi C. Co-immunoprecipitazione studi hanno dimostrato che le mutazioni di chiusura lampo della leucina fanno non compromesso inattivazione formazione. Al contrario, abbiamo dimostrato che la regione cerniera di leucina di hIK1 &egrave; in grado di co-assembly e che questo &egrave; dipendente da una cerniera di leucina intatto. Infine, questa chiusura lampo della leucina &egrave; conservato in un altro membro della famiglia genica, SK3. Tuttavia, mutazione della chiusura lampo della leucina in SK3 ha avuto alcun effetto sulla membrana plasmatica localizzazione o funzione. In conclusione, ci dimostrano che la chiusura lampo della leucina C-terminale &egrave; fondamentale per facilitare il corretto ripiegamento e traffico di membrana plasmatica di hIK1, considerando che questa funzione non &egrave; conservata in altri membri della famiglia genica.

Traffico di Ca2 +-activated K + canale, hIK1, &egrave; dipendente da una chiusura lampo della leucina C-terminale.

C &#x672B;&#x7AEF;&#x5207;&#x65AD; hik1 &#x6A5F;&#x80FD;&#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x306E;&#x640D;&#x5931;&#x306E;&#x7D50;&#x679C;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x306F;&#x5F62;&#x8CEA;&#x819C;&#x3078;&#x306E;&#x30C8;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30AF;&#x3092;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x3044;&#x305A;&#x308C;&#x304B;&#x306E; (i) &#x306E;&#x969C;&#x5BB3;&#x307E;&#x305F;&#x306F; (ii) &#x975E;&#x6A5F;&#x80FD;&#x7684;&#x306A;&#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x306E;&#x5F0F;&#x306B;&#x3088;&#x3063;&#x3066;&#x767A;&#x751F;&#x3059;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x306E;&#x9593;&#x3067;&#x63CF;&#x304F;&#x306B;&#x306F;&#x3001;&#x30D8;&#x30DE;&#x30B0;&#x30EB;&#x30C1;&#x30CB;&#x30F3; &#x30A8;&#x30D4;&#x30C8;&#x30FC;&#x30D7;&#x306F;&#x7D30;&#x80DE;&#x5916;&#x30EB;&#x30FC;&#x30D7;&#x819C;&#x8CAB;&#x901A;&#x30C9;&#x30E1;&#x30A4;&#x30F3; S3 &#x3068; S4 &#x306E;&#x9593;&#x306B;&#x633F;&#x5165;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x8868;&#x9762;&#x767A;&#x73FE;&#x3068;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x6A5F;&#x80FD;&#x86CD;&#x5149;&#x6297;&#x4F53;&#x6CD5;&#x3001;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x306E;&#x514D;&#x75AB;&#x6C88;&#x964D;&#x304A;&#x3088;&#x3073;&#x5168;&#x7D30;&#x80DE;&#x30D1;&#x30C3;&#x30C1;&#x30AF; &#x30E9;&#x30F3;&#x30D7;&#x6CD5;&#x306B;&#x3088;&#x3063;&#x3066;&#x6E2C;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x6700;&#x7D42; 14 &#x30A2;&#x30DF;&#x30CE;&#x9178; hik1 &#x306E;&#x524A;&#x9664; (L414STOP) &#x306E;&#x30D7;&#x30E9;&#x30BA;&#x30DE;&#x819C;&#x767A;&#x73FE;&#x3068;&#x6A5F;&#x80FD;&#x306B;&#x5F71;&#x97FF;&#x306F;&#x3054;&#x3056;&#x3044;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3001;&#x524A;&#x9664;&#x6700;&#x7D42;&#x66F4;&#x65B0; 26 &#x30A2;&#x30DF;&#x30CE;&#x9178; (K402STOP) &#x306E;&#x7D50;&#x679C;&#x3001;&#x819C;&#x5F0F;&#x306E;&#x5B8C;&#x5168;&#x306A;&#x640D;&#x5931;&#x3002;&#x30ED;&#x30A4;&#x30B7;&#x30F3; 7 &#x9023;&#x5B50;&#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;&#x3092;&#x7E70;&#x308A;&#x8FD4;&#x3059; Leu(406) (L399A/L406A) &#x3067;&#x5B8C;&#x5168;&#x306B;&#x7D42;&#x4E86; abrogated &#x306E;&#x819C;&#x306E;&#x30ED;&#x30FC;&#x30AB;&#x30EA;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3002;&#x8FFD;&#x52A0;&#x306E;&#x5909;&#x7570; &#xFF08;L385A/L392A&#x3001;L392A/L406A&#xFF09; &#x306E; 7 &#x9023;&#x5B50;&#x7E70;&#x308A;&#x8FD4;&#x3057;&#x5185;&#x307E;&#x305F;&#x306F;&#x306E;&#x4F4D;&#x7F6E; (I396A/L403A) &#x819C;&#x5C40;&#x5728;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x307B;&#x307C;&#x5B8C;&#x5168;&#x306A;&#x640D;&#x5931;&#x306B;&#x3064;&#x306A;&#x304C;&#x3063;&#x305F;&#x3002;&#x5BFE;&#x7167;&#x7684;&#x306B;&#x3001;&#x500B;&#x3005; &#x306E;&#x30ED;&#x30A4;&#x30B7;&#x30F3;&#x5909;&#x7570;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x3084;&#x95A2;&#x6570;&#x59A5;&#x5354;&#x3057;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x3002;&#x4E21;&#x65B9;&#x306E;&#x819C;&#x306E;&#x30ED;&#x30FC;&#x30AB;&#x30EA;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3068;&#x6A5F;&#x80FD; L399A/L406A &#x306E;&#x90E8;&#x5206;&#x7684; C. Co &#x514D;&#x75AB;&#x6C88;&#x964D;&#x7814;&#x7A76;&#x30ED;&#x30A4;&#x30B7;&#x30F3; &#x30B8;&#x30C3;&#x30D1;&#x30FC;&#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;&#x306F;&#x306A;&#x3044;&#x59A5;&#x5354;&#x306E;&#x591A;&#x91CF;&#x4F53;&#x5F62;&#x6210;&#x3092;&#x884C;&#x3046;&#x5B9F;&#x8A3C; 27 &#x5EA6;&#x3067;&#x5B75;&#x5316;&#x306B;&#x3088;&#x3063;&#x3066;&#x5FA9;&#x5143;&#x3067;&#x304D;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3002;&#x5BFE;&#x7167;&#x7684;&#x306B;&#x3001;hik1 &#x30ED;&#x30A4;&#x30B7;&#x30F3;&#x306E;&#x30B8;&#x30C3;&#x30D1;&#x30FC;&#x306E;&#x5730;&#x57DF;&#x304C;&#x306E; co-assembly &#x304C;&#x53EF;&#x80FD;&#x3067;&#x3042;&#x308A;&#x3001;&#x3053;&#x308C;&#x304C;&#x305D;&#x306E;&#x307E;&#x307E; zipper &#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x3053;&#x306E;&#x30ED;&#x30A4;&#x30B7;&#x30F3; &#x30B8;&#x30C3;&#x30D1;&#x30FC;&#x907A;&#x4F1D;&#x5B50;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x3001;SK3 &#x306E;&#x5225;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x306B;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x7A81;&#x7136;&#x5909;&#x7570;&#x3001;&#x30ED;&#x30A4;&#x30B7;&#x30F3; &#x30B8;&#x30C3;&#x30D1;&#x30FC; SK3 &#x3067;&#x306E;&#x30D7;&#x30E9;&#x30BA;&#x30DE;&#x819C;&#x306E;&#x30ED;&#x30FC;&#x30AB;&#x30EA;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3084;&#x6A5F;&#x80FD;&#x306B;&#x5F71;&#x97FF;&#x304C;&#x306A;&#x304B;&#x3063;&#x305F;&#x3002;&#x7D50;&#x8AD6;&#x3068;&#x3057;&#x3066;&#x306F;&#x3001;C &#x672B;&#x7AEF;&#x30ED;&#x30A4;&#x30B7;&#x30F3; &#x30B8;&#x30C3;&#x30D1;&#x30FC;&#x304C;&#x3053;&#x306E;&#x95A2;&#x6570;&#x306F;&#x3001;&#x4ED6;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x5BB6;&#x65CF;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x306B;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x306B;&#x5BFE;&#x3057;&#x6B63;&#x3057;&#x3044;&#x6298;&#x308A;&#x7573;&#x307F;&#x3068;&#x30D7;&#x30E9;&#x30BA;&#x30DE;&#x819C; hIK1 &#x306E;&#x8F38;&#x9001;&#x3092;&#x5BB9;&#x6613;&#x306B;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;

