Thirty-third Annual Meeting February 12 - 16, 1989 Albert B. Sabin Convention Center Cincinnati, Ohio

1989 Biophysical Journal  
Fluctuation spectroscopy and transient analysis of temporal variations of spatial distributions of fluorescent molecular markers of receptors and ligands map receptor distributions and probe the dynamics of their motion and their reactions. Bright markers and electro-optical image analysis facilitate tracking of the motion of individual receptor molecules on the cell surface. We find that congestion of the dense populations of proteins on cell surfaces alters the statistical thermodynamics of
more » ... ese 2-dimensional solutions, and we infer that diffusion is impeded; consequently, the dynamics of transmembrane signals generated by the binding of extracellular ligands to sparse cell surface receptors appear to be slowed. Fluorescent indicators of intracellular chemistry, second messenger activity, pH and polarizable ion concentration can map the dynamics of the diverse cellular responses to membrane signals. They have revealed spatial waves, temporally coherent spatially inhomogeneous limit cycle oscillations of second messengerq and complex transient vesicle traffic. As time permits we shall illustrate these cellular dynamics with recent results of research in our group and our collaborators sponsored by the NSF, NIH, ONE and the Biotechnology Program at Cornell. We have applied optical microscopy based on light emission (prompt and delayed fluorescence, phosphorescence) in studies of cellular structures and of processes during the cell cycle, differentiation, and development. Two new technologies have been used [1]: (i) a high performance scientific CCD camera system, and (ii) a confocal laser scanning system. A new method (photobleaching FRET-Digital Imaging Microcopy, pbFRET-DIM) has been developed for spatially resolved proximity measurements by exploiting the high dynamic range, sensitivity, and linearity of the CCD camera in the determination of resonance energy transfer (FRET). Only images of the donor emission before and during photobleaching are required to calculate energy transfer images. Data have been acquired for lectin binding sites, cell surface components involved in exocytosis [2], and for chromatin. The CCD camera has also been adapted for the measurement of delayed luminescence (fluorescence, phosphorescence) by incorporating choppers in the excitation and emission paths of the microscope. Sites of DNA replication in living cells can be visualized by this technique. Confocal laser scanning microscopy offers a number of advantages for the localization and quantitation of fluorescence labeled targets and probes. Most important is the rejection of interfering signals emanating from out-of-focus and adjacent structures. The "optical sectioning" of the specimen and 3-D reconstruction of numerous cellular structures and systems have been carried out using laser excitation from 351 to 528 nm. References: [1] T. . Previous studies have shown that the carbon nuclei in glycogen are 100% visible in 13C NMR despite its high molecular weight. This has been used to interpret a variety of animal studies, following glycogen synthesis and degradation in rat hearts and livers. In humans we have been able to observe glycogen in both liver and muscle in the natural abundance 13C NMR spectra. In both tissues the signal-to-noise of the glycogen Cj peak was -30 at 2.1T, in 13 minutes of accumulation, while the muscle glycogen peak with a small coil was "40:1 at 4.7T. It has been possible to follow hepatic glycogen repletion after a fast and muscle glycogen depletion during aerobic exercise. In the exercise experiments the correlation of glycogen consumption with fatigue is being investigated. 1-13C (99% enriched) and insulin have been infused into humans to the hyperglycemic, hyperinsulemic conditions needed for muscle glycogen synthesis. Under these conditions normal healthy subjects start glycogen synthesis within 25 minutes while the type II diabetics do not start glycogen synthesis until much later. The 1-13C glycogen peak gives the rate of glycogen synthesis after quantitating its intensity and correcting for degree of labeling. 