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Identification of a calcium binding protein in the nuclear membrane
J Mel
Cell
Cardiol
24 (Supplement
I) (1992)
O-34-1 IDENTIFICATION
OF A CALCIUM BINDING PROTEIN IN THE NUCLEAR MEMBRANE. James SC. Gilchrist and Grant N. Pierce. Division of Cardiovascular Sciences, St Boniface Hospital Research Centre, University of Manitoba, Winnipeg, Manitoba. Canada. R2H 2A6. Sarco- and endoplasmic reticulum Ca 21 binding proteins are important in mediating transmembraneous Ca2+ movements and intracellular Ca2+ signalling. Recent studies have demonstrated Ca2+ transport across the nuclear membrane. We isolated nuclear enveloue membranes, enriched in nuclear pore complex proteins (lamins A,B and C and devoid of histones, from heart and liver nucleii to investigate the possible association of Ca 1 + bindine oroteins with these membranes. Resolution of liver membrane proteins by SDS-PAGE revealed an’ acidic 93 kDa peptide (~93) that stained blue with the cationic carbocyanine dye “Stains-All”. In heart nuclear membranes, a similar acidic blue staining protein (- 105 kDa) was also observed. Specific 45Ca2+ and ruthemum red binding by p93 on western blots demonstrated that it is a high affinity Ca2+ binding rotein. p93 was resistant to extraction by 1M NaCl, 6M urea, 0.5% CHAPS and 0.5% Triton Xp 100 suggesting that p93 is anchored within a membrane bound rotein complex. However, p93 was completely solubilised with 2% Triton X-100. p93 was enrtc2 ed after the outer nuclear membrane was removed with 1% citric acid treatment indicating ~93 localisation to the inner nuclear membrane. Absence of electro heretic mobility shifts aft&lEndo H treatment indicated that p93 is not a glycoprotein unlike Isndoplasmin (Grp 94) found in sarco- and endoplasmic reticular membranes. These data demonstrate that p93 is a novel hi affinity Ca2+ binding protein localised to the inner nuclear membrane. PSupported by MRC, Canada)
O-34-2
CALCIUM
DEPENDENT
MODULATION
DF CALCIUM
CURRENT
IN GUINEA-PIG
VENTRICULAR
CELL!!?
Aliacn M. Gurney. Susan E. Bates. Dept. of Pharmacotoav. UMDS. St. Thomas’s Hceoital. London. U.K SE1 7EH. In guinea-pkg ventricular myocyt& Ca lnffux throtigh L-G Ca channels Is modulated by feedback from intracellular Ca. We have examined how Ca lnfiuences the Ca current, using the photolablfe caged Ca molecule, nitr-5. Isolated cells. In physiological solution, were vdtage clamped at -70 mV using ths ‘whole-cdl recording c~nftg~ration. Nltr-5 (2mM) was inducted in the pipettefllllng sdution, which contained (mM) CsU 130, C&i, 1, MgCts 1, ATP-Nas 2 and HEPES 15, pH 7.2. Ca currents were activated by a 3OOms step to OmV, after a prep&a to -fOmV. Half way through the test pulse, a 1ms light flash photdysed nftr-5 and raised lntraceflular Ca by -200nM. The flash caused lmmedlate inhibition of the current, reaching a maximum In 1 OOms, fdlowed by potentlatlon. The inhibition measured 1 OOms after the flash was 0.8f 0.4% (s.e.m.. n=5) of the peak, p&lash current, with currents activated at 0.7 Hz. The block was larger (1.8 f 0.8%; n=9) at 0.05M. and increased further (3.9 f 0.3%. n=3; 0.05Hz) when Ca in the pipette was replaced with the chelator BAPTA (12mM). Thus ths largest inhibftion was olxwved when the pre-flash [Cal was lowest, and basal Ca-induced fnactivatlon of the channels was presumably small. Potenttatlon of the current was the most pronounced response to the flash, with increases In amplitude up to 200%. Its time course depended on the stimulus frequency and Ca buffering in the cell. With actlvatlon at 0.05H2, the current increased exponentially with a time constant d 5 f 2 min (n=8). This was accelerated to 1.5 + 0.3 min (n=3) when BAPTA replaced Ca in the pipette. The potentiation induced during faster stimulation (0.7Hz) fdlowed a Mexponential time course, fit with a 20s time-constant in addition to the stow component above. As found previously In frog atria, prestlmulating the Ca current with lsoprenaline (u&M) prevented the flash-induced potentiation, but not the lnit&l blockade (3.0 f 1.9%, n=4; 0.05H2, no BAPTA). This is consistent wkh previous suggestions that augmentation of the current results from phosphoryfation of a &channel protein. The lnhibkory responss Is more likely to reflect a direct interaction between Ca and the channel.