Ca 2 + &#x306E;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;-&#x6D3B;&#x6027;&#x5316; K + &#x30C1;&#x30E3;&#x30CD;&#x30EB;, hIK1&#x3001;C &#x672B;&#x7AEF;&#x30ED;&#x30A4;&#x30B7;&#x30F3; &#x30B8;&#x30C3;&#x30D1;&#x30FC;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#xC6B0;&#xB9AC;&#xB294; Hik1&#xC758; C-&#xD130;&#xBBF8;&#xB110; &#xC798;&#xB9BC; &#xAE30;&#xB2A5; &#xCC44;&#xB110;&#xC758; &#xC190;&#xC2E4;&#xC5D0; &#xACB0;&#xACFC; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xD2B8;&#xB798;&#xD53D; &#xCC44;&#xB110;&#xC758; (i)&#xB294; &#xC2E4;&#xD328; &#xC911; &#xD558;&#xB098; &#xB610;&#xB294; (ii) &#xC791;&#xB3D9; &#xD558;&#xC9C0; &#xC54A;&#xB294; &#xCC44;&#xB110;&#xC758; &#xD45C;&#xD604;&#xC5D0; &#xC758;&#xD574; &#xBC1C;&#xC0DD;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xAC00;&#xB2A5;&#xC131; &#xC911;&#xC5D0;&#xC11C; &#xC724;&#xACFD;&#xC744; &#xADF8;&#xB9AC; &#xB2E4;, &#xC870;&#xB958; epitope &#xB9C9; &#xD6A1;&#xB2E8; &#xB3C4;&#xBA54;&#xC778; S3 &#xBC0F; S4 &#xAC04;&#xC5D0; &#xC138;&#xD3EC; &#xC678; &#xB8E8;&#xD504;&#xC5D0; &#xC0BD;&#xC785; &#xD588;&#xB2E4;. &#xD45C;&#xBA74; &#xD45C;&#xD604; &#xBC0F; &#xCC44;&#xB110; &#xAE30;&#xB2A5; &#xBA74;&#xC5ED; &#xD615;&#xAD11; &#xAC80;&#xC0AC;, &#xC138;&#xD3EC; &#xD45C;&#xBA74; immunoprecipitation &#xBC0F; &#xC804;&#xCCB4; &#xC140; &#xD328;&#xCE58; &#xD074;&#xB7A8;&#xD504; &#xAE30;&#xBC95;&#xC5D0; &#xC758;&#xD574; &#xCE21;&#xC815; &#xB418;&#xC5C8;&#xB2E4;. &#xBE44;&#xB85D; Hik1&#xC758; &#xB9C8;&#xC9C0;&#xB9C9; 14 &#xC544;&#xBBF8;&#xB178;&#xC0B0;&#xC758; &#xC0AD;&#xC81C; (L414STOP) &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xC2DD; &#xBC0F; &#xAE30;&#xB2A5;&#xC5D0;&#xB294; &#xC601;&#xD5A5;&#xC744; &#xC8FC;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;, &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xC2DD;&#xC758; &#xC644;&#xC804; &#xC190;&#xC2E4; &#xADC0;&#xCC29;&#xB418; &#xC5C8; &#xB2E4; &#xB9C8;&#xC9C0;&#xB9C9; 26 &#xC544;&#xBBF8;&#xB178;&#xC0B0; (K402STOP)&#xB97C; &#xC0AD;&#xC81C; &#xD569;&#xB2C8;&#xB2E4;. &#xC2E0; heptad &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xB9C9; &#xC9C0;&#xC5ED;&#xD654; &#xD30C;&#xAE30; Leu(406) (L399A/L406A)&#xC5D0;&#xC11C; &#xC644;&#xC804; &#xD558; &#xAC8C; &#xC885;&#xB8CC;&#xB97C; &#xBC18;&#xBCF5; &#xD569;&#xB2C8;&#xB2E4;. (L385A/L392A, L392A/L406A) heptad &#xBC18;&#xBCF5; &#xB0B4;&#xC5D0;&#xC11C; &#xB610;&#xB294; &#xCD94;&#xAC00; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xB9C9; &#xD654; &#xB41C; &#xCC44;&#xB110;&#xC758; &#xADFC;&#xCC98;-&#xC644;&#xC804; &#xD55C; &#xC190;&#xC2E4; &#xADC0;&#xCC29;&#xB418; &#xC5C8; &#xB2E4; &#xC704;&#xCE58; (I396A/L403A). &#xBC18;&#xBA74;, &#xAC1C;&#xBCC4; leucines &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xCC44;&#xB110; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4; &#xB610;&#xB294; &#xD568;&#xC218; &#xD0C0;&#xD611; &#xD558;&#xC9C0; &#xC54A;&#xC558;&#xB2E4;. C. &#xACF5;&#xB3D9; immunoprecipitation &#xC5F0;&#xAD6C; &#xC2DC;&#xC5F0; &#xC2E0; &#xC9C0;&#xD37C; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xD0C0;&#xD611; multimer &#xD615;&#xC131; &#xD558;&#xC9C0; &#xC54A;&#xB294; 27&#xB3C4;&#xC5D0;&#xC11C; &#xBD80; &#xD654; &#xB9C9; &#xC9C0;&#xC5ED;&#xD654; &#xBC0F; L399A/l406a&#xC758; &#xAE30;&#xB2A5; &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; &#xBCF5;&#xC6D0;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xBC18;&#xBA74;, &#xC6B0;&#xB9AC; &#xC2DC;&#xC5F0; Hik1&#xC758; &#xC2E0; &#xC9C0;&#xD37C; &#xC9C0;&#xC5ED;&#xC740; co-assembly&#xC758;, &#xADF8;&#xB9AC;&#xACE0; &#xC774;&#xAC83;&#xC774; &#xADF8;&#xB300;&#xB85C; &#xC2E0; &#xC9C0;&#xD37C;&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;,&#xC774; &#xC2E0; &#xC9C0;&#xD37C; SK3 &#xC720;&#xC804;&#xC790; &#xAC00;&#xC871;&#xC758; &#xB2E4;&#xB978; &#xAD6C;&#xC131;&#xC6D0;&#xC5D0; &#xBCF4;&#xC874; &#xB429;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; SK3 &#xC2E0; &#xC9C0;&#xD37C;&#xC758; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xC9C0;&#xC5ED;&#xD654; &#xB610;&#xB294; &#xAE30;&#xB2A5;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xC8FC;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4; &#xD588;&#xB2E4;. &#xACB0;&#xB860;&#xC801;&#xC73C;&#xB85C;, C &#xD130;&#xBBF8;&#xB110; &#xC2E0; &#xC9C0;&#xD37C;&#xB294;&#xC774; &#xD568;&#xC218;&#xB294; &#xB2E4;&#xB978; &#xC720;&#xC804;&#xC790; &#xAC00;&#xC871;&#xC5D0; &#xBCF4;&#xC874; &#xD558;&#xC9C0; &#xBC18;&#xBA74; &#xC62C;&#xBC14;&#xB978; &#xD3F4;&#xB529; &#xBC0F; &#xC6D0;&#xD615;&#xC9C8; &#xB9C9;&#xC758; hIK1, &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xB97C; &#xCD09;&#xC9C4; &#xD558;&#xAE30; &#xC704;&#xD558;&#xC5EC; &#xC911;&#xC694; &#xD55C; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;.

Ca2 +&#xC758; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;-&#xD65C;&#xC131;&#xD654; &#xB41C; K + &#xCC44;&#xB110;, hIK1, C &#xD130;&#xBBF8;&#xB110; &#xC2E0; &#xC9C0;&#xD37C;&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4;.

Cystic fibrosis is a common lethal genetic disease among Caucasians. The cystic fibrosis gene encodes a cyclic adenosine monophosphate-activated chloride channel (cystic fibrosis transmembrane conductance regulator (CFTR)) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. Mutations in CFTR fall into two broad categories; those that affect protein biosynthesis/stability and traffic to the cell surface and those that cause altered channel kinetics in proteins that reach the cell surface. Here we report a novel mechanism by which mutations in CFTR give rise to disease. N287Y, a mutation within an intracellular loop of CFTR, increases channel endocytosis from the cell surface without affecting either biosynthesis or channel gating. The sole consequence of this novel mutation is to generate a novel tyrosine-based endocytic sequence within an intracellular loop in CFTR leading to increased removal from the cell surface and a reduction in the steady-state level of CFTR at the cell surface.

