Supplemental Data. Denis B. Tikhonov, Iva Bruhova, Daniel P. Garden, and Boris S. Zhorov

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1 Supplemental Data State-Dependent Interdomain Contacts of Exceptionally Conserved Asparagines in the Inner Helices of Sodium and Calcium Channels Denis. Tikhonov, Iva ruhova, Daniel P. Garden, and oris S. Zhorov Table S1. Inner helices in K +, Na +, and Ca 2+ channels a Channel Repeat i1 i11 i21 i31 KcsA LWGRLVAVVV MVAGITSFGL VTAALATWFV GREQ KvAP PIGKVIGIAV MLTGISALTL LIGTVSNMFQ KILV K v 1.2 IGGKIVGSLC AIAGVLTIAL PVPVIVSNFN YFYH Ca v 1.1 I EWPWIYFVTL ILLGSFFILN LVLGVLSGEF TKER II VLVCIYFIIL FVCGNYILLN VFLAIAVDNL AEAE III VEMAIFFIIY IILIAFFMMN IFVGFVIVTF QEQG IV NFAYYYFISF YMLCAFLIIN LFVAVIMDNF DYLT Ca v 1.2 I ELPWVYFVSL VIFGSFFVLN LVLGVLSGEF SKER II MLVCIYFIIL FISPNYILLN LFLAIAVDNL ADAE III VEISIFFIIY IIIIAFFMMN IFVGFVIVTF QEQG IV SFAVFYFISF YMLCAFLIIN LFVAVIMDNF DYLT Ca v 1.3 I EWPWVYFVSL IILGSFFVLN LVLGVLSGEF SKER II MIVCIYFIIL FICGNYILLK LFLAIAVDNL ADAE III VEISIFFIIY IIIVAFFMMN IFVGFVIVTF QEQG IV NFAIVYFISF YMLCAFLIIN LFVAVIMDNF DYLT Ca v 1.4 I ELPWVYFVSL VIFGSFFVLN LVLGVLSGEF SKER II MLVCVYFIIL FICGNYILLN VFLAIAVDNL ASGD III VEISVFFIVY IIIIAFFMMN IFVGFVIITF RAQG IV NFAIVYFISF FMLCAFLIIN LFVAVIMDNF DYLT Ca v 2.1 I TWNWLYFIPL IIIGSFFMLN LVLGVLSGEF AKER II MVFSIYFIVL TLFGNYTLLN VFLAIAVDNL ANAQ III MEMSIFYVVY FVVFPFFFVN IFVALIIITF QEQG IV EFAYFYFVSF IFLCSFLMLN LFVAVIMDNF EYLT Ca v 2.2 I TWNWLYFIPL IIIGSFFMLN LVLGVLSGEF AKER II MFSSFYFIVL TLFGNYTLLN VFLAIAVDNL ANAQ III MELSIFYVVY FVVFPFFFVN IFVALIIITF QEQG IV DFAYFYFVSF IFLCSFLMLN LFVAVIMDNF EYLT Ca v 2.3 I TWNWLYFIPL IIIGSFFVLN LVLGVLSGEF AKER II MWSAIYFIVL TLFGNYTLLN VFLAIAVDNL ANAQ III MEMSIFYVVY FVVFPFFFVN IFVALIIITF QEQG IV DLAYVYFVSF IFFCSFLMLN LFVAVIMDNF EYLT Ca v 3.1 I FYNFIYFILL IIVGSFFMIN LCLVVIATQF SETK II SWAALYFIAL MTFGNYVLFN LLVAILVEGF QAEG III PWMLLYFISF LLIVAFFVLN MFVGVVVENF HKCR IV VISPIYFVSF VLTAQFVLVN VVIAVLMKHL EESN Ca v 3.2 I FYNFIYFILL IIMGSFFMIN LCLVVIATQF SETK II SWAALYFVAL MTFGNYVLFN LLVAILVEGF QAEG III PWMLLYFISF LLIVSFFVLN MFVGVVVENF HKCR IV ALSPVYFVTF MLVAQFVLVN VVVAVLMKHL EESN 1