4322 Biophysical Journal vol. 55, 1989 PROBING CELLULAR DYNAMICS WITH SPECTROSCOPY W-PM-Sym4 spIN IABEL auCEf. James S. wdel, Witold X. sub9yski, arn Aidhi I i3 lNaticnal Biatedical ESR Oenter, Spin Label Oximetryr is the measurement of axygen ntration, diffusion, arni collisin fr by the effect of Heisenberg exctange betwen le lar xygen ard nitrmoide radical spin labels on the EFR pr ties of the spin labels. Four aspects will be discussed. (1) Cellular respiration. Bimlecular collisions of oxygen with labels that uniformly sample the ectracellular medium permit the detenrination of the effective VMax and KM. (2) The permability of the cell a-a ns to axygen. Usirre sr ets of the bimolecular collision ny y of oxygen with lipid-type labels, the permeability coefficient has been calculated. (3) The effect of bimolecular colli-sions with spin labels is to change the effective spin-lattice relaxa time T1 of the label. The ability to change T1 without affecting other rates in a heterogeneous system has been found to be useful in measurir these other rates. In this third aspect, oxygen serves as a purely technical means for changin T1. (4) We cbserve a wide range of bimolecular oollision frequencies of oxygen with spin labels at various sites in membrane proteins, which may yield information on protein dynamics. There is an analogy between spin label ocimetry and the stiuy of bimDllar collisions of paramagnetic metals with spin labels. Also, collisions between 14Nand 5N-cntainiM spin labels can be m d with our tniques. We are, in fact, develcp1M an EEbased alterative to fluorescence methodology. W-PM-Sym-5X-RAY SPECTROSCOPY AND MAPPING OF SUBCELLULAR COMPOSITION, A.P. Somlyo, PA Musc. Inst. Univ. of PA, Phila., PA, and Dept. of Physiol., Univ. of VA, Charlottesville, VA. X-ray spectra generated by irradiation of a microvolume vith fast electrons provide information about its composition at a spatial resolution of at least lOnm (1-2). In conjunction with rapid freezing and cryoultramicrotomy, electron probe microanalytic spectroscopy (EPMA) permits the in situ measurement of the composition of cell organelles without the translocation of diffusible elements that occur during cell fractionation. The practically attainable limit of detectability (for calcium) in ultra thin cryosections is 0.3mmol/kg (3). The collection of spectra from individual object points, while scanning the electron beam, can be used to generate X-ray maps showing the elemental distribution within cells and organelles. These methods have been used to answer the question whether mitochondri! or the endoplasmic reticulum play a significant role in the regulation of cytoplasmic Ca in nonmuscle cells, to quantitate the monovalent ionic composition of the sarcoplasmic reticulum (SR) and the ionic movements associated with calcium release during contraction in striated (e.g. 4) and in smooth muscle, and to measure subcellular ionic composition related to diverse problems such as visual transduction, pathology of mitochondrial calcium and magnesium transport, localization of calcium in sickle cells, eurokarytic and bacterial membranes and spores. Some of the results of these studies and brief summary of further potential of X-ray and electron energy loss spectroscopy in biology will be presented. Supp. by grant HL15835 to PA Musc Inst. 1) Somlyo, Bacteriorhodopsin is an intrinsic membrane protein that is found in the purple membrane of Halobacteriumz halobium. Light absorption by its all-trans retinal prosthetic group drives the pigment through a photoreaction cycle that pumps protons outside the cell. The primary photochemical reaction is an excitedstate, 13-trans to 13-cis double bond isomerization of the chrcmophore that has recently been directly cbserved using femtosecond (10-15 s) transient absorption spectroscopy (Mathies et al., Science 240:777, 1988). The elucidation of the structure of retinal in the intermediates of bacteriorhodopsin is a prerequisite for defining the molecular mechanism of proton pumping. To accomplish this we have used time-resolved resonance Raman spectrosccpy to obtain vibrational spectra of the chrcmophore in bacteriorhodopsin's intermediates. Our recent work has focused on the structure of the chramophore in the M and N intermediates to define the molecular mechanism of the "reprotonation switch" which controls the connectivity of the Schiff base proton. The M intermediate contains a 13-cis, 14-s-trans, 15-anti Schiff base chrcmophore (Ames et al., Biochem., submitted), and the N intermediate is formed by simply protonating M (Fodor et al., Biochem. 