Conditions Of Low Ca2 Research Articles

Potential impacts of the cardiac troponin I mobile domain on myofilament activation and relaxation

The cardiac thin filament is regulated in a Ca2+-dependent manner through conformational changes of troponin and tropomyosin (Tm). It has been generally understood that under conditions of low Ca2+ the inhibitory peptide domain (IP) of troponin I (TnI) binds to actin and holds Tm over the myosin binding sites on actin to prevent crossbridge formation. More recently, evidence that the C-terminal mobile domain (MD) of TnI also binds actin has made for a more complex scenario. This study uses a computational model to investigate the consequences of assuming that TnI regulates Tm movement via two actin-binding domains rather than one. First, a 16-state model of the cardiac thin filament regulatory unit was created with TnI-IP as the sole regulatory domain. Expansion of this to include TnI-MD formed a 24-state model. Comparison of these models showed that assumption of a second actin-binding site allows the individual domains to have a lower affinity for actin than would be required for IP acting alone. Indeed, setting actin affinities of the IP and MD to 25% of that assumed for the IP in the single-site model was sufficient to achieve precisely the same degree of Ca2+ regulation. We also tested the 24-state model's ability to represent steady-state experimental data in the case of disruption of either the IP or MD. We were able to capture qualitative changes in several properties that matched what was seen in the experimental data. Lastly, simulations were run to examine the effect of disruption of the IP or MD on twitch dynamics. Our results suggest that both domains are required to keep diastolic cross-bridge activity to a minimum and accelerate myofilament relaxation. Overall, our analyses support a paradigm in which two domains of TnI bind with moderate affinity to actin, working in tandem to complete Ca2+-dependent regulation of the thin filament.

The concerted movement of the switch region of Troponin I in cardiac muscle thin filaments as tracked by conventional and pulsed (DEER) EPR.

The absence of a crystal structure of the calcium free state of the cardiac isoform of the troponin complex has hindered our understanding of how the simple binding of Ca2+ triggers conformational changes in troponin which are then propagated to enable muscle contraction. Here we have used continuous wave (CW) and Double Electron-Electron Resonance (DEER) pulsed EPR spectroscopy to measure distances between TnI and TnC to track the movement of the functionally important regulatory 'switch' region of cardiac Tn. Spin labels were placed on the switch region of Troponin I and distances measured to Troponin C. Under conditions of high Ca2+, the interspin distances for one set (TnI151/TnC84) were 'short' (9-10Å) with narrow distance distribution widths (3-8Å) indicating the close interaction of the switch region with the N-lobe of TnC. Additional spin populations representative of longer interspin distances were detected by DEER. These longer distance populations, which were ∼16-19Å longer than the short distance populations, possessed notably broader distance distribution widths (14-29Å). Upon Ca2+ removal, the interspin population shifted toward the longer distances, indicating the release of the switch region from TnC and an overall increase in disorder for this region. Together, our results suggest that under conditions of low Ca2+, the close proximity of the TnI switch region to TnC in the cardiac isoform is necessary for promoting the interaction between the regulatory switch helix with the N-lobe of cardiac Troponin C, which, unlike the skeletal isoform, is largely in a closed conformation.

Step-Wise Truncation of the C-Terminal 14 Residues of Troponin T Reduces the B State of Regulated Actin and Enhances the M State

Actin stimulates the ATPase activity of myosin-S1. The regulatory proteins tropomyosin and troponin prevent this stimulation at conditions of low Ca2+ and low high-affinity myosin binding. At saturating Ca2+ and with strong actin-binding species of myosin (S1-ADP or NEM-S1) the enhancement of ATPase activity exceeds that seen in the absence of regulatory proteins. Inhibition and potentiation of actin activated ATPase activity are dependent on the position of tropomyosin on actin filaments. One position of tropomyosin (M state) leads to high activity whereas the other two positions (B and the intermediate C state) are inactive.We recently showed that the C-terminal 14 residues of troponin T are critical for formation of the inactive B state at low Ca2+. Furthermore, that same region of troponin T limits the formation of the M state at saturating Ca2+. Our present goal is to map the C-terminal region of troponin T to determine if specific residues in the sequence are responsible for each of these activities. The B state was monitored by changes in acrylodan tropomyosin fluorescence and by the rate of rigor S1 binding to regulated actin. The M state was monitored by an ATPase assay. Stepwise deletions within the C-terminal 14 amino acids of Troponin T progressively reduced the population of the inactive B state at low Ca2+. In contrast, the population of the M state was progressively increased at high Ca2+. The net positive charge of the last 14 residues appeared to be a key determinant of activity. Conclusion: The C-terminal region of troponin T plays essential roles in inactivation and in limiting activation. Both of these activities require basic amino acid residues within the COOH terminus.

