Mutations of positively charged amino acids in the S4 transmembrane segment of a voltage-gated ion channel form ion-conducting pathways through the voltage-sensing domain, named ω-current. Here, we used structure modeling and MD simulations to predict pathogenic ω-currents in CaV1.1 and CaV1.3 Ca2+ channels bearing several S4 charge mutations. Our modeling predicts that mutations of CaV1.1-R1 (R528H/G, R897S) or CaV1.1-R2 (R900S, R1239H) linked to hypokalemic periodic paralysis type 1 and of CaV1.3-R3 (R990H) identified in aldosterone-producing adenomas conducts ω-currents in resting state, but not during voltage-sensing domain activation. The mechanism responsible for the ω-current and its amplitude depend on the number of charges in S4, the position of the mutated S4 charge and countercharges, and the nature of the replacing amino acid. Functional characterization validates the modeling prediction showing that CaV1.3-R990H channels conduct ω-currents at hyperpolarizing potentials, but not upon membrane depolarization compared with wild-type channels

Fuchs, JE

Hofer, F

Lieb, A

Liedl, KR

Monteleone, S

Negro, G

Pinggera, A

Striessnig, J

Tuluc, P

UCL Discovery

Biophysical Journal, Volume 113Supplemental InformationMechanisms Responsible for u-Pore Currents in Cav Calcium ChannelVoltage-Sensing DomainsStefania Monteleone, Andreas Lieb, Alexandra Pinggera, Giulia Negro, Julian E.Fuchs, Florian Hofer, Jörg Striessnig, Petronel Tuluc, and Klaus R. Liedl1  Supplemental Information - Figures    Figure 1: Root mean square deviation of wild-type (A), R1H (B) and R1G (C) mutant VSD of Cav1.1 domain II in the resting state α.    2    Figure 2: Root mean square deviation of wild-type, R1S and R2S mutant VSD of Cav1.1 domain III in the resting state α and β.       3    Figure 3: 2d-rmsd of all Cα atoms in wild-type (A) and R2H mutant (B) VSD of Cav1.1 domain IV in the resting state β. The wild-type protein is more stable than the mutant as the simulation converges earlier. Whereas the MD simulation of the mutant requires a longer simulation time to converge.  Figure 4: 2d-rmsd of transmembrane Cα atoms in wild-type (A) and R3H mutant (B) VSD of Cav1.3 domain III in the resting state β.   4    Figure 5: Averaged occupancy values over 100 ns and whole transmembrane segments in the resting state α of the Cav1.1 VSD of domain II R1G (A) and III R1S (B).  A: Compared to the wild-type, segment S3 loses its helical secondary structure and changes toward a beta turn. B: All segments show a prevalent alpha helical secondary structure, but S4 presents also 3-10 helix and beta turn patterns.   5    Figure 6. (A) WT II domain VSD in the resting state α modelled without intra- and extra-cellular linkers. The only difference to the model including loops (Fig. 1) is that R1 forms ionic interactions with E4 because E452 from S1-S2 loop was removed from the structure. (B) The presence or absence of loops does not affect the water occupancy in the center of the VSD.   6   Figure 7: R1S and R2S mutant VSDs of domain III in the resting state β (A) and α (B). The protein is represented as cartoons, colored as rainbow according to the residue number: segment S1, lipids and hydrogen atoms are not shown for clearness. Gating charges are shown as lines, whereas mutated residues are highlighted as sticks. H-bonds are marked with yellow dots. Average water density during the simulation is shown as transparent cyan surface. The average number of water molecules in the VSD is calculated for each frame. A: R1S does not result in a water wire in the resting state β because R0 and R2 seal the center of the VSD. R1S forms H-bond interactions with the backbone of S4 that are conserved in helical secondary structure elements. The ionic interaction between R2 and E2 causes a conformational change of helix S4 that forms a kink at R2 level. B: in the resting state α, R2S does not cause a water wire as water molecules can enter only from the cytoplasm. R2S is close to F and loses the ionic interaction with the counter charges in S2 and S3.    