27:7097, 1988). These results eliminate chranophore based reprotonation switch mechanisms such as C14-C1s bond rotation or inversion of the Schiff base nitrogen. An alternative "C-T Model" for the reprotonation switch in bacteriorhodopsin has been developed which is consistent with the known chrcznophore structures (Fodor et al., Biochem. 27:7097, 1988). This model utilizes isanerization-driven protein conformational changes between a high-free energy "C-form" and a low-free energy "T-form" to regulate the connectivity of the Schiff base proton. This work, together with the results of linear dichroism experiments which indicate that the Schiff base N-H bond points toward the cytoplasm (S.W. Lin and R.A. Mathies, Biophys. Soc. abstract), will be used to discuss possible proton purging mechanisms. Coupling of ATP hydrolysis to the pumping of Ca2+ by the Ca ATPase of SR can be accounted for by changes in the chemical and vectorial specificities for catalysis in different chemical states of the enzyme: E Ca2 catalyzes reversible phosphoryl transfer between ATP and the enzyme and E catalyzes reversible phosphoryl transfer between Pi and the enzyme; the free enzyme binds and dissociates cytoplasmic Ca2+ while phosphoenzyme dissociates and binds calcium inside the vesicle. Phosphorylation of the enzyme by bound ATP requires the binding of 2 Ca2+ and the change in specificity to dephosphorylation by water requires prior dissociation of 2 Ca2+, consistent with the stoichiometry of 2 Ca2+ transported per ATP hydrolyzed. Phosphorylation by ATP occurs in two steps: a ratelimiting conformational change of E ATP Ca2 followed by rapid, reversible phosphoryl transfer. Rapid phosphoryl transfer to ADP in the reverse direction gives a burst of phosphoenzyme disappearance and change in the direction of Ca2+ dissociation. Binding of exterior Ca2+ and phosphorylation by ATP are not inhibited by 20 mM interior Ca2+, which might be expected to form "E2-Ca2' with Ca2+ bound to interior sites. Conformational changes occur in most steps of the reaction. Binding and dissociation of cytoplasmic Ca2+ do not proceed through a free, highaffinity intermediate "E1". These and other properties of the reaction cycle are not consistent with the usual definitions of "E1-E2" and related models for active transport. Functional studies of the sarcoplasmic reticulum (SR) ATPase provide characterization of the catalytic and transport cycle, and allow mechanism-linked analysis of transport energetics. Another important aim is to unveil the roles of specific residues and of the protein conformation in the mechanism of vectorial catalysis. The substrate site is localized by ATP binding inhibition following FITC derivatization of Lys515, and by phosphoryl transfer from ATP to Asp7351. Fluorescence energy transfer measurements with the ATPase covalently labeled at Cys670, Cys674, Cys344, Cys364 and Lys515, as well as TNP-AMP and Pr3+ bound to the nucleotide and calcium sites respectively, suggest that the cytoplasmic portion of the ATPase is folded to formft a crevice for substrate binding. The nucleoside binding moiety of this site is not required for energy transduction, while phosphorylation of Asp351 is crucial. No large changes in secondary or tertiary structure within the cytoplasmic portion of the ATPase have been detected upon enzyme activation. Rather, fluorescence characterization of several tryptophan residues in hydrophobic regions demonstrates an increase of lifetimes and loss of anisotropy upon calcium binding. This reversible phenomenon, likely due to stabilization of clustered membrane hel ices, suggests that an interactive channel is involved in vectorial translocation of calc:ium. Involvement of hydrophobic domains is also evidenced by carbodiimide derivatization. The sensitivity of the transmembrane helices to calcium binding, and the reverse effect by enzyme phosphorylation, constitute an important device in the coupling mechanism. 