Potential risk factors for cardiovascular diseases already tend to cluster in childhood and they are strongly associated with obesity. Our observations suggest that the development of the metabolic cardiovascular syndrome has its origin in childhood.

The effects of MgADP and inorganic phosphate (Pi) on cross-bridge detachment were determined in tonic (rabbit femoral artery) and phasic (rabbit bladder and guinea pig portal vein) smooth muscles permeabilized with staphylococcal alpha-toxin. Relaxation from rigor was induced by photolysis of ATP (1.2-1.5 mM) from caged ATP. The initial one second of relaxation from rigor was resolved into two exponential components: a rapid component with normalized amplitudes, Af, of 8, 15 and 26% and rate constants, kf (in s-1) of 26, 36 and 30 in rabbit femoral artery, guinea pig portal vein, and rabbit bladder; the respective rate constants of the second, slower component, ks, were 0.07, 0.2 and 0.1. Removal of residual endogenous ADP with apyrase treatment increased the amplitude Af and accelerated ks; addition of MgADP reduced Af. The combination of these effects (increases in Af and ks) decreased the t1/2 of relaxation from control values by factors of 2.6 (femoral artery), 6.7 (portal vein) and 10 (bladder). Pi (30 mM) further increased the amplitudes Af. The affinity of MgADP for myosin cross-bridges, estimated as the reduction of the relative amplitude of the rapid component, Af, was significantly higher in tonic than in phasic smooth muscle: the KD of MgADP was 1.1 +/- 0.3 microM in rabbit femoral artery and 4.9 +/- 1.0 microM in rabbit bladder. The higher affinity of tonic smooth muscle myosin for MgADP correlated with its relatively high LC17b isoform content (58 +/- 4.2%) in contrast to the lower affinity of the phasic, bladder detrusor smooth muscle that contained only the LC17a isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

SignificanceExcitatory synapses convert presynaptic action potentials into chemical signals that are sensed by postsynaptic glutamate receptors. To eavesdrop on synaptic transmission, genetically encoded fluorescent sensors for glutamate have been developed. However, even the best available sensors lag behind the very fast glutamate dynamics in the synaptic cleft. Here, we report the development of an ultrafast genetically encoded glutamate sensor, iGluu, which allowed us to image glutamate clearance and synaptic depression during 100-Hz spike trains. We found that only boutons showing paired-pulse facilitation were able to rapidly recover from depression. Thus, presynaptic boutons act as frequency-specific filters to transmit select features of the spike train to specific postsynaptic cells.

The mechanism of the interactions of 2-chloro-(epsilon-amino-Lys75)-[6-[4-(N,N-diethylamino)phenyl]- 1,3,5-triazin-4-yl]calmodulin (TA-calmodulin) with smooth muscle myosin light-chain kinase (MLCK) and two 17-residue peptides, Ac-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-CONH2 (Trp peptide) and Tyr peptide, in which W is replaced by Y, were studied by measurements of equilibrium and transient fluorescence changes in the nanomolar range. Most reactions were carried out in 100 microM CaCl2 at ionic strength 0.15 M, pH 7.0, and 21 degrees C. In each case association of MLCK or peptide to TA-calmodulin could be described by a two-step process, a bimolecular step and an isomerization. In the case of the interaction between TA-calmodulin and Tyr peptide it was shown that the isomerization involved the binary complex of TA-calmodulin and Tyr peptide as opposed to an isomerization of either TA-calmodulin or Tyr peptide in isolation. These distinctions depended in part on development for transient kinetic experiments of a general theory to quantify relative phase amplitudes in two-step mechanisms. The kinetics for all three association reactions were then interpreted in terms of a bimolecular association (rate constants k+1 and k-1) followed by an isomerization of the binary complex (rate constants k+2 and k-2). For the interaction of TA-calmodulin and Tyr peptide, values of the rate constants are k+1, 8.8 x 10(8) M-1 s-1; k-1, 5.7 s-1; k+2, 0.38 s-1; and k-2, 0.65 s-1. The fluorescence intensities (lambda ex 365 nm, lambda ex 365 nm, lambda em > 400 nm) of TA-calmodulin, the initial binary complex of TA-calmodulin and Tyr peptide, and the isomerized binary complex are in the ratio 1:2.8:1.3. Analogous mechanisms were found for TA-calmodulin binding to Trp peptide and to MLCK, but values for the rate constants and relative fluorescence intensities of the binary complexes were generally not so completely defined. Values for the Trp peptide and MLCK, respectively, are k+1, 8.8 x 10(8) M-1 s-1 and 1.1 x 10(8) M-1 s-1; (k+2 + k-2), 0.97 s-1 and 1.3 s-1; and k-1k-2/(k+2 + k-2), 0.0079 s-1 and 0.025-0.056 s-1. Equilibrium dissociation constants (Kd) for interactions of TA-calmodulin and targets determined from these data are Tyr peptide, 4.1 nM; Trp peptide, 0.011 nM; and MLCK, 0.23-0.51 nM.(ABSTRACT TRUNCATED AT 400 WORDS)