7   Figure 8: Mutation of R1 charge to His in CaV1.3 channel does not alter the voltage dependence of the steady-state inactivation. A. Voltage dependence of steady state inactivation (squares) was measured by applying a control test pulse (20 ms to the voltage of maximal inward current, Vmax) followed by 5-s conditioning steps to various potentials and a subsequent 20-ms test pulse to Vmax (30-s recovery between sweeps). Inactivation was calculated as the ratio between the current amplitudes of the test versus control pulse. Whereas the voltage-dependence of activation (circles) of CaV1.3-R990H was significantly shifted by ~3 mV towards more depolarizing potentials compared to WT CaV1.3 channel (same data as in Figure 5B), the voltage dependence of steady-state inactivation (WT V0.5 =-30.8 ± 1.14, n=16; R990H V0.5 = -32.5 ± 1.12, n = 18) or the slope of the voltage dependence of inactivation (WT k=5.38 ±0.30, n=16; R990H k = 6.76 ± 0.39, n=18) were not altered.   8      Figure 9. The amplitude of CaV1.3-R990H guanidinium current (IG) negatively correlates with the number of channels functionally incorporated in the membrane (QON). The linear regression fit of the dependence between IG measured at -100 mV and the QON measured at the reversal potential shows a statistically significant correlation for the CaV1.3-R990H (p <0.05) but not for WT CaV1.3. Since our experimental data shows that the mock transfected cells (no α1 subunit) (N=12) also conduct a guanidinium current we included these values in both WT (N=12) and R990H (N=15) fit functions. Statistics calculated using One Way Repeated Measurements ANOVA with Bonferroni t-test.                    9   Figure 10. CaV1.1 III VSD bearing the V876E mutation modelled in the resting state α (A) and β (B). The modelling predicts the formation of a water wire independent of the gating state. In the resting state α, V876E forms ionic interactions with R2, which moves upwards compared to WT and allows E2 to interact with water molecules. In addition, V876E drives water to the hydrophobic center, which becomes polar due to the introduction of a charged amino acid. In the resting state β, the ionic interactions of R2 with V876E are replaced by R1. 10  Supplemental Information - Tables Table 1: Up-to-date list of mutations in voltage-gated calcium channels that develop or might develop omega currents. Highlighted cells indicate experimentally measured omega-currents. For further mutations at voltage sensors of ion channels we refer to the review of Jurkat-Rott et al. (1). Isoform Domain Mutation N. Disease References Functional Effect Cav1.1 I R174W R4 MHS (2) α-pore gain of function II R528G/H R1 HPP (3-9) α-pore gain or loss of function and ω-current R528C R1 HPP (10) n.c. III  V876E  HPP  n.c. R897S R1 HPP (11) n.c. R900G/S R2 HPP (12, 13) n.c. IV R1239G/H R2 HPP (7-9, 14, 15) α-pore gain of function and leak current  Cav1.3 II R619W R1 APAs (16) n.c. III R990H R3 APAs (17) α-pore not affected but ω-current  Cav2.1 I R192Q R0 FHM (18-21) α-pore gain of function R195K R1 FHM (22) n.c. II R583Q R1 FHM (22-24) α-pore gain of function III R1347Q R1 FHM (25, 26) n.c. R1350Q R2 EA2 (27, 28) α-pore gain of function IV R1661H R1 FHM (29) n.c. R1664Q R2 FHM (30) n.c. R1667W R3 FHM (22) n.c.  