434a The outstanding function-structure problem of cation pumps such as the Na/K-, H/K-and Ca-ATPases is the structure of the cation binding sites and detailed mechanism of cation binding, occlusion and movement through the protein. These pumps show a high degree of primary sequence homology and a similar predicted trans-membrane organization. The recent structural knowledge has lead to interesting (and some wild) speculation as to the nature of cation binding sites but there is as yet little or no real evidence. I shall present experiments utilizing renal Na/K-ATPase and reconstituted vesicles, in conjunction with sensitive assays of cation transport and occlusion, which provide initial information as to the nature of cation binding residues. These include observations on electrogenic potentials and voltage-sensitivity of the reconstituted pumps, and inactivation of Rb(K) and Na occlusion on Na/K-ATPase by the hydrophobic carbodiimide DCCD. Together the experiments suggest that the cation binding and transporting domain of the Na/K-pump contains two negatively charged residues (asp,glu) in a non-aqueous environment and furthermore that the same residues are involved in binding, occlusion and transport of both Na and K(Rb) from either surface. Known features of the cation sites and trans-membrane path will be summarized and where possible these will be compared for the different cation pumps. W-PM-Min-5 LIGANDS CONTROL THE Na/K PUMP'S TRANSPORT REACTION MECHANISM. . I reported previously that injection of 1,4,5 inositol trisphosphate (IP3) caus-ed an increase in cytoplasmic Ca (Cai) that was spatially restricted to certain areas of identified Aplysia bursting neurons (Biophys J 51:424a, 1987). Since I found that the site of largest IP3-induced Ca1 increase wAs generally near the plasma membrane, an important question is whether the sites of mobilized Ca'+ and Ca2+ influx are physically separate. Using double-barrelled Ca selective electrodes and a puffer electrode filled with high K+-seawater, I induced a Ca1 increase by either pressure-injecting IPN intracellularly (using the 2nd barrel of the double-barrelled Ca electrode) or puffing high K+ extracellularly. Both Ca increases were measured at the same site by the Ca selective barrel. The Ca1 increase induced by high K+ was found to be much slower than the 1P2-induced Ca increase. The slower Ca1 increase probably reflects diffusion and buffering pf the Ca + influx fol1lowing depolarization by high K+. This result could imply that entering Ca'+ and mobilized Ca2+ are intended to reach different target molecules. Injection of IP3 was also found to induce distinct changes in holding current (Levy, J Gen Physiol 92:2a, 1988; Scholz et al, J Neurophysiol 60:86, 1988). To investigate whether the changes in holding current were primary or mediated by the Ca2+ released by IP3, we used double-barrelled injecting electrodes which allow delivery of either Ca2+ or IP3 at the same point ( this is because the effects of IP3 were found to be depthand neuron-dependent). Side-by-side comparison of the currents induced by IP3 and Ca4+ show that in most cells they are different both in waveform and time course: for example a rapid inward current induced by IP3 versus an outward current induced by Ca2+. This result implies that changes in membrane currents induced by IP3 are not necessarily caused by mobilized Ca2+. Measurements of the Ca2+ currents in cultured rat pituitary gonadotrophs were made using patch clamp techniques. Analysis of the inward Ca2+ currents, recorded in the presence of 5.2 mM Ca2+ (or Ba2+), revealed a fast component, with activation-inactivation kinetics, and a delayed one with slower activation. The rate of inactivation of the first component was found to be voltage dependent. At -44 mV, a 5.5 mV change in potential induced an e-fold change in the fraction of Ca+ channels available to conduct Ca2+ current. During long-lasting (100-200 msec) low-frequency depolarizing voltage clamp pulses, the size of the delayed component of the Ca2+ current remained constant. A comparison of the time constants for turning off the Ca2+ conductance showed that for brief pulses the tail currents could be described as the sum of two exponentials. For pulses of long duration (100 msec) a single exponential fitted the time course of the current tails. The differential effects of membrane potential on inactivation, and the different time constants for turning off the Ca2+ conductance, suggest the presence of two types of calcium channels in the membrane of the gonadotroph. We propose that the Ca2+ channel with activation-inactivation kinetics plays a major role in the control of Ca2+ entry required for the physiological response of the cell. W-PM-A3 CALCIUM CURRENTS IN RAT PANCREATIC B-CELLS IN CULTURE. J. Hidalgo, M.