Skeletal Muscle Myosin Light Chain Research Articles

Millisecond Time-Resolved Solid-State NMR Reveals a Two-Stage Molecular Mechanism for Formation of Complexes between Calmodulin and a Target Peptide from Myosin Light Chain Kinase.

Calmodulin (CaM) mediates a wide range of biological responses to changes in intracellular Ca2+ concentrations through its calcium-dependent binding affinities to numerous target proteins. Binding of two Ca2+ ions to each of the two four-helix-bundle domains of CaM results in major conformational changes that create a potential binding site for the CaM binding domain of a target protein, which also undergoes major conformational changes to form the complex with CaM. Details of the molecular mechanism of complex formation are not well established, despite numerous structural, spectroscopic, thermodynamic, and kinetic studies. Here, we report a study of the process by which the 26-residue peptide M13, which represents the CaM binding domain of skeletal muscle myosin light chain kinase, forms a complex with CaM in the presence of excess Ca2+ on the millisecond time scale. Our experiments use a combination of selective 13C labeling of CaM and M13, rapid mixing of CaM solutions with M13/Ca2+ solutions, rapid freeze-quenching of the mixed solutions, and low-temperature solid state nuclear magnetic resonance (ssNMR) enhanced by dynamic nuclear polarization. From measurements of the dependence of 2D 13C-13C ssNMR spectra on the time between mixing and freezing, we find that the N-terminal portion of M13 converts from a conformationally disordered state to an α-helix and develops contacts with the C-terminal domain of CaM in about 2 ms. The C-terminal portion of M13 becomes α-helical and develops contacts with the N-terminal domain of CaM more slowly, in about 8 ms. The level of structural order in the CaM/M13/Ca2+ complexes, indicated by 13C ssNMR line widths, continues to increase beyond 27 ms.

Protein Histidine Methylation.

Protein histidine methylation is a rarely studied posttranslational modification in eukaryotes. Although the presence of N-methylhistidine was demonstrated in actin in the early 1960s, so far, only a limited number of proteins containing N-methylhistidine have been reported, including S100A9, myosin, skeletal muscle myosin light chain kinase (MLCK 2), and ribosomal protein Rpl3. Furthermore, the role of histidine methylation in the functioning of the protein and in cell physiology remains unclear due to a shortage of studies focusing on this topic. However, the molecular identification of the first two distinct histidine-specific protein methyltransferases has been established in yeast (Hpm1) and in metazoan species (actin-histidine N-methyltransferase), giving new insights into the phenomenon of protein methylation at histidine sites. As a result, we are now beginning to recognize protein histidine methylation as an important regulatory mechanism of protein functioning whose loss may have deleterious consequences in both cells and in organisms. In this review, we aim to summarize the recent advances in the understanding of the chemical, enzymological, and physiological aspects of protein histidine methylation.

Cyanylated Cysteine Reports Site-Specific Changes at Protein-Protein-Binding Interfaces Without Perturbation.

To investigate the cyanylated cysteine vibrational probe group’s ability to report on binding-induced changes along a protein–protein interface, the probe group was incorporated at several sites in a peptide of the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase. Isothermal titration calorimetry was used to determine the binding thermodynamics between calmodulin and each peptide. For all probe positions, the binding affinity was nearly identical to that of the unlabeled peptide. The CN stretching infrared band was collected for each peptide free in solution and bound to calmodulin. Binding-induced shifts in the IR spectral frequencies were correlated with estimated solvent accessibility based on molecular dynamics simulations. This work generally suggests (1) that site-specific incorporation of this vibrational probe group does not cause major perturbations to its local structural environment and (2) that this small probe group might be used quite broadly to map dynamic protein-binding interfaces. However, site-specific perturbations due to artificial labeling groups can be somewhat unpredictable and should be evaluated on a site-by-site basis through complementary measurements. A fully quantitative, simulation-based interpretation of the rich probe IR spectra is still needed but appears to be possible given recent advances in simulation techniques.

Conformational Ensembles of Calmodulin Revealed by Nonperturbing Site-Specific Vibrational Probe Groups.