APAs: adrenal aldosterone-producing adenomas EA2: episodic ataxia type 2 FHM: familial hemiplegic migraine HPP: hypokalemic periodic paralysis MHS: malignant hyperthermia susceptibility  11  Table 2: Sequence alignment of Cav1.1, Cav1.3 and Cav2.1 voltage sensors. Green highlighted residues represent the gating charges R1 to R4; yellow highlighted residues indicate other positively charged positions of segment S4. Residues for which mutants have been identified are highlighted in red.  12   Table 3: Gating charges and counter charges in voltage sensors of Cav1.1 domains II, III and IV and Cav1.3 domain III.   Cav1.1 Cav1.3 VSD II VSD III VSD IV VSD III F F475 F843 F1161 F930 R0 S525, R469 K894 R1229 K981 R1 R528 R897 R1236 R984 E1/D1 E452 D836 - D923 R2 R531 R900 R1239 R987 E2 E478 E846 E1164 E933 R3 R534 R903 R1242 R990 D3 D500 D872 D1186 D959 E4/D4 E510 - D1196 -  Table 4: Ionic interactions between gating and counter charges in WT voltage sensors at distinct resting states.  Gating charge Counter charge Cav1.1 Cav1.3 II III IV III State α State α State β State β State β R0 E4 D1 D1 D4 - R1 E452 D1 E2 D4 D1 R2 E2 E2, D3 D3 E2, D3 D3 R3 E2 D3 - D3 E2          13  Table 5: Averaged helical secondary structure occupancy over 100 ns of whole transmembrane segments for WT and mutant VSDs in distinct states.  System State S1 S2 S3 S4 Cav1.1 Domain II WT Resting α 98% 99% 56% 67% R1H 98% 99% 58% 77% R1G 100% 99% 29% 93% Domain III WT Resting α 89% 92% 85% 76% Resting β 96% 85% 94% 93% R1S Resting α 79% 94% 95% 59% Resting β 99% 98% 89% 83% R2S Resting α 92% 95% 99% 89% Resting β 80% 76% 95% 66% Domain IV WT Resting β 88% 98% 95% 67% R2H 99% 97% 97% 85% Cav1.3 Domain III WT Resting β 83% 91% 82% 75% R3H 86% 93% 78% 75%  14   Table 6: H-bond counts for WT and R2S mutant of Cav1.1 domain III in distinct states. Gating charges are positively charged amino acids in the transmembrane segment S4. Counter charges are negatively charged residues in the surrounding segments in the voltage sensor. In the WT VSD, R2 forms H-bonds with counter charges (E2 and D3), polar amino acids and water molecules. In the mutant, R2S loses the interactions with counter charges in the resting state α whereas in the resting state β it can still form H-bonds with a negatively charged residue in the helix S3 (D3).  Resting state α Resting state β WT R2S WT R2S Gating charges 0 72% 0 1% Counter charges 100% 0 97% 85% Polar residues 0 1% 67% 0 Water 93% 7% 23% 0   Table 7: H-bond counts for WT and R3H mutant of Cav1.3 domain III in distinct states.  In the WT voltage sensor, R3 forms H-bonds with counter charges and water molecules. In the mutant, R3H loses the interactions with counter charges, but forms H-bonds with polar residues in the active state and water molecules in the resting state β.   Active state Resting state β WT R3H WT R3H Gating charges 0 0 0 17% Counter charges 33% 0 80% 1% Polar residues 0 84% 0 8% Water 55% 0 20% 67%           15  References  1. Jurkat-Rott, K., J. Groome, and F. Lehmann-Horn. 2012. Pathophysiological role of omega pore current in channelopathies. Front Pharmacol 3:112. 2. Striessnig, J., H. J. Bolz, and A. Koschak. 2010. Channelopathies in Cav1.1, Cav1.3, and Cav1.4 voltage-gated L-type Ca2+ channels. Pflugers Arch 460:361-374. 3. Jurkat-Rott, K., F. Lehmann-Horn, A. Elbaz, R. Heine, R. G. Gregg, K. Hogan, P. A. Powers, P. Lapie, J. E. Vale-Santos, J. Weissenbach, and et al. 1994. A calcium channel mutation causing hypokalemic periodic paralysis. Hum Mol Genet 3:1415-1419. 4. Lapie, P., C. Goudet, J. Nargeot, B. Fontaine, and P. Lory. 1996. Electrophysiological properties of the hypokalaemic periodic paralysis mutation (R528H) of the skeletal muscle alpha 1s subunit as expressed in mouse L cells. FEBS Lett 382:244-248. 