SUMMARY1. The effects of AMgADP on cross-bridge kinetics were investigated using laser flash photolysis of caged ATP (P3-1(2-nitrophenyl) ethyladenosine 5'-triphosphate), in guinea-pig portal vein smooth muscle permeabilized with Staphylococcus aureus atoxin. Isometric tension and in-phase stiffness transitions from rigor state were monitored upon photolysis of caged ATP. The estimated concentration of ATP released from caged ATP by high-pressure liquid chromatography (HPLC) was 1P3 mM.2. The time course of relaxation initiated by photolysis of caged ATP in the absence of Ca2+ was well fitted during the initial 200 ms by two exponential functions with tine constants of. respectively, T1 = 34 ms and T2 = 1P2 s and relative amplitudes of 0 14 and 0 86. Multiple exponential functions were needed to fit longer intervals; the half-time of the overall relaxation was 0-8 s. The second order rate constant for cross-bridge detachment by ATP, estimated from the rate of initial relaxation, was 0A4-23 x 104 M-1 s-1.3. MgADP dose dependently reduced both the relative amplitude of the first component and the rate constant of the second component of relaxation. Conversely, treatment of muscles with apyrase. to deplete endogenous ADP, increased the relative amplitude of the first component. In the presence of MgADP, in-phase stiffness decreased during force maintenance, suggesting that the force per crossbridge increased. The apparent dissociation constant (Kd) of MgADP for the crossbridge binding site, estimated from its concentration-dependent effect on the relative amplitude of the first component. was 1P3/M. This affinity is much higher than the previously reported values (50-300 JtM for smooth muscle; 18-400 /tM for skeletal muscle: 7-10 /um for cardiac muscle). It is possible that the high affinity reflects the properties of a state generated during the co-operative reattachment cycle, rather than that of the rigor bridge.4. The rate constant of MgADP release from cross-bridges, estimated from its concentration-dependent effect on the rate constant of the second (T2) component, was 0'35-7'7 s-'. To the extent that reattachment of cross-bridges could slow relaxation even during the initial 200 ms. this rate constant may be an underestimate. § To whom all correspondence should be addressed.Ms 1080 2E. XISHIY-E AND OTHERS 5. Inorganic phosphate (Pi, 30 mM) did not affect the rate of relaxation during the initial -50 ms, but accelerated the slower phase of relaxation, consistent with a cyclic cross-bridge model in which Pi increases the proportion of cross-bridges in detached ('weakly bound') states. In the presence of 100 /tM MgADP, Pi (10-50 mM) predominantly accelerated the final slow phase of relaxation, rather than the initial (T1 and T2) components, and shortened the duration of the plateau phase that followed the initial rapid relaxation.6. Muscles, in which the myosin light chains were thiophosphorylated, relaxed during the initial 40-50 ms after photolysis of caged ATP at the same rate as muscles with non-thiophos...

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Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators.

The activation mechanism of Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) is investigated by steady-state and stopped-flow fluorescence spectroscopies. Lys(75)-labeled TA-cal [Török, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820] is used to measure binding events, and double-labeled AEDANS,DDP-T34C/T110/C-calmodulin [Drum et al. (2000) J. Biol. Chem. 275, 36334-36340] (DA-cal) is used to detect changes in calmodulin conformation. Fluorescence quenching of DA-cal attributed to resonance energy transfer is related to the compactness of the calmodulin molecule. Interprobe distances are estimated by lifetime measurements of Ca(2+)/DA-cal in complexes with unphosphorylated nucleotide-free, nucleotide-bound, and Thr(286)-phospho-alphaCaMKII as well as with alphaCaMKII-derived calmodulin-binding peptides in the presence of Ca(2+). These measurements show that calmodulin can assume at least two spectrally distinct conformations when bound to alphaCaMKII with estimated interprobe distances of 40 and 22-26 A. Incubation with ATP facilitates the assumption of the most compact conformation. Nonhydrolyzable ATP analogues partially replicate the effects of ATP, suggesting that while the binding of ATP induces a conformational change, Thr(286)-autophosphorylation is probably required for the transition of calmodulin into its most compact conformer. The rate constant for the association of Ca(2+)/TA-cal with alphaCaMKII is estimated as 2 x 10(7) M(-1) s(-1) and is not substantially affected by the presence of ATP. The rate of net calmodulin compaction measured by Ca(2+)/DA-cal is markedly slower, occurring with a rate constant of 2.5 x 10(6) M(-1) s(-1), suggesting that unproductive complexes may play a role in the activation mechanism.