A mutation in the cystic fibrosis transmembrane conductance regulator generates a novel internalization sequence and enhances endocytic rates.

Endocytic motifs in the carboxyl terminus of cystic fibrosis transmembrane conductance regulator (CFTR) direct internalization from the plasma membrane by clathrin-mediated endocytosis. However, the fate of such internalized CFTR has remained unknown. Internalized membrane proteins can be either targeted for degradation or recycled back to the plasma membrane. Using cell surface biotinylation and antibody uptake studies, we show that CFTR undergoes constitutive endocytosis and recycling back to the plasma membrane. Expression of dominant negative Rme-1 (a protein that regulates exit from the endosomal recycling compartment) in CFTR-expressing cells results in the expansion of recycling compartments. Transferrin, a marker for the endosomal recycling compartment, and CFTR accumulate in these enlarged recycling endosomes. Such accumulation leads to a loss of cell surface CFTR because it is prevented from being recycled back to the cell surface. In contrast, traffic of the low-density lipoprotein (LDL) is unaffected by the expression of dominant negative Rme-1. In addition, chimeras containing the extracellular domain of the transferrin receptor and the carboxyl terminal tail of CFTR also enter Rme-1-regulated recycling compartments and accumulate in these compartments containing dominant negative Rme-1, suggesting that in addition to endocytic signals, the carboxyl terminal tail of CFTR also contains intracellular traffic information.

Rme-1 regulates the recycling of the cystic fibrosis transmembrane conductance regulator.

NH 2 &#x7EC8;&#x7AEF;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#x548C; dileucine &#x56FE;&#x6848;&#x7684; hIK1 &#x5927;&#x4F1A;&#x3001; &#x8D29;&#x8FD0;&#x548C;&#x901A;&#x9053;&#x7684;&#x51FD;&#x6570;&#x4E2D;&#x7684;&#x4F5C;&#x7528;&#x8FDB;&#x884C;&#x4E86;&#x8C03;&#x67E5;&#xFF0C;&#x4F7F;&#x7528;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x6C89;&#x6DC0;&#x3001; co-&#x6C89;&#x6DC0; (Co-IP)&#x3001; &#x514D;&#x75AB;&#x5370;&#x8FF9;&#x6CD5;&#x548C;&#x5168;&#x7EC6;&#x80DE;&#x4FEE;&#x8865;&#x94B3;&#x6280;&#x672F;&#x3002;NH 2 &#x7EC8;&#x7AEF;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#x9635;&#x5730;&#x6C28;&#x57FA;&#x9178; 18 &#x548C; 25 (L18A/L25A) &#x7684;&#x7A81;&#x53D8;&#x5BFC;&#x81F4;&#x5B8C;&#x5168;&#x4E27;&#x5931;&#x7684;&#x7A33;&#x6001;&#x86CB;&#x767D;&#x7684;&#x8868;&#x8FBE;&#x3001; &#x7EC6;&#x80DE;&#x8868;&#x9762;&#x8868;&#x8FBE;&#x548C;&#x5168;&#x7EC6;&#x80DE;&#x7535;&#x6D41;&#x5BC6;&#x5EA6;&#x3002;Lactacystin &#x86CB;&#x767D;&#x9176;&#x4F53;&#x964D;&#x89E3;&#x7684;&#x6291;&#x5236;&#x8FD8;&#x539F; L18A/L25A &#x86CB;&#x767D;&#x7684;&#x8868;&#x8FBE;&#xFF0C;&#x867D;&#x7136;&#x7531;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x6C89;&#x6DC0;&#x548C;&#x5168;&#x7EC6;&#x80DE;&#x819C;&#x7247;&#x94B3;&#x8BC4;&#x4F30;&#x4E0D;&#x6B64;&#x901A;&#x9053;&#x8868;&#x793A;&#x5728;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x3002;&#x76F8;&#x6BD4;&#x4E4B;&#x4E0B;&#xFF0C;&#x6291;&#x5236;&#x6EB6;&#x9176;&#x4F53;&#x964D;&#x89E3; (leupeptin/pepstatin) &#x548C;&#x5185;&#x541E;&#x4F5C;&#x7528; &#xFF08;&#x6C2F;&#x55B9;&#xFF09; &#x5DF2;&#x7ECF;&#x5BF9; L18A/L25A &#x86CB;&#x767D;&#x8868;&#x8FBE;&#x6216;&#x672C;&#x5730;&#x5316;&#x51E0;&#x4E4E;&#x6CA1;&#x6709;&#x5F71;&#x54CD;&#x3002;&#x8FDB;&#x4E00;&#x6B65;&#x7684;&#x7814;&#x7A76;&#x8BC1;&#x5B9E;&#x6B64;&#x4FE1;&#x9053;&#xFF0C;&#x6709; + /-+ /-0.8 h &#x4E3A;&#x91CE;&#x751F;&#x578B; hIK1 3.2 1.3 &#x5206;&#x949F; 19.0 &#x65F6;&#x95F4;&#x5E38;&#x6570;&#x7684;&#x8FC5;&#x901F;&#x6076;&#x5316;&#x3002;&#x5171;&#x8868;&#x8FBE;&#x7814;&#x7A76;&#x8868;&#x660E; L18A/L25A &#x901A;&#x9053;&#x5C06;&#x91CE;&#x751F;&#x578B;&#x901A;&#x9053;&#xFF0C;&#x4ECE;&#x800C;&#x51CF;&#x8F7B;&#x5176;&#x5728;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x7684;&#x8868;&#x8FBE;&#x4E0E;&#x76F8;&#x5173;&#x8054;&#x3002;Co IP &#x7814;&#x7A76;&#x8BC1;&#x5B9E;&#x8FD9;&#x79CD;&#x5173;&#x8054;&#x3002;&#x7136;&#x800C;&#xFF0C;L18A/L25A &#x9891;&#x9053;&#x5931;&#x8D25;&#x7A97;&#x4F53; homotetrameric &#x6E20;&#x9053;&#xFF0C;&#x5982;&#x6240;&#x8BC4;&#x4F30;&#x7684;&#x516C;&#x53F8; IP&#xFF0C;&#x5EFA;&#x8BAE; NH(2) &#x603B;&#x7AD9;&#x5728; tetrameric &#x6E20;&#x9053;&#x5927;&#x4F1A;&#x4E2D;&#x7684;&#x4F5C;&#x7528;&#x3002;&#x968F;&#x7740;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#xFF0C;&#x7A81;&#x53D8;&#x7684; dileucine &#x5230; alanines&#xFF0C;&#x6BCD;&#x9898;&#x7684; L18A/L19A&#xFF0C;&#x4E0E;&#x540C;&#x6837;&#x6B63;&#x5728;&#x9488;&#x5BF9;&#x86CB;&#x767D;&#x9176;&#x4F53;&#x964D;&#x89E3;&#x7684;&#x86CB;&#x767D;&#x8D28;&#x5BFC;&#x81F4;&#x7A33;&#x6001;&#x86CB;&#x767D;&#x8868;&#x8FBE;&#x51E0;&#x4E4E;&#x5B8C;&#x5168;&#x4E27;&#x5931;&#x3002;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#xFF0C;&#x7136;&#x800C;&#xFF0C;&#x6211;&#x4EEC;&#x7ED3;&#x679C;&#x4E0E;&#x6C2F;&#x55B9;&#x548C;&#x589E;&#x957F;&#x5728;&#x8BB8;&#x53EF;&#x7684;&#x6E29;&#x5EA6; 27 &#x5EA6; C &#x8FD8;&#x539F;&#x8868;&#x8FBE;&#x7684; L18A/L19A &#x5728;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#xFF0C;&#x6697;&#x793A;&#x8D29;&#x8FD0;&#x901A;&#x9053;&#x7F3A;&#x9677;&#x5FAE;&#x5999;&#x7684;&#x7ED3;&#x679C;&#x7684;&#x5355;&#x5143;&#x683C;&#x90FD;&#x6298;&#x53E0;&#x9519;&#x8BEF;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x8BC1;&#x660E; hIK1 &#x7684; NH(2) &#x603B;&#x7AD9;&#x5305;&#x542B;&#x91CD;&#x53E0;&#x4EAE;&#x6C28;&#x9178;&#x62C9;&#x94FE;&#x548C; dileucine &#x56FE;&#x6848;&#x6E20;&#x9053;&#x5927;&#x4F1A;&#x548C;&#x8D29;&#x8FD0;&#x5230;&#x7EC6;&#x80DE;&#x8D28;&#x819C;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;

