Interactions at the NH2-terminal interface of cardiac troponin I modulate myofilament activation.

Cardiac troponin I (cTnI) is an essential element in activation of myofilaments by Ca2+ binding to cardiac troponin C (cTnC). Yet, its role in transduction of the Ca2+ binding signal to cardiac troponin T (cTnT) and tropomyosin-actin remain poorly understood. We have recently discovered that regions of cTnI C-terminal to a previously defined inhibitory peptide are essential for full inhibitory activity and Ca(2+)-sensitivity of cardiac myofilaments (Rarick et al., 1997). However, apart from its role in structural binding to cTnC, there is little knowledge concerning the role of the N-terminus of cTnI in the activation and regulation of cardiac myofilaments. To address this question, we generated wild-type mouse cardiac TnI (WT-cTnI; 211 residues) and two N-terminal deletion mutants of mouse cTnI, cTnI54-211 (missing 53 residues), and cTnI80-211 (missing 79 residues). The cTnI54-211 mutant retained the ability to bind to cTnT, but lost the ability to bind to cTnC, whereas the cTnI80-211 mutant lost the ability to bind to cTnT, but bound weakly to cTnC. Both mutants bound to F-actin. In the absence of Ca2+, cTnI54-211 was able to inhibit the unregulated MgATPase activity of myofibrils lacking endogenous cTnI-cTnC to the same extent as WT-cTnI, whereas cTnI80-211 had some impairment of its inhibitory capability. Reconstitution with cTnI54-211/cTnC complex did not restore Ca(2+)-activation of myofibrillar MgATPase activity at all, however, the cTnI80-211/cTnC complex restored Ca(2+)-activation to nearly 50% of that obtained with WT-cTnI/cTnC. These data provide the first evidence of a significant function of a cTnT-binding domain on cTnI. They also indicate that the structural cTnC binding site on cTnI is required for Ca(2+)-dependent activation of cardiac myofilaments, and that cTnT binding to the N-terminus of cTnI is a negative regulator of activation.

Troponin and tropomyosin: proteins that switch on and tune in the activity of cardiac myofilaments.

We present a current perception of the regulation of activation of cardiac myofilaments with emphasis on troponin (Tn) and tropomyosin (Tm). Activation involves both a Ca2+-regulated molecular switch and a potentiated state, dependent on feedback effects of force-generating crossbridges. Recent developments in the elucidation of the structure and arrangement of the myofilament proteins offer insights into the molecular interactions that constitute the switching and potentiating mechanisms. Transgenic mice overexpressing myofilament proteins, in vitro studies of mutant myofilament proteins, multidimensional multinuclear nuclear magnetic resonance, and fluorescence resonance energy transfer offer important approaches to understanding the molecular signaling processes. These studies reveal special features of the cardiac myofilament proteins that appear specialized for the unique functions of the heart. An important aspect of these special features is their role in mechanical, chemical, and neurohumoral coupling processes that tune myofilament activation to hemodynamics and beating frequency. Understanding these processes has become essential to understanding cardiac pathologies such as heart failure, ischemia and reperfusion injury, stunning, and familial hypertrophic cardiac myopathies.

The C terminus of cardiac troponin I is essential for full inhibitory activity and Ca2+ sensitivity of rat myofibrils.

Although the C terminus of troponin I is known to be important in myofilament Ca2+ regulation in skeletal muscle, the regulatory function of this region of cardiac troponin I (cTnI) has not been defined. To address this question, the following recombinant proteins were expressed in Escherichia coli and purified: mouse wild-type cTnI (WT cTnI; 211 residues), cTnI-(1-199) (missing 12 residues), cTnI-(1-188) (missing 23 residues), and cTnI-(1-151) (missing 60 residues). The inhibitory activity of cTnI and the mutants was tested in myofibrils, from which cTnI.cTnC was extracted by exchanging endogenous cardiac troponin with exogenous cTnT causing the Ca2+ sensitivity of the myofibrils to be lost. Addition of increasing amounts of exogenous WT cTnI or cTnI-(1-199) to cTnT-treated myofibrils at pCa 8 caused a concentration-dependent inhibition of the maximum ATPase activity. However, cTnI-(1-188) and cTnI-(1-151) inhibited this activity to about 75% and 50% of that of the WT cTnI, respectively. We also formed a complex of either WT cTnI or each of the mutants with cTnC, reconstituted the complex into the cTnT-treated myofibrils, and measured the Mg2+-ATPase activity as a function of pCa. We found that the cTnI-(1-188).cTnC complex only partially restored Ca2+ sensitivity, whereas the cTnI-(1-151).cTnC complex did not restore any Ca2+ sensitivity. Each cTnI C-terminal deletion mutant was able to bind to cTnC, as shown by urea-polyacrylamide gel-shift analysis and size exclusion chromatography. Each mutant also co-sedimented with actin. Our results indicate that residues 152-199 (C-terminal to the inhibitory region) of cTnI are essential for full inhibitory activity and Ca2+ sensitivity of myofibrillar ATPase activity in the heart.

An essential myosin light chain peptide induces supramaximal stimulation of cardiac myofibrillar ATPase activity.

