Auto-inhibitory effects of an IQ motif on protein structure and function

The denuded IQ2 domain, i.e. myosin heavy chain not associated with regulatory light chains, exerts an inhibitory effect on myosin ATPase activity. In this study, we elaborated a structural explanation for this auto-inhibitory effect of IQ2 on myosin function. We employed analytical ultracentrifugation, circular dichroism, and surface plasmon resonance spectroscopy to investigate structural and functional properties of a myosin heavy chain (MYH) head-rod fragment aa664-915. MYH(664-915) was monomeric, adopted a closed shape, and bound essential myosin light chains (HIS-MLC-1) with low affinity to IQ1. Deletion of IQ2, however opened MYH(664-915). Four amino acids present in IQ2 could be identified to be responsible for this auto-inhibitory structural effect: alanine mutagenesis of I814, Q815, R819, and W827 stretched MYH(664-915) and increased 30fold the binding affinity of HIS-MLC-1 to IQ1. In this study we show, that denuded IQ2 favours a closed conformation of myosin with a low HIS-MLC-1 binding affinity. The collapsed structure of myosin with denuded IQ2 could explain the auto-inhibitory effects of IQ2 on enzymatic activity of myosin

Ahnak1 modulates L-type Ca(2+) channel inactivation of rodent cardiomyocytes

Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart

Spinophilin is required for normal morphology, Ca(2+) homeostasis and contraction but dispensable for beta-adrenergic stimulation of adult cardiomyocytes

Spinophilin (SPN) is a ubiquitously expressed scaffolding protein that interacts through several binding modules with a variety of target proteins. Thus, SPN bundles F-actin, targets protein phosphatase 1 to the ryanodine receptor, and targets regulators of G-protein signaling to G-protein coupled receptors in cardiomyocytes. In this work we studied the role of SPN on cardiomyocyte morphology, function, and beta-adrenergic responsiveness using a homozygous SPN knock-out mouse model (SPN-/-). We show that spinophilin deficiency significantly (1) reduced cardiomyocyte length, (2) increases both Ca(2+) amplitude and maximal rate of Ca(2+) rise during systole, and (3) decreased shortening amplitude and maximal rate of shortening, while (4) beta-adrenergic stimulation remained intact. Our data suggest that spinophilin is an upstream regulator required for normal growth and excitation-contraction coupling, but is dispensable for beta-adrenergic stimulation of adult cardiomyocytes

Distinct interactions between actin and essential myosin light chain isoforms

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein-protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with {alpha}-actin as well as {alpha}-actin with alanin-mutated ELC binding site ({alpha}-actin(ala3)) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of {alpha}-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to {alpha}-actin (KD = 575 nM) was significantly (p<0.01) lower compared with the affinity of hVLC-1 to {alpha}-actin (KD = 186 nM). The reduced affinity of hALC-1 to {alpha}-actin was mainly due to a significantly (p<0.01) lower association rate (kon: 1018 M(-1)s(-1)) compared with kon of the hVLC-1/{alpha}-actin complex interaction (2908 M(-1)s(-1)). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct {alpha}-actin interactions

Human essential myosin light chain isoforms revealed distinct myosin binding, sarcomeric sorting, and inotropic activity

Aims: In this paper we tested the hypothesis that different binding affinities of the C-terminus of human cardiac alkali (essential) myosin light chain (A1) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity. Methods and Results: We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of a recombinant head-rod fragments of rat cardiac beta-myosin heavy chain aa664-915 with alanin mutated IQ2 domain (rbeta-MYH(664-915)IQ(ala4)) and A1 isoforms (human atrial -hALC1- and human ventricular -hVLC-1- light chains). Double epitope tagging competition was used to monitor the intracellular localization of exogenously introduced hALC-1 and hVLC-1 constructs in neonatal rat cardiomyocytes. Contractile functions of A1 isoforms were investigated by monitoring shortening and intracellular free Ca(2+) (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using hALC-1 or beta-galactosidase as expression cassettes. hALC-1 bound more strongly (>3fold lower K(D)) to rbeta-MYH(664-915) than did hVLC-1. Sorting specificity of A1 isoforms to sarcomeres of cardiomyocytes rose in the order hVLC-1 to hALC-1. Replacement of endogenous VLC-1 by hALC-1 in adult rat cardiomyocytes increased contractility while the systolic Ca(2+) signal remained unchanged. Conclusions: Intense myosin binding of hALC-1 provides a mechanism for preferential sarcomeric sorting and Ca(2+)-independent positive inotropic activity

Reconstitution of ventricular myosin with atrial light chains-1 improves its functional properties

Atrial light chain 1 (ALC-1) is expressed in embryonic and hypertrophied human ventricles but not in normal adult human ventricles. We investigated the effects of recombinant human atrial light chains (hALC-1) on the structure and enzymatic activity of synthetic filaments of ventricular myosin. The endogenous ventricular myosin light chain 1 (VLC-1) was partially replaced by recombinant hALC-1 yielding hALC-1 levels of 12%, 24% and 42%. This reconstitution of ventricular myosin with hALC-1 did not change the length of synthetic myosin filaments but led to more rounded myosin heads in comparison with those of control filaments. Actin-activated ATPase activity of myosin, a parameter of functional activity of molecular motor, amounted to 79.5 nmol P i/ mg per min in control myosin filaments. Reconstitution with hALC-1 caused a profound increase of the actin-activated myosin ATPase activity in a dose dependent manner, for example, synthetic myosin filaments formed with 12%, 24% and 42% hALC-1 reconstituted myosin revealed the actin-activated ATPase activity increased by 18%, 26% and 36%, respectively, as compared to control. These results strongly suggest that in vivo expression of ALC-1 enhances ventricular myosin function, thereby contributing to cardiac compensation

Constriction velocities of renal afferent and efferent arterioles of mice are not related to SMB expression

Background. Constriction of renal arterioles contributes significantly to the control of perfusion and glomerular filtration. Afferent but not efferent arterioles express smooth muscle myosin heavy chain B (SMB) (with a 5′-insert of seven amino acids). The aim of the present study was to investigate (1) the constriction characteristics of afferent and efferent arterioles under physiologic load and (2) whether expression of SMB may causally contribute to these constriction characteristics. Methods. We compared constriction parameters [constriction amplitude, maximal rate of constriction velocity ("dc/dt max"), and time to half-maximal constriction (t 1/2) of in vitro perfused renal afferent and efferent arterioles of wild-type (smb(+/+)] and homozygous SMB knockout [smb(-/-)] mice upon stimulation with angiotensin II (Ang II) (10 -8 mol/L) and potassium chloride (KCl) (100 mmol/L). SMB expression was investigated by double-labeling immunofluorescence. Results. Contraction amplitude and dc/dt max of mouse afferent arterioles upon Ang II stimulation were significantly greater compared to efferent arterioles. However, constriction amplitudes, dc/dt max, and t 1/2 of afferent as well as efferent arterioles upon Ang II stimulation were similar in smb(+/+) and smb(-/-) mice. Constriction amplitudes upon KCl stimulation of afferent arterioles were similar in both smb(+/+) and smb(-/-) mice. Furthermore, KCl-induced dc/dt max and t 1/2 of afferent arterioles were similar in both smb(+/+) and smb(-/-) mice. SMB expression could be detected in afferent but not efferent arterioles in smb(+/+) mice. No SMB expression in either arteriole could be observed in smb(-/-) mice. Conclusion. Our results suggest that the presence of different alternatively 5′-spliced smooth muscle-myosin heavy chain (SM-MHC) isoforms does not dominate the different contractile features of physiologically loaded renal afferent or efferent arterioles