&#x5927;&#x4F1A;&#x548C;&#x8D29;&#x8FD0;&#x7684;&#x4E2D;&#x95F4;&#x7684;&#x7535;&#x5BFC; Ca2 + NH2 &#x603B;&#x7AD9;&#x7684;&#x4F5C;&#x7528;-&#x6FC0;&#x6D3B; K + &#x901A;&#x9053; hIK1&#x3002;

The role of the NH(2)-terminal leucine zipper and dileucine motifs of hIK1 in the assembly, trafficking, and function of the channel was investigated using cell surface immunoprecipitation, co-immunoprecipitation (Co-IP), immunoblot, and whole-cell patch clamp techniques. Mutation of the NH(2)-terminal leucine zipper at amino acid positions 18 and 25 (L18A/L25A) resulted in a complete loss of steady-state protein expression, cell surface expression, and whole-cell current density. Inhibition of proteasomal degradation with lactacystin restored L18A/L25A protein expression, although this channel was not expressed at the cell surface as assessed by cell surface immunoprecipitation and whole-cell patch clamp. In contrast, inhibitors of lysosomal degradation (leupeptin/pepstatin) and endocytosis (chloroquine) had little effect on L18A/L25A protein expression or localization. Further studies confirmed the rapid degradation of this channel, having a time constant of 19.0 +/- 1.3 min compared with 3.2 +/- 0.8 h for wild type hIK1. Co-expression studies demonstrated that the L18A/L25A channel associates with wild type channel, thereby attenuating its expression at the cell surface. Co-IP studies confirmed this association. However, L18A/L25A channels failed to form homotetrameric channels, as assessed by Co-IP, suggesting the NH(2) terminus plays a role in tetrameric channel assembly. As with the leucine zipper, mutation of the dileucine motif to alanines, L18A/L19A, resulted in a near complete loss in steady-state protein expression with the protein being similarly targeted to the proteasome for degradation. In contrast to our results on the leucine zipper, however, both chloroquine and growing the cells at the permissive temperature of 27 degrees C restored expression of L18A/L19A at the cell surface, suggesting that the defect in the channel trafficking is the result of a subtle folding error. In conclusion, we demonstrate that the NH(2) terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.

Role of the NH2 terminus in the assembly and trafficking of the intermediate conductance Ca2+-activated K+ channel hIK1.

Le r&ocirc;le de l&#39;h&eacute;misph&egrave;re Nord (2)-terminal leucine zipper et dileucine motifs de hIK1 dans l&#39;ensemble, le trafic et la fonction du canal a &eacute;tudi&eacute; en utilisant cell surface immunopr&eacute;cipitation, co-immunopr&eacute;cipitation (Co-IP), immunoblot et techniques de cellules enti&egrave;res de patch clamp. Mutation de l&#39;h&eacute;misph&egrave;re Nord (2)-tirette de leucine terminal aux positions d&#39;acides amin&eacute;s 18 et 25 (L18A/L25A) a entra&icirc;n&eacute; une perte compl&egrave;te de l&#39;expression des prot&eacute;ines de l&#39;&eacute;quilibre et expression superficielle de cellules de densit&eacute; de courant de cellule enti&egrave;re. L&#39;inhibition de la d&eacute;gradation prot&eacute;asomale avec lactacystin restaur&eacute;e L18A/L25A expression de la prot&eacute;ine, bien que ce canal ne s&#39;est pas exprim&eacute; &agrave; la surface cellulaire, tel qu&#39;&eacute;valu&eacute; par immunopr&eacute;cipitation de surface de cellules et de cellules enti&egrave;res de patch-clamp. En revanche, inhibiteurs de d&eacute;gradation lysosomale (leupeptine/pepstatine) et l&#39;endocytose (chloroquine) ont eu peu d&#39;effet sur l&#39;expression de la prot&eacute;ine L18A/L25A ou localisation. D&#39;autres &eacute;tudes ont confirm&eacute; la d&eacute;gradation rapide de ce canal, ayant une constante de temps de 19,0 +/-1,3 min comparativement &agrave; 3,2 +/-0,8 h pour hIK1 de type sauvage. Co-expression &eacute;tudes ont d&eacute;montr&eacute; que le canal L18A/L25A associe &agrave; canal de type sauvage, att&eacute;nuant ainsi son expression &agrave; la surface cellulaire. Co-IP &eacute;tudes ont confirm&eacute; cette association. Cependant, L18A/L25A canaux a &eacute;chou&eacute; pour former des canaux homot&eacute;tram&egrave;res, tel qu&#39;&eacute;valu&eacute; par Co-IP, sugg&eacute;rant que le terminus NH(2) joue un r&ocirc;le dans Assembl&eacute;e canal t&eacute;tram&egrave;re. &Agrave; l&#39;instar de la tirette de leucine, mutation du motif dileucine &agrave; biog&eacute;n&eacute;tiquement, L18A/L19A, a entra&icirc;n&eacute; une perte compl&egrave;te pr&egrave;s dans l&#39;expression des prot&eacute;ines de l&#39;&eacute;tat d&#39;&eacute;quilibre avec la prot&eacute;ine cibl&eacute;e de m&ecirc;me pour le prot&eacute;asome pour la d&eacute;gradation. Contrairement &agrave; nos r&eacute;sultats sur la tirette de leucine, cependant, la chloroquine et la croissance des cellules &agrave; la temp&eacute;rature permissive d&#39;expression 27 degr&eacute;s C restaur&eacute; de L18A/L19A &agrave; la surface des cellules, ce qui sugg&egrave;re que l&#39;irr&eacute;gularit&eacute; dans le trafic du canal est le r&eacute;sultat d&#39;un subtil pliage d&#39;erreur. En conclusion, nous d&eacute;montrons que le terminus NH(2) de hIK1 contient chevauchements leucine zipper et dileucine motifs essentiels pour assemblage de canal et le trafic vers la membrane plasmique.

R&ocirc;le de l&#39;extr&eacute;mit&eacute; NH2 dans l&#39;assembly et le trafic de la conductance interm&eacute;diaire Ca2 +-activ&eacute; hIK1 de canaux K +.

Die Rolle der NH 2-Terminal Leucin Zipper und Dileucine Motive der hIK1 bei der Montage, Menschenhandel und Funktion des Kanals wurde mit Zelle Oberfl&auml;che Immunopr&auml;zipitation, co-Immunopr&auml;zipitation (Co-IP) und Immunoblot ganze Zellen Patch Clamp Techniken untersucht. Mutation der NH 2-Terminal des Leucins an Aminos&auml;ure-Positionen 18 und 25 (L18A/L25A) f&uuml;hrte zu einem v&ouml;lligen Verlust von station&auml;ren Proteinexpression, Zelle Oberfl&auml;che Ausdruck und ganze Zellen Stromdichte. Hemmung der Proteasomal Verschlechterung mit Lactacystin wiederhergestellt L18A/L25A Proteinexpression, obwohl dieser Kanal nicht an der Zelloberfl&auml;che zum Ausdruck kam, die Zelle Oberfl&auml;che Immunopr&auml;zipitation und ganze Zellen Patch Clamp zu bewerten. Inhibitoren der lysosomalen Abbau (Leupeptin/Pepstatin) und Endozytose (Chloroquin) hatte dagegen kaum Auswirkungen auf L18A/L25A Proteinexpression oder Lokalisierung. Weitere Studien best&auml;tigt den raschen Abbau dieses Kanals, nachdem eine Zeitkonstante von 19,0 +/-1,3 min gegen&uuml;ber 3.2 +/-0,8 h f&uuml;r Wildtyp hIK1. Koexpression Studien gezeigt, dass der Kanal L18A/L25A Wildtyp-Kanal, abgeschw&auml;cht und damit seinen Ausdruck an der Zelloberfl&auml;che zuordnet. Co-IP Studien best&auml;tigten diese Zuordnung. Allerdings gelang es L18A/L25A Kan&auml;le, Form Homotetrameric Kan&auml;le, durch Co-IP, was darauf hindeutet, dass das NH(2) Terminus in tetrameren Kanal Versammlung eine Rolle spielt. Wie bei der des Leucins, Mutation des Motivs Dileucine, Alanines, L18A/L19A, resultierte ein nahe vollst&auml;ndige Verlust in station&auml;ren Proteinexpression mit dem Protein wird in &auml;hnlicher Weise gezielt an das Proteasom Leistungsabfall. Im Gegensatz zu unseren Ergebnissen auf Leucins, jedoch sowohl Chloroquin und wachsende Zellen bei der permissive Temperatur von 27 Grad C wiederhergestellt Ausdruck der L18A/L19A an der Zelloberfl&auml;che, die darauf hindeutet, dass der Fehler in den Kanal-Handel eine subtile f&uuml;hrt Falten Fehler. Abschlie&szlig;end zeigen wir, dass der NH(2)-Endpunkt der hIK1 &uuml;berlappende Leucin Zipper und Dileucine Motive f&uuml;r Kanal-Montage und Handel an der Plasmamembran enth&auml;lt.