Seven native residues on the regulatory protein calmodulin, including three key methionine residues, were replaced (one by one) by the vibrational probe amino acid cyanylated cysteine, which has a unique CN stretching vibration that reports on its local environment. Almost no perturbation was caused by this probe at any of the seven sites, as reported by CD spectra of calcium-bound and apo calmodulin and binding thermodynamics for the formation of a complex between calmodulin and a canonical target peptide from skeletal muscle myosin light chain kinase measured by isothermal titration. The surprising lack of perturbation suggests that this probe group could be applied directly in many protein–protein binding interfaces. The infrared absorption bands for the probe groups reported many dramatic changes in the probes’ local environments as CaM went from apo- to calcium-saturated to target peptide-bound conditions, including large frequency shifts and a variety of line shapes from narrow (interpreted as a rigid and invariant local environment) to symmetric to broad and asymmetric (likely from multiple coexisting and dynamically exchanging structures). The fast intrinsic time scale of infrared spectroscopy means that the line shapes report directly on site-specific details of calmodulin’s variable structural distribution. Though quantitative interpretation of the probe line shapes depends on a direct connection between simulated ensembles and experimental data that does not yet exist, formation of such a connection to data such as that reported here would provide a new way to evaluate conformational ensembles from data that directly contains the structural distribution. The calmodulin probe sites developed here will also be useful in evaluating the binding mode of calmodulin with many uncharacterized regulatory targets.

Selective Excitation of Cyanophenylalanine Fluorophores for Multi-Site Binding Studies.

Recently, it has been shown that nitrile-derivatized phenylalanines possess distinct fluorescent properties depending on the position of the cyano-group within the aromatic ring. These fluorophores have potential as probes for studying protein dynamics due to their sensitivity to local environment. Herein, we demonstrate that 2-cyanophenylalanine (Phe2CN) and Phe4CN can independently monitor multiple sites during the Ca2+ dependent binding of a skeletal muscle myosin light chain kinase (MLCK) peptide fragment to the protein calmodulin (CaM). These cyano-probes were incorporated at two different positions along the peptide chain and monitored simultaneously via selective excitation of the two chromophores. The peptide was labeled with Phe4CN at a residue known to bind to a hydrophobic binding pocket of CaM, while Phe2CN was designed to acquire dynamics external to the binding pocket. By selectively exciting each of the chromophores, it was determined that the fluorescence emission of Phe4CN located at position 581 of MLCK was quenched in the presence of CaM, while no significant change in Phe2CN emission was observed at exposed position 594. The CaM binding affinity (Kd) of the double labeled MLCK peptide was calculated to be approximately 64 nM, which is in agreement with previous measurements. These results indicate that multiple PheCN reporters within the same peptide can simultaneously detect variations in the local environment, and that these fluorophores could be utilized to investigate a wide variety of biological problems.

Shortening Speed Dependence of Concentric Force Potentiation in the Absence of Myosin Phosphorylation

Contraction‐induced elevation of myoplasmic Ca2+ leads to sequential activation of skeletal muscle myosin light chain kinase (skMLCK), an enzyme responsible for phosphorylating the myosin regulatory light chain (RLC). In wildtype (WT) mouse skeletal muscle containing skMLCK, force potentiation correlates with RLC phosphate content; although, these muscles may contain an RLC phosphorylation‐independent component. In this study, extensor digitorum longus (EDL) muscles from knockout (KO) C57BL/6 mice, devoid of skMLCK, were activated in vitro (25°C) across a range of stimulation frequencies (10, 25, 45, 70 and 100 Hz) and subjected to shortening ramps at 0.1, 0.3 or 0.5 of maximal shortening velocity (Vmax). This was performed before and after a standard conditioning stimulus (CS) that potentiated mean concentric force at 0.3 and 0.5 Vmax to 1.20 ± 0.03 and 1.30 ± 0.02 of unpotentiated (pre‐CS) values, respectively; however, mean forces were depressed to 0.84 ± 0.03 of pre‐CS values at 0.1 Vmax (n = 3, P < 0.05). Increasing shortening speed augmented the frequency at which concentric force potentiation was maximal: 25 and 45 Hz at 0.3 and 0.5 Vmax, respectively. These results mirrored previous results from WT muscles (Gittings et al., 2012), except that concentric force potentiation was attenuated by ~50% across all frequencies at 0.3 and 0.5 Vmax, while force at 0.1 Vmax was reduced rather than unchanged. Thus, our data indicates the presence of an RLC phosphorylation‐independent mechanism for potentiation in skMLCK KO muscles that may complement the intact RLC phosphorylation mechanism in WT muscles.Supported by NSERC (2014‐05122).

