1. K812 30 Oct 07 LQT 9 and 1

2. 16 Oct 072 LQT 1 I Ks (  ) KVLQT1aK V 7.1potassium LQT 2 I Kr (  ) HERGK V 11.1potassium LQT 3 I Na (  ) SCN 5ANa V 1.5sodium LQT 4Ankyrin Bnot a channel LQT 5 I Ks (  ) Min Kpotassium LQT 6 I Kr (  ) MiRPpotassium LQT 7I K1 KCNJ2K ir 2.1potassium LQT 8 I Ca (  ) CACNA1cCa V 1.2calcium LQT 9Caveolin 3not a channel LQT 10 I Na (  ) SCN 4Bsodium Long QT syndrome associated genes

3. Mutant caveolin-3 induces persistent late sodium current and is associated with long-QT syndrome Vatta M, Ackerman MJ, Ye B, Makielski JC, Ughanze EE, Taylor EW, Tester DJ, Balijepalli RC, Foell JD, Li Z, Kamp TJ, Towbin JA. Circulation 114:2104-12, 2006

4. Caveolin-3 Caveolin-3 (cav-3), protein marker of caveolae in muscle cells. 151a.a., 17kDa. Caveolae are narrow- necked invaginations on the sarcolemma (SL) enriched in sphingolipids and cholesterol. Recent studies have shown NCX, RyR and sodium channel are associated with cav-3 Parton R.G. Science 2001

5. Vatta, M. et al. Circulation 2006;114:2104-2112 CAV3 mutations in LQTS Figure 1. CAV3 mutations in LQTS. Schematic represen- tation of the linear topology of the caveolin-3 protein shows both the location of critical domains and the non- synonymous single nucleotide polymorphisms (common and rare) identified in LQTS patients (A). Sequencing analysis showed the novel nucleotide variants leading to non- synonymous changes, which have been identified in each patient (B), and the amino acid conservation analysis identified that all LQTS variants modified highly conserved amino acids in caveolin-3 (C).

6. CaseSex Age at Diagnosis, yRace Nucleotide Change CAV3 Variant Other LQTS Mutations Presenting Symptom QTc, ms Other ECG Abnormalities Family History Other Comments 1F14W233 C>TT78M * A913V- KCNH2 (LQT2) Nonexertional syncope 405U waves, sinus bradycardia PosSeizure-like presentation 2M8B233 C>TT78M *... Nonexertional syncope 433Marked sinus bradycardia Pos... 3M40W233 C>TT78M *... Asymptomatic 456...Pos... 4F36W253 G>AA85T *... Cardiac arrest in sleep NA Neg... 5F10W290 T>GF97C *... Shortness of breath, chest pain 532Neg... 6M16W423 C>GS141R *... Nonexertional syncope 480NANeg W indicates white; Pos, positive; B, black; and Neg, Negative. *Novel variant. QTc of 532 ms recorded in the setting of albuterol metered-dose inhaler therapy for asthma. TABLE 1. Summary of Putative CAV3 Mutations Found in Patients With LQTS (Absent in 1000 control alleles)

7. Copyright ©2006 American Heart Association Vatta, M. et al. Circulation 2006;114:2104-2112 ECG of patient with the LQTS-associated mutation F97C-CAV3

8. Copyright ©2006 American Heart Association Vatta, M. et al. Circulation 2006;114:2104-2112 Caveolin-3 and hNa V 1.5 localization in human myocardium Figure 3. Caveolin-3 and hNa v 1.5 localization in human myocardium. Immunohistochemical analysis on human tissue from right ventricular free wall performed with anti-caveolin-3 and anti-hNa v 1.5 antibodies demonstrates that both caveolin-3 (green) and hNa v 1.5 (red) show sarcolemmal localization. Colocalization is confirmed in the merge panel, along with the cell nuclei (blue).