Rolle &uuml;ber die NH2-Terminus in der Versammlung und den Handel mit den mittleren Leitwert Ca2 +-aktivierte K +-Kanal hIK1.

Il ruolo del NH (2)-terminale della leucina zipper e dileucine motivi di hIK1 nell&#39;Assemblea, il traffico e la funzione del canale &egrave; stato studiato utilizzando cellule superficiali immunoprecipitazione, co-immunoprecipitazione (IP-Co), immunoblot e tecniche di cellule intere patch clamp. Mutazione di NH (2)-chiusura lampo della leucina terminale nelle posizioni dell&#39;amminoacido 18 e 25 (L18A/L25A) ha provocato una perdita completa di espressione della proteina dello stato stazionario, espressione di superficie delle cellule e cellule intere densit&agrave; di corrente. L&#39;inibizione della degradazione proteosomale con lactacystin restaurato L18A/L25A l&#39;espressione della proteina, anche se questo canale non &egrave; stato espresso alla superficie delle cellule, come valutato da immunoprecipitazione di superficie delle cellule e cellule intere patch clamp. Al contrario, gli inibitori della degradazione lisosomiale (leupeptin/pepstatin) e l&#39;endocitosi (clorochina) erano poco effetto sulla espressione della proteina L18A/L25A o localizzazione. Ulteriori studi hanno confermato il rapido degrado di questo canale, avendo una costante di tempo di 19,0 + 1,3 min rispetto ai 3.2 + /-0,8 h per hIK1 wild type. Co-Expression studi hanno dimostrato che il canale L18A/L25A associa con canale di tipo selvatico, attenuando quindi la sua espressione alla superficie delle cellule. Co-IP studi hanno confermato questa associazione. Tuttavia, L18A/L25A canali non &egrave; riuscito a canali di forma homotetrameric, come valutato da Co-IP, suggerendo che il capolinea NH(2) svolge un ruolo nell&#39;assembly canale tetramerica. Come con la chiusura lampo della leucina, mutazione del motivo dileucine a alanines, L18A/L19A, ha provocato una perdita completa vicino di espressione della proteina stazionario con la proteina similmente essere mirata al proteasoma per la degradazione. In contrasto con i nostri risultati sulla cerniera di leucina, tuttavia, sia clorochina e crescono le celle alla temperatura di 27 gradi C restaurato espressione di L18A/L19A alla superficie delle cellule, suggerendo che il difetto del traffico del canale &egrave; il risultato di un sottile permissiva pieghevole errore. In conclusione, ci dimostrano che il capolinea NH(2) di hIK1 contiene sovrapposte leucina zipper e dileucine motivi essenziali per l&#39;assemblaggio di canale e traffico di membrana del plasma.

Ruolo di capolinea in assembly e il traffico della conduttanza intermedia Ca2 + NH2-attivato K + canale hIK1.

NH (2) &#x306E;&#x5F79;&#x5272;-&#x30BF;&#x30FC;&#x30DF;&#x30CA;&#x30EB; &#x30ED;&#x30A4;&#x30B7;&#x30F3;&#x306E;&#x30B8;&#x30C3;&#x30D1;&#x30FC;&#x3068;&#x30B8;&#x30ED;&#x30A4;&#x30B7;&#x30F3;&#x30BD;&#x30FC;&#x30C6;&#x30A3;&#x30F3;&#x30B0;&#x306E;&#x30E2;&#x30C1;&#x30FC;&#x30D5; hik1 &#x3067;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x3001;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x3001;&#x304A;&#x3088;&#x3073;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x6A5F;&#x80FD;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x514D;&#x75AB;&#x6C88;&#x964D;&#x3001;co-&#x514D;&#x75AB;&#x6C88;&#x964D; (Co-IP), &#x30A4;&#x30E0;&#x30CE;&#x30D6;&#x30ED;&#x30C3;&#x30C8;&#x3001;&#x5168;&#x7D30;&#x80DE;&#x30D1;&#x30C3;&#x30C1;&#x30AF; &#x30E9;&#x30F3;&#x30D7;&#x6CD5;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x8ABF;&#x67FB;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;NH (2) &#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;-&#x30BF;&#x30FC;&#x30DF;&#x30CA;&#x30EB; &#x30ED;&#x30A4;&#x30B7;&#x30F3; &#x30B8;&#x30C3;&#x30D1;&#x30FC; &#x30A2;&#x30DF;&#x30CE;&#x9178;&#x4F4D;&#x7F6E; 18 &#x3068; 25 &#xFF08;L18A/L25A) &#x5B9A;&#x5E38;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x767A;&#x73FE;&#x3001;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x767A;&#x73FE;&#x304A;&#x3088;&#x3073;&#x5168;&#x4F53;&#x30BB;&#x30EB;&#x96FB;&#x6D41;&#x5BC6;&#x5EA6;&#x306E;&#x5B8C;&#x5168;&#x306A;&#x640D;&#x5931;&#x306B;&#x8D77;&#x56E0;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306F;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x306B;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x514D;&#x75AB;&#x6C88;&#x964D;&#x3068;&#x5168;&#x7D30;&#x80DE;&#x30D1;&#x30C3;&#x30C1;&#x30AF; &#x30E9;&#x30F3;&#x30D7;&#x306B;&#x3088;&#x308B;&#x8A55;&#x4FA1;&#x3068;&#x3057;&#x3066;&#x767A;&#x73FE;&#x3057;&#x3066;&#x3044;&#x306A;&#x304B;&#x3063;&#x305F;&#x304C;&#x30E9;&#x30AF;&#x30BF;&#x30B7;&#x30B9;&#x30C1;&#x30F3; &#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30BD;&#x30FC;&#x30E0;&#x52A3;&#x5316;&#x306E;&#x6291;&#x5236; L18A/L25A &#x86CB;&#x767D;&#x8CEA;&#x306E;&#x8868;&#x73FE;&#x3092;&#x5FA9;&#x5143;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x5BFE;&#x7167;&#x7684;&#x306B;&#x3001;&#x30EA;&#x30BD;&#x30BD;&#x30FC;&#x30E0;&#x52A3;&#x5316; (leupeptin/&#x30DA;&#x30D7;&#x30B9;&#x30BF;&#x30C1;&#x30F3;) &#x3068;&#x30A8;&#x30F3;&#x30C9;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9; (&#x30AF;&#x30ED;&#x30ED;&#x30AD;&#x30F3;) &#x306E;&#x963B;&#x5BB3;&#x5264; L18A/L25A &#x86CB;&#x767D;&#x8CEA;&#x306E;&#x767A;&#x73FE;&#x3084;&#x5C40;&#x5728;&#x306B;&#x306F;&#x307B;&#x3068;&#x3093;&#x3069;&#x5F71;&#x97FF;&#x3092;&#x6301;&#x3063;&#x3066; &#x3002;&#x3055;&#x3089;&#x306A;&#x308B;&#x7814;&#x7A76;&#x306F; 19.0 3.2 &#x91CE;&#x751F;&#x578B; hIK1 &#x306E; 0.8 h +/-&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066; 1.3 &#x5206; +/-&#x306E;&#x6642;&#x5B9A;&#x6570;&#x3092;&#x6301;&#x3064;&#x3053;&#x306E;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x6025;&#x6FC0;&#x306A;&#x4F4E;&#x4E0B;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;L18A/L25A &#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x306F;&#x305D;&#x308C;&#x306B;&#x3088;&#x308A;&#x305D;&#x306E;&#x767A;&#x73FE;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x3092;&#x6E1B;&#x8870;&#x3001;&#x91CE;&#x751F;&#x578B;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x3092;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x307E;&#x3059;&#x5171;&#x7814;&#x7A76;&#x3092;&#x5B9F;&#x8A3C;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;Co IP &#x7814;&#x7A76;&#x306F;&#x3053;&#x306E;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;L18A/L25A &#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x3067;&#x306F;&#x3001;Co-IP NH(2) &#x30C6;&#x30EB;&#x30DF;&#x30CA;&#x30B9; 4 &#x91CF;&#x4F53;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x306E;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3067;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x3068;&#x304C;&#x30D5;&#x30A9;&#x30FC;&#x30E0; homotetrameric &#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x306B;&#x5931;&#x6557;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30ED;&#x30A4;&#x30B7;&#x30F3; &#x30B8;&#x30C3;&#x30D1;&#x30FC;&#x3068;&#x3057;&#x3066;&#x3001;&#x30A2;&#x30E9;&#x30CB;&#x30F3;&#x3078;&#x30B8;&#x30ED;&#x30A4;&#x30B7;&#x30F3;&#x30BD;&#x30FC;&#x30C6;&#x30A3;&#x30F3;&#x30B0; &#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;&#x306E; L18A/L19A &#x3068;&#x540C;&#x69D8;&#x306B;&#x52A3;&#x5316;&#x3092;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30BD;&#x30FC;&#x30E0;&#x306B;&#x5BFE;&#x8C61;&#x3068;&#x306A;&#x308B;&#x86CB;&#x767D;&#x8CEA;&#x3068;&#x5B9A;&#x5E38;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x8868;&#x73FE;&#x306E;&#x8FD1;&#x304F;&#x5B8C;&#x5168;&#x306A;&#x640D;&#x5931;&#x306E;&#x7D50;&#x679C;&#x3057;&#x307E;&#x3059;&#x3002;&#x30ED;&#x30A4;&#x30B7;&#x30F3;&#x306E;&#x30B8;&#x30C3;&#x30D1;&#x30FC;&#x306F;&#x3001;&#x3057;&#x304B;&#x3057;&#x3001;&#x6211;&#x3005; &#x306E;&#x7D50;&#x679C;&#x3068;&#x306F;&#x5BFE;&#x7167;&#x7684;&#x30A8;&#x30E9;&#x30FC; &#x30AF;&#x30ED;&#x30ED;&#x30AD;&#x30F3;&#x3068;&#x5BDB;&#x5BB9;&#x306A;&#x6E29;&#x5EA6; 27 &#x2103; &#x3068;&#x5FA9;&#x5143;&#x5F0F; L18A/L19A &#x306E;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306E;&#x6B20;&#x9665;&#x306E;&#x5FAE;&#x5999;&#x306A;&#x7D50;&#x679C;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x6210;&#x9577;&#x3092;&#x6298;&#x308B;&#x3002;&#x7D50;&#x8AD6;&#x3068;&#x3057;&#x3066;&#x3001;&#x6211;&#x3005; NH(2) &#x30C6;&#x30EB;&#x30DF;&#x30CA;&#x30B9; hik1 &#x91CD;&#x306A;&#x308A;&#x5408;&#x3046;&#x30ED;&#x30A4;&#x30B7;&#x30F3; &#x30B8;&#x30C3;&#x30D1;&#x30FC;&#x3068;&#x30B8;&#x30ED;&#x30A4;&#x30B7;&#x30F3;&#x30BD;&#x30FC;&#x30C6;&#x30A3;&#x30F3;&#x30B0; &#x30E2;&#x30C1;&#x30FC;&#x30D5; &#x30C1;&#x30E3;&#x30CD;&#x30EB; &#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x3068;&#x539F;&#x5F62;&#x8CEA;&#x819C;&#x306B;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306E;&#x91CD;&#x8981;&#x306A;&#x542B;&#x307E;&#x308C;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