Site-Specific Conformational Diversity in Calmodulin using Cyanylated Cysteine Vibrational Probes along its Binding Interfaces

Vibrational spectroscopy was used to probe the modular binding interfaces of calmodulin. Two hydrophobic amino acid patches allow calmodulin great flexibility in binding to target partners, and these patches contain key methionine residues. Site-specific mutagenesis was used to generate numerous single cysteine variants of calmodulin, including three mutations at key methionine residues, which were expressed, purified and cyanylated at cysteine to generate a vibrational probe group at each site. Infrared spectroscopy was used to assess the environment surrounding each probe in apo- and calcium saturated conditions, as well as complexed with the binding peptide from skeletal muscle myosin light chain kinase. Circular dichroism (CD) experiments were conducted to ensure the SCN vibrational probe did not perturb calmodulin's secondary structure, and isothermal calorimetry was performed for each labeled variant with the skMLCK peptide to ensure the probe did not disrupt complex formation. Surprisingly, the methionine to cyanylated cysteine mutation at key methionine residues did not lead to a large perturbation in binding thermodynamics. This suggests that the cyanylated cysteine probe group is relatively innocent even when placed directly in the binding interface and can report directly on structural dynamics along the binding interface between calmodulin and its many targets.

Munc13-Like skMLCK Variants Cannot Mimic the Unique Calmodulin Binding Mode of Munc13 as Evidenced by Chemical Cross-Linking and Mass Spectrometry

Among the neuronal binding partners of calmodulin (CaM) are Munc13 proteins as essential presynaptic regulators that play a key role in synaptic vesicle priming and are crucial for presynaptic short-term plasticity. Recent NMR structural investigations of a CaM/Munc13-1 peptide complex have revealed an extended structure, which contrasts the compact structures of most classical CaM/target complexes. This unusual binding mode is thought to be related to the presence of an additional hydrophobic anchor residue at position 26 of the CaM binding motif of Munc13-1, resulting in a novel 1-5-8-26 motif. Here, we addressed the question whether the 1-5-8-26 CaM binding motif is a Munc13-related feature or whether it can be induced in other CaM targets by altering the motif's core residues. For this purpose, we chose skeletal muscle myosin light chain kinase (skMLCK) with a classical 1-5-8-14 CaM binding motif and constructed three skMLCK peptide variants mimicking Munc13-1, in which the hydrophobic anchor amino acid at position 14 was moved to position 26. Chemical cross-linking between CaM and skMLCK peptide variants combined with high-resolution mass spectrometry yielded insights into the peptides' binding modes. This structural comparison together with complementary binding data from surface plasmon resonance experiments revealed that skMLCK variants with an artificial 1-5-8-26 motif cannot mimic CaM binding of Munc13-1. Apparently, additional features apart from the spacing of the hydrophobic anchor residues are required to define the functional 1-5-8-26 motif of Munc13-1. We conclude that Munc13 proteins display a unique CaM binding behavior to fulfill their role as efficient presynaptic calcium sensors over broad range of Ca2+ concentrations.

The complex between Ca-Calmodulin and skeletal muscle myosin light chain kinase from combination of NMR and aqueous and contrast-matched SAXS data

The complex between Ca-Calmodulin and skeletal muscle myosin light chain kinase from combination of NMR and aqueous and contrast-matched SAXS data

Nuclear Calcium Signaling in Spinal Neurons Drives a Genomic Program Required for Persistent Inflammatory Pain

Persistent pain induced by noxious stimuli is characterized by the transition from normosensitivity to hypersensitivity. Underlying mechanisms are not well understood, although gene expression is considered important. Here, we show that persistent nociceptive-like activity triggers calcium transients in neuronal nuclei within the superficial spinal dorsal horn, and that nuclear calcium is necessary for the development of long-term inflammatory hypersensitivity. Using a nucleus-specific calcium signal perturbation strategy in vivo complemented by gene profiling, bioinformatics, and functional analyses, we discovered a pain-associated, nuclear calcium-regulated gene program in spinal excitatory neurons. This includes C1q, a modulator of synaptic spine morphogenesis, which we found to contribute to activity-dependent spine remodelling on spinal neurons in a manner functionally associated with inflammatory hypersensitivity. Thus, nuclear calcium integrates synapse-to-nucleus communication following noxious stimulation and controls a spinal genomic response that mediates the transition between acute and long-term nociceptive sensitization by modulating functional and structural plasticity.