Voltage Dependence of a Neuromodulator-Activated Ionic Current.

The neuromodulatory inward current (IMI) generated by crab Cancer borealis stomatogastric ganglion neurons is an inward current whose voltage dependence has been shown to be crucial in the activation of oscillatory activity of the pyloric network of this system. It has been previously shown that IMI loses its voltage dependence in conditions of low extracellular calcium, but that this effect appears to be regulated by intracellular calmodulin. Voltage dependence is only rarely regulated by intracellular signaling mechanisms. Here we address the hypothesis that the voltage dependence of IMI is mediated by intracellular signaling pathways activated by extracellular calcium. We demonstrate that calmodulin inhibitors and a ryanodine antagonist can reduce IMI voltage dependence in normal Ca2+, but that, in conditions of low Ca2+, calmodulin activators do not restore IMI voltage dependence. Further, we show evidence that CaMKII alters IMI voltage dependence. These results suggest that calmodulin is necessary but not sufficient for IMI voltage dependence. We therefore hypothesize that the Ca2+/calmodulin requirement for IMI voltage dependence is due to an active sensing of extracellular calcium by a GPCR family calcium-sensing receptor (CaSR) and that the reduction in IMI voltage dependence by a calmodulin inhibitor is due to CaSR endocytosis. Supporting this, preincubation with an endocytosis inhibitor prevented W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride)-induced loss of IMI voltage dependence, and a CaSR antagonist reduced IMI voltage dependence. Additionally, myosin light chain kinase, which is known to act downstream of the CaSR, seems to play a role in regulating IMI voltage dependence. Finally, a Gβγ-subunit inhibitor also affects IMI voltage dependence, in support of the hypothesis that this process is regulated by a G-protein-coupled CaSR.

The Transcription Factors Islet and Lim3 Combinatorially Regulate Ion Channel Gene Expression

Expression of appropriate ion channels is essential to allow developing neurons to form functional networks. Our previous studies have identified LIM-homeodomain (HD) transcription factors (TFs), expressed by developing neurons, that are specifically able to regulate ion channel gene expression. In this study, we use the technique of DNA adenine methyltransferase identification (DamID) to identify putative gene targets of four such TFs that are differentially expressed in Drosophila motoneurons. Analysis of targets for Islet (Isl), Lim3, Hb9, and Even-skipped (Eve) identifies both ion channel genes and genes predicted to regulate aspects of dendritic and axonal morphology. Significantly, some ion channel genes are bound by more than one TF, consistent with the possibility of combinatorial regulation. One such gene is Shaker (Sh), which encodes a voltage-dependent fast K(+) channel (Kv1.1). DamID reveals that Sh is bound by both Isl and Lim3. We used body wall muscle as a test tissue because in conditions of low Ca(2+), the fast K(+) current is carried solely by Sh channels (unlike neurons in which a second fast K(+) current, Shal, also contributes). Ectopic expression of isl, but not Lim3, is sufficient to reduce both Sh transcript and Sh current level. By contrast, coexpression of both TFs is additive, resulting in a significantly greater reduction in both Sh transcript and current compared with isl expression alone. These observations provide evidence for combinatorial activity of Isl and Lim3 in regulating ion channel gene expression.

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis.

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.

GTP and noradrenaline‐induced force in isolated toxin‐permeabilized rat anococcygeus and guinea‐pig portal vein.