NH2 &#x672B;&#x7AEF;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x3001;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306E;&#x4E2D;&#x9593;&#x30B3;&#x30F3;&#x30C0;&#x30AF;&#x30BF;&#x30F3;&#x30B9; ca 2 + &#x306E;&#x5F79;&#x5272;-K + &#x30C1;&#x30E3;&#x30CD;&#x30EB; hIK1 &#x3092;&#x6D3B;&#x6027;&#x5316;&#x3057;&#x307E;&#x3059;&#x3002;

&#xB274; &#xD584;&#xD504;&#xC154; (2)-&#xD130;&#xBBF8;&#xB110; &#xC2E0; &#xC9C0;&#xD37C;&#xC640; dileucine &#xBAA8;&#xD2F0;&#xBE0C; hIK1 &#xC5B4;&#xC148;&#xBE14;&#xB9AC;, &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;, &#xADF8;&#xB9AC;&#xACE0; &#xCC44;&#xB110;&#xC758; &#xAE30;&#xB2A5;&#xC5D0;&#xC11C;&#xC758; &#xC5ED;&#xD560;&#xC740; &#xC138;&#xD3EC; &#xD45C;&#xBA74; immunoprecipitation, &#xACF5;&#xB3D9;-immunoprecipitation (Co-IP), immunoblot, &#xC804;&#xCCB4; &#xC140; &#xD328;&#xCE58; &#xD074;&#xB7A8;&#xD504; &#xAE30;&#xBC95;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC870;&#xC0AC; &#xD558; &#xACE0; &#xC788;&#xB2E4;. &#xB274; &#xD584;&#xD504;&#xC154; (2)-&#xD130;&#xBBF8;&#xB110; &#xC2E0; &#xC9C0;&#xD37C; &#xC544;&#xBBF8;&#xB178;&#xC0B0; &#xC704;&#xCE58; 18&#xACFC; 25 (L18A/L25A)&#xC758; &#xBCC0;&#xC774; &#xC815;&#xC0C1; &#xC0C1;&#xD0DC;&#xC758; &#xB2E8;&#xBC31;&#xC9C8; &#xBC1C;&#xD604;, &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xC2DD; &#xBC0F; &#xC804;&#xCCB4; &#xC140; &#xC804;&#xB958; &#xBC00;&#xB3C4;&#xC758; &#xC644;&#xC804; &#xC190;&#xC2E4; &#xADC0;&#xCC29;&#xB418; &#xC5C8; &#xB2E4;. Lactacystin&#xC640; proteasomal &#xC800;&#xD558;&#xC758; &#xC5B5;&#xC81C; &#xC138;&#xD3EC; &#xD45C;&#xBA74; immunoprecipitation &#xBC0F; &#xC804;&#xCCB4; &#xC140; &#xD328;&#xCE58; &#xD074;&#xB7A8;&#xD504;&#xC5D0; &#xC758;&#xD574; &#xD3C9;&#xAC00;&#xC774; &#xCC44;&#xB110; &#xC138;&#xD3EC; &#xD45C;&#xBA74;&#xC5D0; &#xD45C;&#xD604; &#xD558;&#xC9C0;&#xB294; &#xD558;&#xC9C0;&#xB9CC; L18A/L25A &#xB2E8;&#xBC31;&#xC9C8; &#xBC1C;&#xD604;&#xC744; &#xBCF5;&#xC6D0;. &#xBC18;&#xBA74;, lysosomal &#xC800;&#xD558; (leupeptin/pepstatin) &#xBC0F; endocytosis (&#xD074;&#xB85C;&#xB85C;&#xD038;) &#xC5B5;&#xC81C;&#xC81C; L18A/L25A &#xB2E8;&#xBC31;&#xC9C8; &#xC2DD; &#xB610;&#xB294; &#xC9C0;&#xC5ED;&#xD654;&#xC5D0; &#xAC70;&#xC758; &#xC601;&#xD5A5;&#xC744; &#xD588;&#xB2E4;. &#xCD94;&#xAC00; &#xC5F0;&#xAD6C; 19.0 &plusmn; 0.8 h &#xC57C;&#xC0DD; &#xD0C0;&#xC785; hIK1 + 3.2&#xC5D0; &#xBE44;&#xD574; 1.3 &#xBD84;&#xC758; &#xC2DC;&#xAC04; &#xC0C1;&#xC218;&#xB97C; &#xAC16;&#xB294;&#xC774; &#xCC44;&#xB110;&#xC758; &#xAE09;&#xACA9; &#xD55C; &#xC800;&#xD558;&#xB97C; &#xD655;&#xC778; &#xD588;&#xB2E4;. Co-expression &#xC5F0;&#xAD6C; L18A/L25A &#xCC44;&#xB110; &#xC57C;&#xC0DD; &#xD0C0;&#xC785; &#xCC44;&#xB110;, &#xADF8;&#xB85C; &#xC778;&#xD558;&#xC5EC; &#xC138;&#xD3EC; &#xD45C;&#xBA74;&#xC5D0;&#xB294; &#xC2DD;&#xC744; &#xAC10;&#xC1E0;&#xC640; &#xC5F0;&#xACB0;&#xC744; &#xC2DC;&#xC5F0; &#xD588;&#xB2E4;. &#xACF5;&#xB3D9; IP &#xC5F0;&#xAD6C;&#xAC00;&#xC774; &#xD611;&#xD68C;&#xB97C; &#xD655;&#xC778; &#xD588;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, L18A/L25A &#xCC44;&#xB110; &#xACF5;&#xB3D9;-IP, NH(2) &#xC885;&#xC810; tetrameric &#xCC44;&#xB110; &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC5D0; &#xC5ED;&#xD560;&#xC744; &#xC81C;&#xC548; &#xD558; &#xC5EC; &#xD3C9;&#xAC00; &#xC591;&#xC2DD; homotetrameric &#xCC44;&#xB110;&#xC744; &#xD558;&#xC9C0; &#xBABB;&#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC2E0; &#xC9C0;&#xD37C;&#xB97C; alanines, dileucine &#xBAA8;&#xD2F0;&#xBE0C;&#xC758; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC640; &#xB9C8;&#xCC2C;&#xAC00;&#xC9C0;&#xB85C; L18A/L19A, &#xB9C8;&#xCC2C;&#xAC00;&#xC9C0;&#xB85C; &#xC131;&#xB2A5; &#xC800;&#xD558; &#xD504;&#xB85C;&#xD14C;&#xC544;&#xC880;&#xC5D0; &#xB300;&#xC0C1; &#xB2E8;&#xBC31;&#xC9C8;&#xC73C;&#xB85C; &#xC815;&#xC0C1; &#xC0C1;&#xD0DC;&#xC758; &#xB2E8;&#xBC31;&#xC9C8; &#xC2DD;&#xC5D0; &#xAC00;&#xAE4C;&#xC6B4; &#xC644;&#xC804; &#xC190;&#xC2E4; &#xADC0;&#xCC29;&#xB418; &#xC5C8; &#xB2E4;. &#xADF8;&#xB7EC;&#xB098; &#xC2E0; &#xC9C0;&#xD37C;,&#xC5D0; &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; &#xB2EC;&#xB9AC; &#xD074;&#xB85C;&#xB85C;&#xD038;&#xACFC; &#xCC44;&#xB110; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC758; &#xACB0;&#xD568;&#xC744; &#xBBF8;&#xBB18;&#xD55C; &#xACB0;&#xACFC; &#xC81C;&#xC548; &#xD558;&#xB294; &#xC138;&#xD3EC; &#xD45C;&#xBA74;&#xC5D0; L18A/l19a&#xC758; 27 &#xC12D;&#xC528; &#xC628;&#xB3C4; &#xBCF5;&#xC6D0; &#xC2DD;&#xC758; &#xD5C8;&#xC6A9; &#xC628;&#xB3C4;&#xC5D0;&#xC11C; &#xC138;&#xD3EC;&#xB97C; &#xC131;&#xC7A5; &#xD558; &#xACE0; &#xC811;&#xB294; &#xC624;&#xB958;. &#xB05D;&#xC73C;&#xB85C;, Hik1&#xC758; NH(2) &#xC885;&#xC810; &#xACB9;&#xCE58;&#xB294; &#xC2E0; &#xC9C0;&#xD37C;&#xC640; dileucine &#xBAA8;&#xD2F0;&#xBE0C; &#xCC44;&#xB110; &#xC870;&#xB9BD; &#xBC0F; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778;&#xC744; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218; &#xD3EC;&#xD568; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;.