2 Channel Repeat i1 i11 i21 i31 Ca v 3.3 I FYNFIYFILL IIVGSFFMIN LCLVVIATQF SETK II PWASLYFVAL MTFGNYVLFN LLVAILVEGF QAEG III PWMLLYFISF LLIVSFFVLN MFVGVVVENF HKCR IV FVSPLYFVSF VLTAQFVLIN VVVAVLMKHL DDSN Na v 1.1 I KTYMIFFVLV IFLGSFYLIN LILAVVAMAY EEQN II AMCLTVFMMV MVIRNLVVLN LFLALLLSSF SADN III LYMYLYFVIF IIFGSFFTLN LFIGVIIDNF NQQK IV SVGIFFFVSY IIISFLVVVN MYIAVILENF SVAT Na v 1.2 I KTYMIFFVLV IFLGSFYLIN LILAVVAMAY EEQN II TMCLTVFMMV MVIGNLVVLN LFLALLLSSF SSDN III LYMYLYFVIF IIFGSFFTLN LFIGVIIDNF NQQK IV SVGIFFFVSY IIISFLVVVN MYIAVILENF SVAT Na v 1.3 I KTYMIFFVLV IFLGSFYLVN LILAVVAMAY EEQN II TMCLIVFMLV MVIGNLVVLN LFLALLLSSF SSDN III LYMYLYFVIF IIFGSFFTLN LFIGVIIDNF NQQK IV SVGIFFFVSY IIISFLVVVN MYIAVILENF SVAT Na v 1.4 I KTYMIFFVVI IFLGSFYLIN LILAVVAMAY AEQN II AMCLTVFLMV MVIGNLVVLN LFLALLLSSF SADS III LYMYLYFVIF IIFGSFFTLN LFIGVIIDNF NQQK IV SIGICFFCSY IIISFLIVVN MYIAIILENF NVAT Na v 1.5 I KIYMIFFMLV IFLGSFYLVN LILAVVAMAY EEQN II SLCLLVFLLV MVIGNLVVLN LFLALLLSSF SADN III LYMYIYFVVF IIFGSFFTLN LFIGVIIDNF NQQK IV AVGILFFTTY IIISFLIVVN MYIAIILENF SVAT Na v 1.6 I KTYMIFFVLV IFVGSFYPVN LILAVVAMAY EEQN II AMCLIVFMMV MVIGNLVVLN LFLALLLSSF SADN III IYMYIYFVIF IIFGSFFTLN LFIGVIIDNF NQQK IV SVGIFFFVSY IIISFLIVVN MCIAIILENF SVAT Na v 1.7 I KTYMIFFVVV IFLGSFYLIN LILAVVAMAY EEQN II TMCLIVYMMV MVIGNLVVLN LFLALLLSSF SSDN III LYMYIYFVIF IIFGSFFTLN LFIGVIIDNF NQQK IV SVGIFYFVSY IIISFLVVVN MYIAVILENF SVAT rna v 1.8 I KMYMVFFVLV IFLGSFYLVN LILAVVTMAY EEQS II SICLILFLTV MVLGNLVVLN LFIALLLNSF SADN III LYMYLYFVVF IIFGGFFTLN LFVGVIIDNF NQQK IV AVGIIFFTTY IIISFLIVVN MYIAVILENF NVAT rna v 1.9 I IYFVFFFVVV IFLGSFYLLN LTLAVVTMAY EEQN II PLCIIVFVLI MVIGKLVVLN LFIALLLNSF SNEE III LYAYLYFVVF IIFGSFFTLN LFIGVIIDNF NQQQ IV QIAVVYFVSY IIISFLIVVN MYIAVILENF NTAT NaChac WWSWIYFVIF ILVGTFIVFN LFIGVIVNNV EKAN a Sequences of eukaryotic Ca 2+ and Na + channels correspond to rabbit proteins 2

