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L Vs10 V Directly A346 ISK (MINK), KVLQT1 POTASSIUM CHANNEL Th-PM-H7 Th-PM-H8 COEXPRESSION OF minK MODIFIES hKV1.5 ACTIVATION IN IKs channels: minK/KvLQT1 heteromultimers have a larger unitary MAMMALIAN CELLS. ((Tao Yang, Sabina Kupershmidt, Dan M. Roden, Dirk conductance than homomeric KvLQT1 channels. J. Snyders)) Vanderbilt University, Nashville, TN 37232. ((Federico Sesti, and Steve A. N. Goldstein)) Section of Developmental Biology and Biophysics, Departments of Pediatrics and Cellular & Molecular Physiology, Coexpression of minK modifies K+ currents obtained with expression of Yale University School of Medicine, New Haven, CT. KvLQTI (resulting in slowed activation and an increase in current) and ofHERG IKs is a voltage-dependent K+ channel in human heart that opens during (resulting in increased current with no change in gating kinetics). In this study, we contraction to repolarize the myocardium. The channel is a heteromeric complex have examined the interaction ofminK with hKvl .5 stably transfected in L-cells, of the single P loop subunit KvLQTI and minK, a non-P loop protein with a single transmembrane segment (Barhanin et al., 1996. Nature. 384:78-80; Sanguinetti et and transiently transfected in HEK-293 cells. Current obtained with transfection al., 1996. ibid. 384:80-83). Mutations in both subunits have now been identified to by green fluorescence protein (GFP) alone was compared to that with minK + GFP. cause inherited Long QT syndrome (LQTS), a cardiac dysrrhythmia that In both systems, minK shifted the voltage dependence of activation - 10-15 mV predisposes to sudden death (Splawski et al. Nature Genetics. 1997. in press). positive, and attenuated slow inactivation. MinK strikingly slowed activation, Recently, we showed that the IKs pore is formed by contributions of both without altering deactivation: for example, at +50 mV, control activation T was subunits (cf., Tai and Goldstein, this volume) using cysteine mutation and Cd2+ or 2.3±0.1 ms vs 5.4±0.6 ms with minK Usual hKvl .5 currents were observed with Zn2+ to probe side-chain exposure. Here we use heterologous expression of expression of an hKvl .5 mutation lacking the C-terminal 57 amino acids, but no wildtype and engineered subunits in Xenopus oocytes to explore the single channel effect of coexpression with minK was found. We conclude that minK modifies characteristics of IKs and homomeric KvLQTl channels. Application of transition function of hKvl.5 IL cell metals to channels carrying substituted cysteines is used to confirm the identity of Kvl.5+GFP IL cell Kv1.5+mimK+G the channels under study. We find the unitary current through KvLQTI homomers channels expressed in is too small to be resolved by direct measurement and can only be evaluated by mammalian cells, and that variance analysis; in contrast, currents through single IKs channels are observed the interaction between y i-I mV l Vs10 V directly. Our findings contradict those of Romey et al. (JBC. 1997. 272:16713-6). the two proteins likely IKs channels formed with minK mutants that lead to LQTS in humans are now involves the C-terminal under study. domain of hKvl .5. bOrM SMOOTH MUSCLE REGULATORY PROTEINS II Th-Posl Th-Pos2 TRANSGENIC MOUSE HEARTS WITH A POINT MUTATION IN a- VISUALIZATION OF TROPOMYOSIN TURNOVER AND INCORPORATION IN ADULT TROPOMYOSIN SHOW ALTERED MYOFILAMENT SENSITIVITY TO CARDIAC MYOCYTES ((Daniel Lt Michele and Joseph M. Metzger)) Department of CALCIUM, INDEPENDENT OF PHOSPHORYLATION STATE. ((C.C. Evans,+ Physiology, University ofMichigan, Ann Arbor, Mt 48109. B.M. Wolska+, M. Muthuchamy*, D.F. Wieczorek*, R.J. Solaro+)) 'University of Illinois at Chicago, College of Medicine, Chicago, IL 60612, *University of Adult contractile cells turn over their contractile proteins with average half lives of several Cincinnati, College of Medicine, Cincinnati, OH 45267. days while maintaining the ability to produce force. Utilizing adenoviral gene transfer into adult cardiac myocytes in Wvno, we have remodeled the tropomyosin (Tm) within the We studied from adult mouse contractile apparatus with ectopically expressed tropomyosin isoforms. An adenoviral vector myofilaments nontransgenic (NTG) hearts that express (AdaxTnFLAG) was constructed with a CMV promoter driving expression ofhuman ea-Tm wild type a-tropomyosin (a-Tm) and from hearts ofadult transgenic mice (TG) over with a C-terminal FLAG. Western blot analysis oftotal protein from adult rat cardiac expressing a mutant a-Tm (Aspl75Asn) linked to familial hypertrophic myocytes infected with AdaxTnmFLAG showed no detectable aTmFLAG protein at day 1, but cardiomyopathy (FHC). Transgene expression was driven by the murine a-myosin aTmFLAG/total Tm increased to 40%/ by 6 days post gene transfer. Dual monoclonal heavy chain promoter and was restricted to the heart. TG mouse hearts demonstrated immunfluorescence and confocal microscopy using a FLAG epitope Ab and a troponin I or an a