&#xC5B4;&#xC148;&#xBE14;&#xB9AC; &#xBC0F; &#xC911;&#xAC04; &#xC804;&#xB3C4;&#xB3C4; Ca2 +&#xC758; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; NH2 &#xC885;&#xCC29;&#xC5ED;&#xC758; &#xC5ED;&#xD560;-K + &#xCC44;&#xB110; Hik1&#xB97C; &#xD65C;&#xC131;&#xD654; &#xD569;&#xB2C8;&#xB2E4;.

El papel de los NH 2-terminal leucina cremallera y dileucine motivos de hIK1 en la Asamblea, el tr&aacute;fico y la funci&oacute;n del canal fue investigado mediante inmunoprecipitaci&oacute;n de superficie celular, co-inmunoprecipitaci&oacute;n (Co-IP), immunoblot y t&eacute;cnicas de abrazadera de parche de c&eacute;lulas completas. Mutaci&oacute;n de la cremallera de leucina NH 2-terminal en las posiciones de amino&aacute;cido 18 y 25 (L18A/L25A) dio lugar a una p&eacute;rdida completa de la expresi&oacute;n de la prote&iacute;na de estado estacionario, expresi&oacute;n superficial de la c&eacute;lula y la densidad de corriente de c&eacute;lulas completas. Inhibici&oacute;n de la degradaci&oacute;n proteasomal con lactacystin hab&iacute;a restaurado L18A/L25A expresi&oacute;n de la prote&iacute;na, aunque este canal no fue expresado en la superficie celular seg&uacute;n lo determinado por inmunoprecipitaci&oacute;n de superficie celular y la abrazadera del remiendo de c&eacute;lulas completas. En contraste, los inhibidores de la degradaci&oacute;n lisosomal (leupeptin/pepstatin) y la endocitosis (cloroquina) tuvieron poco efecto sobre la expresi&oacute;n de la prote&iacute;na de L18A/L25A o localizaci&oacute;n. Otros estudios confirmaron la r&aacute;pida degradaci&oacute;n de este canal, que tiene una constante de tiempo de 19.0 +/-1,3 min en comparaci&oacute;n con 3.2 +/-0,8 h para hIK1 de tipo salvaje. Coexpresi&oacute;n estudios demostraron que el canal L18A/L25A se asocia con canal de tipo salvaje, atenuando as&iacute; su expresi&oacute;n en la superficie celular. Co-IP estudios confirmaron esta asociaci&oacute;n. Sin embargo, L18A/L25A canales no canales de forma homotetrameric, seg&uacute;n lo determinado por la Co-IP, sugiriendo que la terminal NH(2) desempe&ntilde;a un papel en la Asamblea de canal tetram&eacute;rica. Como con la cremallera de leucina, mutaci&oacute;n del motivo dileucine a alanines, L18A/L19A, resultado en una p&eacute;rdida casi completa en expresi&oacute;n de la prote&iacute;na de estado estacionario con la prote&iacute;na est&aacute; igualmente dirigida al proteasoma para su degradaci&oacute;n. En contraste con nuestros resultados en la cremallera de leucina, sin embargo, la cloroquina y crecimiento de las c&eacute;lulas a la temperatura permisiva de 27 grados C restaurado expresi&oacute;n de L18A/L19A en la superficie celular, sugiriendo que el defecto en el tr&aacute;fico del canal es el resultado de una sutil plegable error. En conclusi&oacute;n, demostramos que la terminal NH(2) de hIK1 contiene superpuestos leucina cremallera y dileucine motivos esenciales para el montaje del canal y el tr&aacute;fico a la membrana plasm&aacute;tica.

Papel del NH2 terminal en la Asamblea y el tr&aacute;fico de la conductancia intermedia Ca2 +-activa el canal hIK1 de K +.

Freedom of expression. Focus on "Endoplasmic reticulum stress and the unfolded protein response regulate cystic fibrosis transmembrane conductance regulator expression".

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl-selective anion channel expressed in epithelial tissues. Mutations in CFTR lead to the genetic disease cystic fibrosis (CF). Within each epithelial cell, CFTR interacts with a large number of transient macromolecular complexes, many of which are involved in the trafficking and targeting of CFTR. Understanding how these complexes regulate the trafficking and fate of CFTR, provides a singular insight not only into the patho-physiology of cystic fibrosis, but also provides potential drug targets to help cure this debilitating disease.