3 Table S2. Effect of sodium channel mutations on activation, inactivation, and block Mutant Isoform ΔV 0.5, mv locker lock change a Ref. Acti- vation Inactivation Resting Use-dependent Inactivation N 1i20 A Etidocaine (1) N 1i20 K upivacaine x1.5 x3 (2,3) N 1i20 R upivacaine x1.5 x3 (3) N 1i20 A upivacaine x 2 = (3) N 1i20 F upivacaine x 6 x3 (3) N 1i20 W upivacaine x 7 x3 (3) N 1i20 Y upivacaine x 12 x3 (3) N 1i20 T upivacaine x 2 (3) N 1i20 C upivacaine x 5 x1.5 (3) N 1i20 D upivacaine x 2 x2 (3) N 1i20 S Pilsicainide (4) N 1i20 S Lidocaine (4) N 1i20 S Quinidine (4) N 1i20 K 1.4 enzocaine x 2 (2) N 1i20 K 1.4 Lidocaine x 3 (2) N 1i20 K 1.4 Etidocaine x 7 x2 x11 (2) N 1i20 K 1.4 QX-314 x 17 (2) N 1i20 D 1.4 enzocaine x 2 (2) N 1i20 D 1.4 Lidocaine x 2 (2) N 1i20 D 1.4 Etidocaine x 2 (2) N 1i20 K upivacaine x 3 x7 (5,6) N 1i20 C Lidocaine x2 x1.5 (7) N 1i20 A Mibefradil x 1.5 (6) N 1i20 C Mibefradil x 7 (6) N 1i20 F Mibefradil x4 (6) N 1i20 D Mibefradil (6) N 1i20 E Mibefradil (6) N 1i20 R Mibefradil x 2 (6) N 1i20 K Mibefradil x 4 (6) N 2i20 A Etidocaine (1) N 2i20 C (7) N 3i20 A Etidocaine x 3 x 8 (8) N 4i20 A Etidocaine x 15 (1,9) N 4i20 K upivacaine x 3 x 40 (5,6) N 4i20 K Cocaine x 3 x27 (9-11) a, lock increases;, lock decreases;,large decrease of the block;, small change of the block; xn, Effect changes N-fold 3

4 Table S3. Linker-helices S4-S5 and S5 helices in Cav2.1 channel Channel Repeat domain ====S4-S5==== ==============S5=============== k1 k11 o1 o11 o21 SKGLQILGQT LK ASMRELGLLI FFLFIGVILF SSAVYFAEA K v 1.2 b Ca v 2.1 I IPSLQVVLKS IM KAMIPLLQIG LLLFFAILIF AIIGLEFYM II WASLRNLVVS LL NSMKSIISLL FLLFLFIVVF ALLGMQLFG III LPKLKAVFDC VV NSLKNVFNIL IVYMLFMFIF AVVAVQLFK IV GYTIRILLWT FV QSFKALPYVC LLIAMLFFIY AIIGMQVFG 4

5 References 1. Yarov-Yarovoy, V., McPhee, J. C., Idsvoog, D., Pate, C., Scheuer, T., and Catterall, W. A. (2002) The Journal of biological chemistry 277(38), Wang, G. K., Quan, C., and Wang, S. Y. (1998) Molecular pharmacology 54(2), Nau, C., Wang, S. Y., Strichartz, G. R., and Wang, G. K. (1999) Molecular pharmacology 56(2), Itoh, H., Shimizu, M., Takata, S., Mabuchi, H., and Imoto, K. (2005) Journal of cardiovascular electrophysiology 16(5), Nau, C., Wang, S. Y., Strichartz, G. R., and Wang, G. K. (2000) Anesthesiology 93(4), McNulty, M. M., Kyle, J. W., Lipkind, G. M., and Hanck, D. A. (2006) Molecular pharmacology 70(5), Kondratiev, A., and Tomaselli, G. F. (2003) Molecular pharmacology 64(3), Yarov-Yarovoy, V., rown, J., Sharp, E. M., Clare, J. J., Scheuer, T., and Catterall, W. A. (2001) The Journal of biological chemistry 276(1), Ragsdale, D. S., McPhee, J. C., Scheuer, T., and Catterall, W. A. (1994) Science (New York, N.Y 265(5179), Wright, S. N., Wang, S. Y., and Wang, G. K. (1998) Molecular pharmacology 54(4), Wang, S. Y., Tikhonov, D.., Zhorov,. S., Mitchell, J., and Wang, G. K. (2007) Pflugers Arch 454(2),

6 A C D Figure S1. X-ray structures of open cation channels. Hydrogen atoms are added with PYMOL. P-loops are removed for clarity. A model of batrachotoxin is shown in the same scale at each panel. A C, Cytoplasmic views of the open potassium channels Kv1.2 (A), KvAP (), and MthK (C). In respective homology models of Nav1.4, TX would pass into the pore through the open activation gate. D and E, Cytoplasmic and side views of prokaryotic sodium channel NavMs. In the NavMs-based homology model of Nav1.4, openings at the activation gate (D) and the membrane-exposed fenestration (E) are too small to let TX through into the inner pore. E