5. Jurkat-Rott, K., U. Uetz, U. Pika-Hartlaub, J. Powell, B. Fontaine, W. Melzer, and F. Lehmann-Horn. 1998. Calcium currents and transients of native and heterologously expressed mutant skeletal muscle DHP receptor α1 subunits (R528H). FEBS Letters 423:198-204. 6. Morrill, J. A., R. H. Brown, Jr., and S. C. Cannon. 1998. Gating of the L-type Ca channel in human skeletal myotubes: an activation defect caused by the hypokalemic periodic paralysis mutation R528H. J Neurosci 18:10320-10334. 7. Morrill, J. A., and S. C. Cannon. 1999. Effects of mutations causing hypokalaemic periodic paralysis on the skeletal muscle L-type Ca2+ channel expressed in Xenopus laevis oocytes. J Physiol 520 Pt 2:321-336. 8. Kuzmenkin, A., C. Hang, E. Kuzmenkina, and K. Jurkat-Rott. 2007. Gating of the HypoPP-1 mutations: I. Mutant-specific effects and cooperativity. Pflugers Arch 454:495-505. 9. Jurkat-Rott, K., M.-A. Weber, M. Fauler, X.-H. Guo, B. D. Holzherr, A. Paczulla, N. Nordsborg, W. Joechle, and F. Lehmann-Horn. 2009. K+-dependent paradoxical membrane depolarization and Na+ overload, major and reversible contributors to weakness by ion channel leaks. Proceedings of the National Academy of Sciences of the United States of America 106:4036-4041. 10. Yang, B., Y. Yang, W. L. Tu, Y. Shen, and Q. Dong. 2014. A rare case of unilateral adrenal hyperplasia accompanied by hypokalaemic periodic paralysis caused by a novel dominant mutation in CACNA1S: features and prognosis after adrenalectomy. Bmc Urol 14. 11. Chabrier, S., N. Monnier, and J. Lunardi. 2008. Early onset of hypokalaemic periodic paralysis caused by a novel mutation of the CACNA1S gene. Journal of Medical Genetics 45:686-688. 12. Matthews, E., R. Labrum, M. G. Sweeney, R. Sud, A. Haworth, P. F. Chinnery, G. Meola, S. Schorge, D. M. Kullmann, M. B. Davis, and M. G. Hanna. 2009. Voltage sensor charge loss accounts for most cases of hypokalemic periodic paralysis. Neurology 72:1544-1547. 13. Hirano, M., Y. Kokunai, A. Nagai, Y. Nakamura, K. Saigoh, S. Kusunoki, and M. P. Takahashi. 2011. A novel mutation in the calcium channel gene in a family with hypokalemic periodic paralysis. J Neurol Sci 309:9-11. 14. Ptacek, L. J., R. Tawil, R. C. Griggs, A. G. Engel, R. B. Layzer, H. Kwiecinski, P. G. McManis, L. Santiago, M. Moore, G. Fouad, and et al. 1994. Dihydropyridine receptor mutations cause hypokalemic periodic paralysis. Cell 77:863-868. 15. Lehmann-Horn, F., I. Sipos, K. Jurkat-Rott, R. Heine, H. Brinkmeier, B. Fontaine, L. Kovacs, and W. Melzer. 1995. Altered calcium currents in human hypokalemic periodic paralysis myotubes expressing mutant L-type calcium channels. Soc Gen Physiol Ser 50:101-113. 16. Nishimoto, K., S. A. Tomlins, R. Kuick, A. K. Cani, T. J. Giordano, D. H. Hovelson, C.-J. Liu, A. R. Sanjanwala, M. A. Edwards, C. E. Gomez-Sanchez, K. Nanba, and W. E. Rainey. 2015. Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands. 16  Proceedings of the National Academy of Sciences 112:E4591-E4599. 17. Azizan, E. A., H. Poulsen, P. Tuluc, J. Zhou, M. V. Clausen, A. Lieb, C. Maniero, S. Garg, E. G. Bochukova, W. Zhao, L. H. Shaikh, C. A. Brighton, A. E. Teo, A. P. Davenport, T. Dekkers, B. Tops, B. Kusters, J. Ceral, G. S. Yeo, S. G. Neogi, I. McFarlane, N. Rosenfeld, F. Marass, J. Hadfield, W. Margas, K. Chaggar, M. Solar, J. Deinum, A. C. Dolphin, I. S. Farooqi, J. Striessnig, P. Nissen, and M. J. Brown. 2013. Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension. Nat Genet 45:1055-1060. 18. Ophoff, R. A., G. M. Terwindt, M. N. Vergouwe, R. van Eijk, P. J. Oefner, S. M. Hoffman, J. E. Lamerdin, H. W. Mohrenweiser, D. E. Bulman, M. Ferrari, J. Haan, D. Lindhout, G. J. van Ommen, M. H. Hofker, M. D. Ferrari, and R. R. Frants. 