Endocytic trafficking of CFTR in health and disease.

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.

Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells.

The initial entry of papillomaviruses into their target cells has been shown to occur by clathrin-mediated endocytosis and caveolae-mediated endocytosis. These mechanisms entail the formation of nascent-coated vesicles at the plasma membrane. Such coated vesicles, clathrin or caveolin, form and pinch-off in a controlled mechanism that involves several proteins including dynamin. Dynamin is a GTPase that forms a dynamin ring at the stem connecting the nascent vesicle to the plasma membrane. In a still not fully characterized mechanism, dynamin's contraction and twisting results in the scission of the vesicle. In an effort to better characterize the role and molecular mechanisms of dynamin's function, researchers have identified dynasore, a dynamin GTPase inhibitor that prevents the scission of dynamin-dependent endocytic vesicles. Here, we have tested if infection by pseudovirus corresponding to the oncogenic human papillomavirus type 16 and bovine papillomavirus type 1 can be blocked by dynasore. We present data demonstrating that dynasore can block infection of human papillomavirus type 16 and bovine papillomavirus type 1 pseudovirions in a dose- and time-dependent manner with equal efficiency. Presently, there is no available therapy that can block infection by a wide range of papillomavirus regardless of species or genotypes. Targeting dynamin may lead to the rational design of drug able to prevent infection by papillomaviruses, and by other infectious agents dependent on this protein for initial internalization into target cells. Whether such an approach will prove successful needs further investigation.

HPV16 and BPV1 infection can be blocked by the dynamin inhibitor dynasore.

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, DeltaF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

Rab11b regulates the apical recycling of the cystic fibrosis transmembrane conductance regulator in polarized intestinal epithelial cells.

Dynasore, a small molecule inhibitor of dynamin, was used to probe the role of dynamin in the endocytosis of wild-type and mutant CFTR (cystic fibrosis transmembrane conductance regulator). Internalization of both wild-type and 'temperature-corrected' DeltaF508 CFTR was markedly inhibited by a short exposure to dynasore, implicating dynamin as a key element in the endocytic internalization of both wild-type and mutant CFTR. The inhibitory effect of dynasore was readily reversible upon washout of dynasore from the growth media. Corr-4 ({2-(5-chloro-2-methoxy-phenylamino)-4'-methyl-[4,5']-bithiazolyl-2'-yl}-phenyl-methanonone), a pharmacological corrector of DeltaF508 CFTR biosynthesis, caused a marked increase in the cell surface expression of mutant CFTR. Co-incubation of DeltaF508 CFTR expressing cells with Corr-4 and dynasore caused a significantly greater level of cell surface CFTR than that observed in the presence of Corr-4 alone. These results argue that inhibiting the endocytic internalization of mutant CFTR provides a novel therapeutic target for augmenting the benefits of small molecule correctors of mutant CFTR biosynthesis.

Dynasore inhibits removal of wild-type and DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) from the plasma membrane.

Lemur tyrosine kinase 2 (LMTK2) is a novel membrane-anchored kinase reported to be involved in several normal and pathophysiological conditions, including endosomal membrane recycling, prostate cancer, and neurodegeneration. In this study, we have investigated the topology and orientation of LMTK2 within cellular membranes utilizing fluorescence protease protection. Appending the green fluorescent protein to either the amino or carboxyl terminus of LMTK2, we were able to determine which side of intracellular membrane these regions were located. Our results indicate that LMTK2 is an integral membrane protein in which both the amino and carboxyl termini are exposed to the cytoplasm. Moreover, this topology places the kinase active site within the cytoplasm.

Determination of the membrane topology of lemur tyrosine kinase 2 (LMTK2) by fluorescence protease protection.

Do you know the sex of your cells? Not a question that is frequently heard around the lab bench, yet thanks to recent research is probably one that should be asked. It is self-evident that cervical epithelial cells would be derived from female tissue and prostate cells from a male subject (exemplified by HeLa and LnCaP, respectively), yet beyond these obvious examples, it would be true to say that the sex of cell lines derived from non-reproductive tissue, such as lung, intestine, kidney, for example, is given minimal if any thought. After all, what possible impact could the presence of a Y chromosome have on the biochemistry and cell biology of tissues such as the exocrine pancreatic acini? Intriguingly, recent evidence has suggested that far from being irrelevant, genes expressed on the sex chromosomes can have a marked impact on the biology of such diverse tissues as neurons and renal cells. It is also policy of AJP-Cell Physiology that the source of all cells utilized (species, sex, etc.) should be clearly indicated when submitting an article for publication, an instruction that is rarely followed (http://www.the-aps.org/mm/Publications/Info-For-Authors/Composition). In this review we discuss recent data arguing that the sex of cells being used in experiments can impact the cell's biology, and we provide a table outlining the sex of cell lines that have appeared in AJP-Cell Physiology over the past decade.

Do you know the sex of your cells?

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel composed of 1480 amino acids. The major mutation responsible for cystic fibrosis results in loss of amino acid residue, F508 (F508del). Loss of F508 in CFTR alters the folding pathway resulting in endoplasmic-reticulum-associated degradation. This study investigates the role of synonymous codon in the expression of CFTR and CFTR F508del in human HEK293 cells. DNA encoding the open reading frame (ORF) for CFTR containing synonymous codon replacements was expressed using a heterologous vector integrated into the genome. The results indicate that the codon usage greatly affects the expression of CFTR. While the promoter strength driving expression of the ORFs was largely unchanged and the mRNA half-lives were unchanged, the steady-state levels of the mRNA varied by as much as 30-fold. Experiments support that this apparent inconsistency is attributed to nonsense mediated decay independent of exon junction complex. The ratio of CFTR/mRNA indicates that mRNA containing native codons was more efficient in expressing mature CFTR as compared to mRNA containing synonymous high-expression codons. However, when F508del CFTR was expressed after codon optimization, a greater percentage of the protein escaped endoplasmic-reticulum-associated degradation resulting in considerable levels of mature F508del CFTR on the plasma membrane, which showed channel activity. These results indicate that codon usage has an effect on mRNA levels and protein expression, for CFTR, and likely on chaperone-assisted folding pathway, for F508del CFTR.

Article	Year
Analysis of CFTR endocytosis by cell surface biotinylation. Methods in molecular medicine\| PubMed ID: 11917535	2002
Trafficking of the Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal leucine zipper. The Journal of biological chemistry\| PubMed ID: 12493744	2003
A mutation in the cystic fibrosis transmembrane conductance regulator generates a novel internalization sequence and enhances endocytic rates. The Journal of biological chemistry\| PubMed ID: 12529365	2003
Rme-1 regulates the recycling of the cystic fibrosis transmembrane conductance regulator. American journal of physiology. Cell physiology\| PubMed ID: 12839834	2003
Role of the NH2 terminus in the assembly and trafficking of the intermediate conductance Ca2+-activated K+ channel hIK1. The Journal of biological chemistry\| PubMed ID: 14754884	2004
Freedom of expression. Focus on "Endoplasmic reticulum stress and the unfolded protein response regulate cystic fibrosis transmembrane conductance regulator expression". American journal of physiology. Cell physiology\| PubMed ID: 17005602	2007
Endocytic trafficking of CFTR in health and disease. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 17098482	2007
Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells. Molecular biology of the cell\| PubMed ID: 17686995	2007
HPV16 and BPV1 infection can be blocked by the dynamin inhibitor dynasore. American journal of therapeutics\| PubMed ID: 18645330	2008
Rab11b regulates the apical recycling of the cystic fibrosis transmembrane conductance regulator in polarized intestinal epithelial cells. Molecular biology of the cell\| PubMed ID: 19244346	2009
Dynasore inhibits removal of wild-type and DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) from the plasma membrane. The Biochemical journal\| PubMed ID: 19442237	2009
Determination of the membrane topology of lemur tyrosine kinase 2 (LMTK2) by fluorescence protease protection. American journal of physiology. Cell physiology\| PubMed ID: 23114966	2013
Do you know the sex of your cells? American journal of physiology. Cell physiology\| PubMed ID: 24196532	2014
Synonymous Codon Usage Affects the Expression of Wild Type and F508del CFTR. Journal of molecular biology\| PubMed ID: 25676312	2015

Neil A. Bradbury

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