The N-terminal region of skeletal myosin light chain-1 (MLC-1) binds to the C terminus of actin, yet the functional significance of this interaction is unclear. We studied a fragment (MLC-pep; residues 5-14) of the ventricular MLC-1. When added to rat cardiac myofibrils, 10 nM MLC-pep induced a supramaximal increase in the MgATPase activity at submaximal Ca2+ levels with no effect at low and maximal Ca2+ levels. A nonsense, scrambled sequence peptide had no effect at any pCa value. MLC-pep did not affect myosin KEDTA and CaATPase activities or actin-activated MgATPase activities in the absence or presence of tropomyosin. The MLC-pep did not alter the ability of troponin I to inhibit MgATPase activity. Moreover, when troponin I and troponin C were extracted from the myofibrils, the MLC-pep lost its ability to stimulate the ATPase rate. This effect was fully restored upon reconstitution of the extracted myofibrils with troponin I-troponin C complex. Thus, activation of MgATPase activity by the peptide required a full complement of thin filament regulatory proteins. Interestingly, the stimulatory effect occurred at a ratio of 4 peptides to 1 thin filament, suggesting that the peptide engages in a highly cooperative process that may involve activation of the entire thin filament.

Sites of interaction between rod G-protein alpha-subunit and cGMP-phosphodiesterase gamma-subunit. Implications for the phosphodiesterase activation mechanism.

In photoreceptor cells of vertebrates light activates a series of protein-protein interactions resulting in activation of a cGMP-phosphodiesterase (PDE). Interaction between the GTP-bound form of rod G-protein alpha-subunit (alpha t) and PDE inhibitory gamma-subunit (P gamma) is a key event for effector enzyme activation. This interaction has been studied using P gamma labeled with the fluorescent probe, lucifer yellow vinyl sulfone, at Cys-68 (P gamma LY) and sites of interaction on alpha t and P gamma have been investigated. Addition of alpha tGTP gamma S to P gamma LY produced a 3.2-fold increase in the fluorescence of P gamma LY. The Kd for alpha tGTP gamma S.P gamma LY interaction was 36 nM. Addition of 1 microM alpha tGDP had no effect, but in the presence of A1F4-, alpha tGDP increased P gamma LY fluorescence by 85%. When P gamma LY was reconstituted with P alpha beta to form fluorescent holo-PDE, alpha tGTP gamma S increased the fluorescence of holo-PDE with a K0.5 = 0.7 microM. Also, alpha tGTP gamma S stimulated the activity of this PDE over an identical range of concentrations with a similar K0.5 (0.6 microM). alpha tGTP gamma S enhanced the fluorescence of a COOH-terminal P gamma fragment, P gamma LY-46-87, as well (Kd = 1.5 microM). We demonstrate that an alpha t peptide, alpha t-293-314, which activated PDE (Rarick, H. M., Artemyev, N. O., and Hamm, H. E. (1992) Science 256, 1031-1033), mediates PDE activation by interacting with the P gamma-46-87 region. Peptide alpha t-293-314 bound to P gamma LY (K0.5 = 1.2 microM) as well as to the carboxyl-terminal P gamma fragment, P gamma LY-46-87 (K0.5 = 1.7 microM) as measured by fluorescence increase, while other alpha t peptides had no effect. A peptide from the P gamma central region, P gamma-24-46, blocked the interaction between alpha tGTP gamma S and P gamma LY. The Kd for alpha tGTP gamma S.P gamma-24-46 interaction was 0.7 microM. On the other hand, P gamma-24-46 had no effect on alpha t-293-314 interaction with P gamma LY. Our data suggest that there are at least two distinct sites of interaction between alpha tGTP gamma S and P gamma. The interaction between alpha t-293-314 and P gamma-46-87 is important for PDE activation.(ABSTRACT TRUNCATED AT 400 WORDS)

A site on rod G protein alpha subunit that mediates effector activation.

The heterotrimeric guanine nucleotide binding proteins (G proteins) are activated by sensory or hormone receptors. In turn, the G proteins activate effector proteins such as adenylyl cyclase, cyclic guanosine 3',5'-monophosphate phosphodiesterase (cGMP PDE), phospholipase C, and potassium and calcium ion channels by mechanisms that are poorly understood. A site on the alpha subunit of the G protein transducin (alpha t) has been identified that interacts with and activates cGMP phosphodiesterase, the effector enzyme in rod photoreceptors. A 22-amino acid peptide, corresponding to residues 293 to 314 from the COOH-terminal region of alpha t, fully mimicked alpha t and potently activated PDE. This region is adjacent to the receptor activation domain; thus, the alpha subunit of this G protein has a site for interaction with both its effector and receptor that maps near the COOH-terminus.

Differential limb regeneration in diploid and triploid Rana pipiens larvae with reference to spinal motor neuron development.

Developmental manipulations that can alter nerve-limb relationships can assist in understanding the neural control of limb regeneration. Pressure-induced triploidy in Rana pipiens tadpoles results in alterations of the quantitative characteristics of the spinal motor neurons that innervate the limbs, whereas the limbs appear unaltered. Unilateral midthigh amputations at larval stages IX, XI, and XIII of diploid and triploid animals resulted in complete regeneration for only stage IX animals regardless of ploidy. Nevertheless, triploid limbs regenerated much faster than did diploids, an event that can be related to the differential dynamics of nerve fiber extension and/or the altered numbers and sizes of triploid spinal motor neurons. Although normal limb development from stage IX to the endpoint at stage XVIII was the same in diploids and triploids, the rate of regeneration in triploids was nearly twice that of diploids. The data of this noninvasive means of altering the quantitative relationship of nerve-to-peripheral target suggest a unique means of studying nerve-dependent limb regeneration in an animal that progressively loses its regenerative capability during development.