1996. Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell 87:543-552. 19. Kraus, R. L., M. J. Sinnegger, H. Glossmann, S. Hering, and J. Striessnig. 1998. Familial hemiplegic migraine mutations change alpha1A Ca2+ channel kinetics. J Biol Chem 273:5586-5590. 20. Hans, M., S. Luvisetto, M. E. Williams, M. Spagnolo, A. Urrutia, A. Tottene, P. F. Brust, E. C. Johnson, M. M. Harpold, K. A. Stauderman, and D. Pietrobon. 1999. Functional consequences of mutations in the human alpha1A calcium channel subunit linked to familial hemiplegic migraine. J Neurosci 19:1610-1619. 21. van den Maagdenberg, A. M., D. Pietrobon, T. Pizzorusso, S. Kaja, L. A. Broos, T. Cesetti, R. C. van de Ven, A. Tottene, J. van der Kaa, J. J. Plomp, R. R. Frants, and M. D. Ferrari. 2004. A Cacna1a knockin migraine mouse model with increased susceptibility to cortical spreading depression. Neuron 41:701-710. 22. Ducros, A., C. Denier, A. Joutel, M. Cecillon, C. Lescoat, K. Vahedi, F. Darcel, E. Vicaut, M. G. Bousser, and E. Tournier-Lasserve. 2001. The clinical spectrum of familial hemiplegic migraine associated with mutations in a neuronal calcium channel. N Engl J Med 345:17-24. 23. Battistini, S., S. Stenirri, M. Piatti, C. Gelfi, P. G. Righetti, R. Rocchi, F. Giannini, N. Battistini, G. C. Guazzi, M. Ferrari, and P. Carrera. 1999. A new CACNA1A gene mutation in acetazolamide-responsive familial hemiplegic migraine and ataxia. Neurology 53:38-43. 24. Kraus, R. L., M. J. Sinnegger, A. Koschak, H. Glossmann, S. Stenirri, P. Carrera, and J. Striessnig. 2000. Three new familial hemiplegic migraine mutants affect P/Q-type Ca(2+) channel kinetics. J Biol Chem 275:9239-9243. 25. Alonso, I., J. Barros, A. Tuna, A. Seixas, P. Coutinho, J. Sequeiros, and I. Silveira. 2004. A novel R1347Q mutation in the predicted voltage sensor segment of the P/Q-type calcium-channel alpha-subunit in a family with progressive cerebellar ataxia and hemiplegic migraine. Clin Genet 65:70-72. 26. Stam, A., K. Vanmolkot, H. Kremer, J. Gärtner, J. Brown, E. Leshinsky-Silver, R. Gilad, E. Kors, W. Frankhuizen, H. Ginjaar, J. Haan, R. Frants, M. Ferrari, A. Van Den Maagdenberg, and G. Terwindt. 2008. CACNA1A R1347Q: a frequent recurrent mutation in hemiplegic migraine. Clinical Genetics 74:481-485. 27. Miki, T., T. A. Zwingman, M. Wakamori, C. M. Lutz, S. A. Cook, D. A. Hosford, K. Herrup, C. F. Fletcher, Y. Mori, W. N. Frankel, and V. A. Letts. 2008. Two novel alleles of tottering with distinct Ca(v)2.1 calcium channel neuropathologies. Neuroscience 155:31-44. 28. Blumkin, L., M. Michelson, E. Leshinsky-Silver, S. Kivity, D. Lev, and T. Lerman-Sagie. 2010. Congenital Ataxia, Mental Retardation, and Dyskinesia Associated With a Novel CACNA1A Mutation. J Child Neurol 25:892-897. 29. Friend, K. L., D. Crimmins, T. G. Phan, C. M. Sue, A. Colley, V. S. C. Fung, J. G. L. Morris, G. R. Sutherland, and R. I. Richards. 1999. Detection of a novel missense mutation and second 17  recurrent mutation in the CACNA1A gene in individuals with EA-2 and FHM. Human Genetics 105:261-265. 30. Tonelli, A., M. G. D'Angelo, R. Salati, L. Villa, C. Germinasi, T. Frattini, G. Meola, A. C. Turconi, N. Bresolin, and M. T. Bassi. 2006. Early onset, non fluctuating spinocerebellar ataxia and a novel missense mutation in CACNA1A gene. J Neurol Sci 241:13-17.   

Mechanisms Responsible for omega-Pore Currents in Ca-v Calcium Channel Voltage-Sensing Domains

http://discovery.ucl.ac.uk/10033694/7/Monteleone_Mechanisms_Responsible_Oomega-Pore_S1.pdf

Mechanisms Responsible for omega-Pore Currents in Ca-v Calcium Channel Voltage-Sensing Domains

Abstract

Similar works

Full text

Available Versions

UCL Discovery