O-34-3FAST
DIGITAL IMAGING OF INTRACELLULAR FREE CALCIUM DISTRIBUTION IN CULTURED SINGLE VENTRICULAR MYOCYTE Shin-ichi Momomura, Osami Kohmoto, Hiroshi Matsui, Takashi Serizawa, Masahiko lizuka. The 2nd Department of Internal Medicine, University of Tokyo, Tokyo 113, Japan It has been reported that fluorescence image from furaloaded ventricular myocytes could be detected using image intensified CCD camera. We developed a system which enables beat to beat digital imaging of intracellular free calcium ([Ca’+]i) distribution from a cultured single ventricular myocyte. Spontaneously contracting ventricular myocytes were cultured from 1 O-day-old chick embryos. These ceils were incubated with Ca” fluorescent dye indo-l for 30 minutes and washed for 15 minutes. Fluorescence images at 405nm and 480nm were obtained on the same image intensified CCD camera (ARGUS 50, Hamamatsu Photonics) as fast as 60 images/set. After geometric correction, the ratio of 405nrrtI480nm fluorescence was calculated, then frame-by-frame digital color imaging of [Ca**]i was obtained. Time course of [Ca’+]i transient in a single cell was also calculated from the digitalized data. This was consistent with previous results by photomultiplier tube method. This system may become a useful tool for investigating intracellular calcium handling and distribution in excitable cells. 5.134
Cell
Cardiol
24 (Supplement
I) (1992)
O-34-1 IDENTIFICATION
OF A CALCIUM BINDING PROTEIN IN THE NUCLEAR MEMBRANE. James SC. Gilchrist and Grant N. Pierce. Division of Cardiovascular Sciences, St Boniface Hospital Research Centre, University of Manitoba, Winnipeg, Manitoba. Canada. R2H 2A6. Sarco- and endoplasmic reticulum Ca 21 binding proteins are important in mediating transmembraneous Ca2+ movements and intracellular Ca2+ signalling. Recent studies have demonstrated Ca2+ transport across the nuclear membrane. We isolated nuclear enveloue membranes, enriched in nuclear pore complex proteins (lamins A,B and C and devoid of histones, from heart and liver nucleii to investigate the possible association of Ca 1 + bindine oroteins with these membranes. Resolution of liver membrane proteins by SDS-PAGE revealed an’ acidic 93 kDa peptide (~93) that stained blue with the cationic carbocyanine dye “Stains-All”. In heart nuclear membranes, a similar acidic blue staining protein (- 105 kDa) was also observed. Specific 45Ca2+ and ruthemum red binding by p93 on western blots demonstrated that it is a high affinity Ca2+ binding rotein. p93 was resistant to extraction by 1M NaCl, 6M urea, 0.5% CHAPS and 0.5% Triton Xp 100 suggesting that p93 is anchored within a membrane bound rotein complex. However, p93 was completely solubilised with 2% Triton X-100. p93 was enrtc2 ed after the outer nuclear membrane was removed with 1% citric acid treatment indicating ~93 localisation to the inner nuclear membrane. Absence of electro heretic mobility shifts aft&lEndo H treatment indicated that p93 is not a glycoprotein unlike Isndoplasmin (Grp 94) found in sarco- and endoplasmic reticular membranes. These data demonstrate that p93 is a novel hi affinity Ca2+ binding protein localised to the inner nuclear membrane. PSupported by MRC, Canada)
O-34-2
CALCIUM
DEPENDENT
MODULATION
DF CALCIUM
CURRENT
IN GUINEA-PIG
VENTRICULAR
CELL!!?