Differences in two-dimensional gel electrophoresis patterns of skeletal muscle myosin light chain isoforms between Bos taurus, Sus scrofa and selected poultry species

In this study the interspecies differences in two-dimensional electrophoresis patterns of skeletal muscle myosin light chain (MLC) isoforms between Bos taurus (cattle), Sus scrofa (pig), Gallus gallus (chicken), Meleagris gallopavo (turkey), Anas platyrhynchos (duck) and Anser anser (goose) were characterised on the basis of specific properties of MLCs associated with their structure and mobility in gel. Two-dimensional electrophoresis separations revealed species-specific differences in the molecular weight and pI of individual MLC isoforms (MLC1f, MLC2f and MLC3f). In the case of closely related animal species such as goose and duck or turkey and chicken, significant differences occurred in MLC1f. For MLC2f, differences between cattle and turkey and between pig and chicken were around 1 and 0.3 kDa respectively. It appeared from the comparison of amino acid sequences that even MLCs with only 2% difference in sequences have different electrophoretic mobilities. Interspecies differences in skeletal MLC isoforms appeared between cattle, pig, chicken, turkey, duck and goose. The slight changes observed in the course of the aging process confirmed that these proteins are relatively little susceptible to proteolytic enzymes during meat aging.

Skeletal muscle myosin light chain composition of highly‐trained endurance runners

Individual slow (MHC I) and fast (MHC IIa) twitch muscle fibers from highly-trained competitive runners display a greater contractile velocity (Vo) compared to moderately-active individuals. Myosin light chains (MLC) have been implicated in altering myofiber Vo, but this relationship is ambiguous in human skeletal muscle. Therefore, the purpose of this study was to examine MLC composition and its association with single muscle fiber contractile function in competitive (RUN; 71±1 ml·kg−1·min−1, 95±2 km/wk running) and recreational runners (REC; 47±2 ml·kg−1·min−1, <25 km/wk). SDS-PAGE, silver staining, and densitometry were used to assess MLC composition in mixed muscle homogenates and individual myofibers (MHC I and IIa) obtained from skeletal muscle biopsy samples. At the mixed muscle level, MLC 2f was lower (p<0.05) in RUN compared to REC with trends (p<0.08) for greater 1s, 2s and lower 3f ratios. The MLC profile of MHC I fibers was similar between RUN and REC. However, the MLC profile of the MHC IIa fibers from RUN contained less MLC 1s and greater amounts of 1f and 2s (p<0.05) compared to REC. These data suggest that only subtle differences exist in the MLC profile between RUN and REC. Therefore, it is likely that MLC composition contributes only a minor, if any, role in the robust differences in contractile function between competitive and recreational runners. Supported by NIH Grant AG032127

Fast Photochemical Oxidation of Proteins for Comparing Structures of Protein−Ligand Complexes: The Calmodulin−Peptide Model System

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based protein footprinting method that modifies proteins on the microsecond time scale. Highly reactive (•)OH, produced by laser photolysis of hydrogen peroxide, oxidatively modifies the side chains of approximately one-half the common amino acids on this time scale. Because of the short labeling exposure, only solvent-accessible residues are sampled. Quantification of the modification extent for the apo and holo states of a protein-ligand complex provides structurally sensitive information at the amino-acid level to compare the structures of unknown protein complexes with known ones. We report here the use of FPOP to monitor the structural changes of calmodulin in its established binding to M13 of the skeletal muscle myosin light chain kinase. We use the outcome to establish the unknown structures resulting from binding with melittin and mastoparan. The structural comparison follows a comprehensive examination of the extent of FPOP modifications as measured by proteolysis and LC-MS/MS for each protein-ligand equilibrium. The results not only show that the three calmodulin-peptide complexes have similar structures but also reveal those regions of the protein that became more or less solvent-accessible upon binding. This approach has the potential for relatively high throughput, information-dense characterization of a series of protein-ligand complexes in biochemistry and drug discovery when the structure of one reference complex is known, as is the case for calmodulin and M13 of the skeletal muscle myosin light chain kinase, and the structures of related complexes are not.