significant increase in Ca2" sensitivity in a Ca2"-activated myofibrillar MgATPase a-actinin Ab was used to visualize the incorporation ofthe cxTmFLAG into the contractile assay (A pCa50 = 0.1 1) compared to NTG mice. Force measurements from Triton X- apparatus. At day 2 post infection, aTmFLAG was first detected evenly expressed throughout 100 skinned fiber bundles prepared from the TG mouse hearts also had increased Ca2" the myocyte in a narrow band near the free end (pointed end) ofthe thin filament. This was sensitivity compared to fibers from NTG hearts (A pCa5o= 0.08). Phosphorylation in contrast to the troponin I Ab which labelled the entire thin filament from barbed to pointed oftroponin I with cyclic-AMP dependent protein kinase induced an equal decrease end. In addition, we have constructed an adenovirus vector containing a CMV promoter in Ca2" sensitivity of force in fibers from NTG (ApCa55 = 0.06) and TG (ApCa50 = driving expression of rat P-Tm (AdpTm). Western blot analysis indicated that 30-35% ofthe 0.07) hearts. Our preliminary studies also show no difference in maximum Ca2 - total Tm was the expressed P-Tm 5-7 days post infection with no P-Tm protein detected in activated force in fiber bundles from NTG and TG mouse hearts. These results control myocytes. Immunfluorescence using an Ab that recognizes both Tm isoforms showed indicate that although there is no difference in the maximum force generated by periodic staining was maintained in infected cells 6 days post infection suggesting that the myofilaments from TG hearts as compared to NTG hearts, TG heart myofilaments expressed Tm is incorporated into sarcomeres. We conclude that 1) adenoviral mediated are more sensitive to Ca2" whether they are phosphorylated or not. Increased Ca2+ gene transfer can be used to remodel Tm in the context ofthe adult cardiac myocyte, 2) Tm turnover and incorporation in adult cardiac myocytes appears to occur most readily at the free sensitivity, particularly under phosphorylating conditions as occurs during J3- end ofthe adrenergic stimulation, may predispose the myocardium to impaired relaxation and thin filament. contribute to altered cardiac function in patients with FHC during exercise or stress. Th-Pos3 Th-Pos4 CHARACTERIZATION OF TN EXCHANGE IN MYOFIBRILS AND SOLUTION. HIGH RESOLUTION OF THE pCa/FLUORESCENCE RELATION OF DANZ- ((M. She', S. Frisbie', D. C. Trimble', L. C. Yu', and J. M. Chalovich')) 'NIAMS/NIH, TNC SUBSTITUTED RABBIT PSOAS SINGLE FIBERS. ((M Huang, D Bethesda MD 20892; 'E. Carolina Med. School, Greenville, NC 27834. Burkhoff, F Schachat, & P Brandt)) Depts. of Anatomy & Cell Biology, It has been shown earlier that fluorescently labeled troponin (Tn) can be exchanged into Biomedical Engineering and Medicine, Columbia U., and Cell Biology, Duke U. rabbit psoas fibers in order to simultaneously measure mechanical properties and the state of the regulatory proteins (Brenner & Chalovich, 1995 Biophys. J. A142). We We constructed an apparatus to continued to characterize this exchange process. X-ray diffraction patterns of single 1.0- measure fluorescence ratio fibers in rigor showed intensification of 385 A after 12 h of incubation with avidin- o/° change (AF) in single muscle attached Tn, an indication of Tn exchange. Fluorescence microscopy of myofibrils 0.8- fibers with further showed that the exchange of Tn under rigor conditions occurred mostly in the small changes in overlap region, as in muscle fibers. However, in the presence of exogenous S1, the I t 1 pCa. AF (0) of DANZ-TnC exchange occurred along the entire thin filament, requiring more than 1 h of incubation. 0 0.6i substituted fibers increases In relaxing condition, little exchange was observed. These results suggest that the from the first decrease in pCa exchange process is slow but can be facilitated by myosin binding to actin. Several 80o.4- Cy j(8 to until saturation at methods were attempted to estimate the rate of Tn exchange in solution. When the thin 7.7) pCa filaments were prepared using a radioactively labeled TnI, in - 3 h at 20 OC, the ° 0.2- / e 6. Fitting the AF points with one radioactivity could be totally displaced by a large excess of unlabeled Tn. The time c binding constant fails to fit the course of labeled-Tn exchange in the presence of excess Tn was also monitored via the 0.0- upper half well while fitting with decrease of fluorescence of lANBD-Tn and by the change in fluorescence energy two binding constants (3 transfer between IAANS-Tn (donor) and DABMI-actin (acceptor). Both systems 8.0 7.5 7.0 6.56 r.05.5 0.0 variable parameters) includes reported a similar characteristic time of exchange, - 40 min at 20 IC. The exchange pca all the points. The fitted pKs for process appeared to be faster in the presence of SI but this could not be accurately AF in the figure (fiber F0918A) are 7.21 and 6.41 and the nH's are 1.0 and 2.33 measured due to a decrease in the fluorescence change.
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