Aliacn M. Gurney. Susan E. Bates. Dept. of Pharmacotoav. UMDS. St. Thomas’s Hceoital. London. U.K SE1 7EH. In guinea-pkg ventricular myocyt& Ca lnffux throtigh L-G Ca channels Is modulated by feedback from intracellular Ca. We have examined how Ca lnfiuences the Ca current, using the photolablfe caged Ca molecule, nitr-5. Isolated cells. In physiological solution, were vdtage clamped at -70 mV using ths ‘whole-cdl recording c~nftg~ration. Nltr-5 (2mM) was inducted in the pipettefllllng sdution, which contained (mM) CsU 130, C&i, 1, MgCts 1, ATP-Nas 2 and HEPES 15, pH 7.2. Ca currents were activated by a 3OOms step to OmV, after a prep&a to -fOmV. Half way through the test pulse, a 1ms light flash photdysed nftr-5 and raised lntraceflular Ca by -200nM. The flash caused lmmedlate inhibition of the current, reaching a maximum In 1 OOms, fdlowed by potentlatlon. The inhibition measured 1 OOms after the flash was 0.8f 0.4% (s.e.m.. n=5) of the peak, p&lash current, with currents activated at 0.7 Hz. The block was larger (1.8 f 0.8%; n=9) at 0.05M. and increased further (3.9 f 0.3%. n=3; 0.05Hz) when Ca in the pipette was replaced with the chelator BAPTA (12mM). Thus ths largest inhibftion was olxwved when the pre-flash [Cal was lowest, and basal Ca-induced fnactivatlon of the channels was presumably small. Potenttatlon of the current was the most pronounced response to the flash, with increases In amplitude up to 200%. Its time course depended on the stimulus frequency and Ca buffering in the cell. With actlvatlon at 0.05H2, the current increased exponentially with a time constant d 5 f 2 min (n=8). This was accelerated to 1.5 + 0.3 min (n=3) when BAPTA replaced Ca in the pipette. The potentiation induced during faster stimulation (0.7Hz) fdlowed a Mexponential time course, fit with a 20s time-constant in addition to the stow component above. As found previously In frog atria, prestlmulating the Ca current with lsoprenaline (u&M) prevented the flash-induced potentiation, but not the lnit&l blockade (3.0 f 1.9%, n=4; 0.05H2, no BAPTA). This is consistent wkh previous suggestions that augmentation of the current results from phosphoryfation of a &channel protein. The lnhibkory responss Is more likely to reflect a direct interaction between Ca and the channel.
O-34-3FAST
DIGITAL IMAGING OF INTRACELLULAR FREE CALCIUM DISTRIBUTION IN CULTURED SINGLE VENTRICULAR MYOCYTE Shin-ichi Momomura, Osami Kohmoto, Hiroshi Matsui, Takashi Serizawa, Masahiko lizuka. The 2nd Department of Internal Medicine, University of Tokyo, Tokyo 113, Japan It has been reported that fluorescence image from furaloaded ventricular myocytes could be detected using image intensified CCD camera. We developed a system which enables beat to beat digital imaging of intracellular free calcium ([Ca’+]i) distribution from a cultured single ventricular myocyte. Spontaneously contracting ventricular myocytes were cultured from 1 O-day-old chick embryos. These ceils were incubated with Ca” fluorescent dye indo-l for 30 minutes and washed for 15 minutes. Fluorescence images at 405nm and 480nm were obtained on the same image intensified CCD camera (ARGUS 50, Hamamatsu Photonics) as fast as 60 images/set. After geometric correction, the ratio of 405nrrtI480nm fluorescence was calculated, then frame-by-frame digital color imaging of [Ca**]i was obtained. Time course of [Ca’+]i transient in a single cell was also calculated from the digitalized data. This was consistent with previous results by photomultiplier tube method. This system may become a useful tool for investigating intracellular calcium handling and distribution in excitable cells. 5.134