9. Vatta, M. et al. Circulation 2006;114:2104-2112 Effect of WT and mutant CAV3 expression on INa in stable hNav1.5-expressing HEK293 Cells Figure 4. Effect of WT and mutant CAV3 expression on I Na in stable hNa v 1.5-expressing HEK293 Cells. Whole-cell I Na traces were recorded with test potentials of 24-ms duration from –120 to 60 mV from a holding potential of –140 mV. Representative I Na traces were recorded from pcDNA3 (A), WT CAV3 (B), F97C-CAV3 (C), and S141R-CAV3 (D) transiently expressed in stable hNa v 1.5 cell lines.

10. pcDNA3CAV3F97CS141R I Na density pA/pF–360±89–348±139–416±120–409±77 Activation V1/2, mV–44±8–43±9–49±4–52±3 Slope factor5555 Inactivation V1/2, mV–82±3–84±2–85±3–83±2 Recovery f, ms2±0.23±0.42±0.82±0.7 s, ms52±1251±1128±9*38±6 As, %88±478±582±179±1 n89109 pA/pF indicates current density; V1/2, voltage of half-maximal activation/inactivation; f, current fast inactivation; and s, current slow inactivation. The fitted kinetic parameters and I Na density from n experiments were averaged and are reported as mean±SD. pcDNA3 (empty vector), WT-CAV3, and 2 CAV3 mutants (F97C and S141R) were transiently expressed in hNa v 1.5-stable cell line. All parameters were analyzed by Kruskal-Wallis test across pcDNA3, WT CAV3, and 2 CAV3 mutants. There is no statistically significant difference for I Na density, activation, inactivation from recovery, rate of recovery, and time constant for slow component when pcDNA3, WT CAV3, or 2 CAV3 mutants were expressed in hNa v 1.5-stable cell lines. TABLE 2. Kinetic parameters for Na v 1.5 alone, with WT CAV3, and with mutant CAV3

11. Vatta, M. et al. Circulation 2006;114:2104-2112 F97C- and S141R-CAV3 increase late sodium current Figure 5. F97C- and S141R-CAV3 increase late sodium current. I Na traces in response to a step to –20 mV for 700-ms duration from a holding potential of – 140 mV (see protocol inset) are shown with peak current off-scale to better show the late currents (A). The cell capacitance is as follows: pcDNA3=11 pF, CAV3=1 2 pF, F97C=12 pF, and S141R=12 pF. Summary data for late I Na represented as percent of peak I Na were significantly increased when F97C and S141R were expressed (B). *Statistically significant differences between mutant CAV3 and experiments with WT CAV3 and without CAV3 (pcDNA3) (P>0.001).

12. Vatta, M. et al. Circulation 2006;114:2104-2112 hNav1.5 and either WT or mutant caveolin-3 coimmunoprecipitate Figure 6. hNa v 1.5 and either WT or mutant caveolin-3 coimmunopre- cipitate. HEK293 cells were transfected with pcDNA3- WT hNa v 1.5+WT CAV3, pcDNA3-hNa v 1.5+P104L- CAV3, pcDNA3-WT hNa v 1.5+S141R-CAV3, or pcDNA3-WT hNa v 1.5+F97C-CAV3. The lysates were subjected to immunoprecipitation using anti–caveolin-3 antibody and were analyzed by immunoblotting. hNa v 1.5, WT caveolin-3, and mutant caveolin-3 are detected in the immunoprecipitates, whereas control immunoglobulin G does not immunoprecipitate the proteins.