1. Strips of smooth muscle from rat anococcygeus and guinea-pig portal vein were treated with solutions containing crude alpha-toxin from the bacterium Staphylococcus aureus. This rendered the surface membrane permeable to small molecular weight substances, but left functional sarcolemmal adrenoceptors. Tension measurements from these preparations were used to investigate the effects of guanosine-5'-triphosphate (GTP) on the noradrenaline-induced Ca2+ release from the sarcoplasmic reticulum (SR) of the smooth muscle of rat anococcygeus and guinea-pig portal vein. 2. Under conditions of low Ca2+ buffering (0.2 mM-EGTA), applying a maximal dose of noradrenaline (30 microM) to a toxin-permeabilized strip of anococcygeus muscle and longitudinal muscle of guinea-pig portal vein caused a transient contracture. Subsequent exposures to noradrenaline resulted in progressively smaller contractures. However, the rate of decline in the size of the noradrenaline-induced contracture was greater in rat anococcygeus muscle than in guinea-pig portal vein preparations. The decline in the size of the contracture in toxin-permeabilized anococcygeus muscle was not due to a fall in the Ca2+ content of the SR or a reduced Ca2+ release from the SR in response to myo-inositol 1,4,5-trisphosphate (IP3). 3. The tension transients due to noradrenaline were enhanced and maintained in the presence of 100 microM-GTP in toxin-permeabilized guinea-pig portal vein. Addition of 100 microM-GTP caused a transient contracture in permeabilized rat anococcygeus muscle and only promoted the next noradrenaline response, thereafter the amplitude of the contractures decayed to zero. 4. Addition of guanosine-5'-O-(2 thiodiphosphate) (GDP-beta-S, 100 microM) would be expected to cause a reversible reduction of the noradrenaline response by binding to the intermediary G-protein. This was observed in toxin-permeabilized portal vein, but in rat anococcygeus muscle, GDP-beta-S caused slowing of the response to noradrenaline, thereafter the response to noradrenaline was absent. The noradrenaline response did not recover when GDP-beta-S was removed. 5. The non-metabolizable form of GTP, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S, 100 microM), caused a transient contracture in both toxin-permeabilized rat anococcygeus muscle and guinea-pig portal vein. In both these tissues, the addition of GTP-gamma-S resulted in the irreversible inhibition of the response to noradrenaline. 6. In the presence of a high concentration (10 mM) of the Ca2+ buffer EGTA, GTP (100 microM) and noradrenaline (30 microM) increased Ca(2+)-activated force in both tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Potassium currents in canine airway smooth muscle cells

The electrical properties of dissociated canine tracheal smooth muscle cells were examined using the whole cell patch-clamp technique. In current clamp mode, current clamp steps did not initiate action potentials but showed clear outward rectification, which was abolished when cells were loaded with Cs+ ions and when tetraethylammonium (TEA+) ions replaced Na+ in the bath solution. In voltage-clamp experiments, depolarizations positive to -45 mV evoked brief voltage-dependent inward Ca2+ currents [Am. J. Physiol. 254 (Cell Physiol. 23): C793-C801, 1988], followed by sustained outward currents, which did not completely inactivate. Outward currents were identified as K+ currents on the basis of the reversal potential of the current and by ion-substitution experiments. The currents were further defined as Ca2(+)-insensitive delayed rectifier currents, since they were unaltered under conditions in which 1) the Ca2+ current was completely blocked by Mn2+ or nifedipine (10 microM); 2) Ba2+ ions were substituted for Ca2+ as the inward current charge carrier; or 3) charybdotoxin (40 nM) or TEA+ (up to 10 mM) were added to the bath. A Ca2(+)-activated potassium [K(Ca)] current was activated by application of methacholine (100 microM), or A23187 (1 microM), under conditions of low Ca2+ buffering capacity in the internal solution [0.3 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)]. The K(Ca) current was blocked by 10 mM TEA+ and was not observed under conditions of high intracellular Ca2+ buffering (11 mM EGTA). These data indicate that canine airway smooth muscle cells contain voltage-dependent delayed rectifier channels that underlie membrane rectification and K(Ca) channels that are activated by agents which release intracellular Ca2+ stores.