7 A V i17 L k11 F i16 I i25 V o2 V o2 V i17 I i19 I i19 L k11 V i23 I i25 V i23 Figure S2. Cytoplasmic (A) and side () views of the N i20 contacts in the X-ray structure of the closed sodium channel NavAb (PD code 3RVW). Hydrogen atoms are added with PYMOL. N i20 (space-filled) is tightly surrounded by hydrophobic residues and only its cytoplasm-oriented side lacks close contacts. Semitransparent pink and gray/green surfaces show inter- and intra-subunit contacts, respectively. L i20 F i29 Figure S3. Intersubunit contact of L i29 and F i29 in the X-ray structure of Kv1.2

8 A L Xi20 L Xi20 F (X-1)i29 F (X-1)i29 Figure S4. Cytoplasmic (A) and side () views of the X-ray structure of the open Kv1.2 channel with the inner helices of two neighboring subunits shown as cylinders joined by rods. The cytoplasmic halves of the inner helices cross each other at a big angle. The only intersubunit contact in this region is that between residues i20 and i29 whose side chains are space-filled. Note that these residues flank the PVP motif, which determines the S6 kink (shown by rod).

9 A Q 1o1 K 2i20 C D N 2o1 K 3i20 Figure S5. Inter-repeat H-bonds K 2i20 ---Q 1o1 (A,) and K 3i20 ---N 2o1 (C,D) in the MC-minimized NavAb-based models of the closed-state Nav1.4 mutants N 2i20 K (A,) and N 3i20 K (C,D). The H-bonds may lock the channels in the closed state in agreement with the data that the mutants express no or little currents (see text). The backbones were kept rigid during MC-minimization. The MC-minimized energy of the mutant is close to that of the wild-type channels indicating that the H-bond formation is energetically possible.

10 A N 1i20 N 1i20 N 4i29 N 4i29 T 4k6 N 4i29 T 4k6 N 4i29 Figure S6. H-bond N 4i29 ---T 4k6 in the Nav1.4 mutant N 1i20 A. P-loops are shown as cylinders, S6s as helices, and S4-S5 linkers as rods. A and, Cytoplasmic and side views at the superimposition of the wild-type (red) and mutant (green) models. Red and green labels indicate residues in the wild-type and mutant channel, respectively. For clarity, helix IVS6 is shown at A by strands. Yellow arrows indicate displacement of N 4i29 in the mutant vs. the wild-type channel.

11 A T 2i25 N 3i20 N 2i29 C D T 2i25 N 3i20 Figure S7. Interdomain H-bonds in the MC-minimized Kv1.2-based models of the open-state Cav1.2 mutants. A and, In the mutant I 2i25 T two H-bonds hyper-stabilize the open state. C and D, The rescue mutations I 2i25 T/N 2i29 A eliminates the N 3i20 ---N 2i29 H-bond, and remaining H-bond N 3i29 ---N 2i29 stabilizes the open state.

12 N 2i20 S 1i29 Figure S8. H-bond N 2i20 ---C 1i29 in the MC-minimized model of the open Cav2.1 channel. The backbones were kept rigid during MC-minimization. A N 3i20 N 3o9 N 3o5 A 3i24 m C 2i29 A 2i33 E 2i34 Figure S9. Open Cav2.1 channel with MTS-modified engineered cysteine, m C 2i29. The open state is stabilized by multiple interactions, including an H-bond between N 3i20 and a sulfur atom MTS (section 3.5.1). Linker IIL45 is remover at A for clarity.

13 K 1i20 N 4i29 D 4i28 Figure S10. Interdomain H-bonds N 4i29 ---K 1i20 ---D 4i28 in the MC-minimized Kv1.2- based model of the open-state Cav1.2 mutant N 1i20 K. The H-bonds would lock the channels in the open state in agreement with the data that the mutants do not inactivate. The structures are MC-minimized with rigid backbones implying that formation of the H-bonds in the real channel does not require significant shifts of IS6 and IVS6 helices.

14 A N 4i29 K 1i20 Figure S11. Interdomain H-bond K 1i N 4i29 in the MC-minimized Kv1.2-based homology model of Nav1.4 mutant N 1i20 K. The backbones were kept rigid during MCminimization. The MC-minimized energy of the mutant is close to that of the wild-type channel indicating that the H-bond formation is energetically possible. A, side view with two residues in the linker-helix IVL45 linker removed for clarity., a close-up view.