13. Clinical aspects of type-1 long-QT syndrome by location, coding type, and biophysical function of mutations involving the KCNQ1 gene Moss AJ, Shimizu W, Wilde AA, Towbin JA, Zareba W, Robinson JL, Qi M, Vincent GM, Ackerman MJ, Kaufman ES, Hofman N, Seth R, Kamakura S, Miyamoto Y, Goldenberg I, Andrews ML, McNitt S Circulation 115:2481-9, 2007

14. Location and Coding * No. of SubjectsType of MutationFunctional Effect N-terminus M1V1MissenseUnknown G57V1MissenseUnknown Transmembrane W120C2MissenseUnknown T144A7MissenseUnknown A150fs/133 [del CT 451-452]2FrameshiftHaploinsufficiency E160K3MissenseUnknown G168R44MissenseUnknown Y171X [513 C>G]6NonsenseHaploinsufficiency R174H2MissenseUnknown A178P5MissenseDominant-negative effect (a) Y184S18MissenseUnknown G185S10MissenseUnknown G189E2MissenseUnknown G189R4MissenseDominant-negative effect (b) R190Q4MissenseHaploinsufficiency (b, c) L191fs/90 [del TGCGC 572- 576] 8FrameshiftHaploinsufficiency R195fs/40 [del G 585]2FrameshiftHaploinsufficiency S225L13MissenseDominant-negative effect (d) A226V3MissenseUnknown R237P1MissenseUnknown D242N3MissenseUnknown R243C13MissenseHaploinsufficiency (e) V254 mol/L59MissenseDominant-negative effect (b, f) R258C1MissenseHaploinsufficiency R259C1MissenseHaploinsufficiency (g) L266P15MissenseUnknown G269D35MissenseDominant-negative effect (h) G269S25MissenseHaploinsufficiency (i) L273F6MissenseDominant-negative effect (a) I274V1MissenseUnknown S277L3MissenseUnknown Y278H2MissenseUnknown E284K2MissenseUnknown G292D3MissenseUnknown F296S2MissenseUnknown G306R2MissenseDominant-negative effect (b, j) V310I1MissenseUnknown T312I14MissenseDominant-negative effect (a) G314S8MissenseDominant-negative effect (h, k, l, m) Y315C10MissenseDominant-negative effect (d, n) Y315S1MissenseDominant-negative effect (h, m) D317G3MissenseUnknown P320H1MissenseUnknown T322 mol/L2MissenseUnknown G325R3MissenseUnknown delF340 [del CTT 1017-1019]7In-frame deletionHaploinsufficiency A341E9MissenseDominant-negative effect (b) A341V20MissenseDominant-negative effect (o)

15. Copyright ©2007 American Heart Association Moss, A. J. et al. Circulation 2007;115:2481-2489 Frequency and location of 74 different mutations in the KCNQ1 potassium channel involving 581 subjects

16. Copyright ©2007 American Heart Association Moss, A. J. et al. Circulation 2007;115:2481-2489 Kaplan-Meier estimate of the cumulative probability of a first cardiac event by location (A), type (B), and biophysical function of the mutation (C)

17. KCNQ1 assembly and function is blocked by long-QT syndrome mutations that disrupt interaction with calmodulin Ghosh S, Nunziato DA, Pitt GS Circ Res. 98:1048-54, 2006

18. Ghosh, S. et al. Circ Res 2006;98:1048-1054 The KCNQ1 CT binds CaM Figure 1. The KCNQ1 CT binds CaM. A, Schematic of KCNQ1 showing the CT construct expressed in Escherichia coli, the locations of the consensus CaM- binding IQ motifs (IQ 1 and IQ 2 ), and the LQTS mutations studied. B, Coomassie-stained gel showing expression and purification of the KCNQ1 CT constructs in the presence and absence of CaM or CaM 1234. Ex indicates bacterial cell extract; Sup, 100 000g supernatant; and P, metal-affinity purified protein.

19. Ghosh, S. et al. Circ Res 2006;98:1048-1054 The KCNQ1 CT/CaM complex assembles into a tetrameric complex

20. Ghosh, S. et al. Circ Res 2006;98:1048-1054 LQTS mutations disrupt CaM interaction with the KCNQ1 CT

21. Ghosh, S. et al. Circ Res 2006;98:1048-1054 KCNQ1 channels are Ca 2+ -sensitive

22. Ghosh, S. et al. Circ Res 2006;98:1048-1054 KCNQ1 channels are CaM sensitive