-Y. Li, I. Atwater and E. Rojas. LCBG, NIH, Bethesda, MD 20892 (Introd. bv V. Cena). The patch voltage clamp technique was used to measure Ca2+ currents across the membrane of rat pancreatic B-cells. Cell cultures maintained for 2-10 days in a medium (CMRL 1066 from Gibco) supplemented with glucose (5.6 mM) secreted insulin in response to the sugar (11-33 mM) and to high K+ (50 mM) in a bicarbonate-buffered Krebs solution at 37 OC. Whole cell membrane currents (pipet solution (mM): 70 CsCl, Cs-glutamate, 10 Cs-Pipes, 5 Mg-ATP, 10 Cs-EGTA at pH 7) exhibited two distinct components, an early transient one and a delayed non-inactivating one. In the presence of a modified Krebs solution (mM: 120 Choline-Cl or TMA-Cl, 5 KC1, 1 MgCl2, 2-6 CaC12, 5 CsHepes, pH = 7.4), the mid-point for the activation of the component with activation-inactivation kinetics (Ca-l) was ca. -45 mV and that of the delayed component (Ca-2) was 10 mV. With Ba2+ in place of Ca2+ in the external solution, only the delayed, non-inactivating, component was present in the records. The mean value of the maximum transient Ca2+ current (21 I-V curves from 5 cells) was -75 pA (Ca2+ = 4 mM) and that of the noninactivating component was -125 pA. In the presence of glucose (11 mM) in the external solution single Ca2+-channel currents (cell-attached configuration) measured with high (100 mM) Ca2+ or Ba2+ in the pipet ranged between 0.5 and 1.1 pA. Therefore, from the ratio "maximum whole cell membrane current/single Ca2+-channel current" (i.e. 200/0.5) we estimate that an islet B-cell is equipped with about 400 Ca2+-channels. Differentiated PC12 cells and sympathetic neurons show a non-inactivating, dihydropyridine (DHP)-sensitive current at relatively positive holding potentials (40 mV) and a partially inactivating, DHP-inensitive current at more negative holding potentials (-90 mV) when 20 mM Ba2+ is the charge carrier. 16 pMw-Conotoxin (GV1A) inhibits the barium currents (by 50% in PC12 and by >70% in neonatal day 1-3 srmpathetic neurons) and reveals three components: 1) Conotoxin-sensitive; 2) Conotoxin-resistant, DHP-sensitive, non-inactivating; and 3) Conotoxin-resistant, DHP-resistant, inactivating. The proportions of these three currents differ in the PC12 and sympathetic cells, but all three components can be found. Single channel recordings reveal at least two types of calcium channels. The first is the U-type channel (conductance = 26 pS, DHP-sensitive). The second channel is DHP resistant and is strongly inactivated at positive holding potentials but differs from the previously described N-type channel by its larger conductance (19-21 pS) and its much slower rate of inactivation. The difference in conductance may be explained by our finding that this channel can, especially in the sympathetic neurons, spend prolonged periods of time in a subconductance state of approximately 13 pS (arrows). 20 mV L-type NSF grants DMB-8517812 and DMR-8806975 (S.K.) and NIH grant 1ROlGM30741-07 (S.A.A.). Efforts to establish structure-function relationships involving nucleic acids have focused attention upon a variety of non-B conformations of DNA, for example A-, Z,-, bent, and hairpin-looped DNA. When such features are embedded within B-DNA, as would be the case in vivo, spectroscopic structural assignment is complicated because the region of interest constitutes only a sn,all portion of the macromolecule. We have developed a new nitroxide spin labeled analog of thy*ine (T ) which may be incorporated by automated chemical synthesis into deoxyoligonucleotides. EPR spectra of the,spin labeled synthetic
doi:10.1016/s0006-3495(89)82829-2 fatcat:i4evaytibbhq7pzf4zcfox2bp4