Single-molecule force spectroscopy distinguishes target binding modes of calmodulin

The eukaryotic signaling protein calmodulin (CaM) can bind to more than 300 known target proteins to regulate numerous functions in our body in a calcium-dependent manner. How CaM distinguishes between these various targets is still largely unknown. Here, we investigate fluctuations of the complex formation of CaM and its target peptide sequences using single-molecule force spectroscopy by AFM. By applying mechanical force, we can steer a single CaM molecule through its folding energy landscape from the fully unfolded state to the native target-bound state revealing equilibrium fluctuations between numerous intermediate states. We find that the prototypical CaM target sequence skMLCK, a fragment from skeletal muscle myosin light chain kinase, binds to CaM in a highly cooperative way, while only a lower degree of interdomain binding cooperativity emerges for CaMKK, a target peptide from CaM-dependent kinase kinase. We identify minimal binding motifs for both of these peptides, confirming that affinities of target peptides are not exclusively determined by their pattern of hydrophobic anchor residues. Our results reveal an association mode for CaMKK in which the peptide binds strongly to only partially Ca(2+)-saturated CaM. This binding mode might allow for a fine-tuning of the intracellular response to changes in Ca(2+) concentration.

Distinct Mechanisms of Regulation by Ca2+/Calmodulin of Type 1 and 8 Adenylyl Cyclases Support Their Different Physiological Roles

Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are stimulated by Ca(2+)/calmodulin (CaM), have discrete neuronal functions. Although the Ca(2+) sensitivity of AC1 is higher than that of AC8, precisely how these two ACs are regulated by Ca(2+)/CaM remains elusive, and the basis for their diverse physiological roles is quite unknown. Distinct localization of the CaM binding domains within the two enzymes may be essential to differential regulation of the ACs by Ca(2+)/CaM. In this study we compare in detail the regulation of AC1 and AC8 by Ca(2+)/CaM both in vivo and in vitro and explore the different role of each Ca(2+)-binding lobe of CaM in regulating the two enzymes. We also assess the relative dependence of AC1 and AC8 on capacitative Ca(2+) entry. Finally, in real-time fluorescence resonance energy transfer-based imaging experiments, we examine the effects of dynamic Ca(2+) events on the production of cAMP in cells expressing AC1 and AC8. Our data demonstrate distinct patterns of regulation and Ca(2+) dependence of AC1 and AC8, which seems to emanate from their mode of regulation by CaM. Such distinctive properties may contribute significantly to the divergent physiological roles in which these ACs have been implicated.

The Fast Skeletal Muscle Myosin Light Chain Is Differentially Expressed in Smooth Muscle Cells of OVA-challenged Mouse Trachea

The Fast Skeletal Muscle Myosin Light Chain Is Differentially Expressed in Smooth Muscle Cells of OVA-challenged Mouse Trachea

Enhanced Skeletal Muscle Contraction with Myosin Light Chain Phosphorylation by a Calmodulin-sensing Kinase

Repetitive low frequency stimulation results in potentiation of twitch force development in fast-twitch skeletal muscle due to myosin regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin (CaM)-dependent skeletal muscle myosin light chain kinase (skMLCK). We generated transgenic mice that express an skMLCK CaM biosensor in skeletal muscle to determine whether skMLCK or CaM is limiting to twitch force potentiation. Three transgenic mouse lines exhibited up to 22-fold increases in skMLCK protein expression in fast-twitch extensor digitorum longus muscle containing type IIa and IIb fibers, with comparable expressions in slow-twitch soleus muscle containing type I and IIa fibers. The high expressing lines showed a more rapid RLC phosphorylation and force potentiation in extensor digitorum longus muscle with low frequency electrical stimulation. Surprisingly, overexpression of skMLCK in soleus muscle did not recapitulate the fast-twitch potentiation response despite marked enhancement of both fast-twitch and slow-twitch RLC phosphorylation. Analysis of calmodulin binding to the biosensor showed a frequency-dependent activation to a maximal extent of 60%. Because skMLCK transgene expression is 22-fold greater than the wild-type kinase, skMLCK rather than calmodulin is normally limiting for RLC phosphorylation and twitch force potentiation. The kinase activation rate (10.6 s(-1)) was only 3.6-fold slower than the contraction rate, whereas the inactivation rate (2.8 s(-1)) was 12-fold slower than relaxation. The slower rate of kinase inactivation in vivo with repetitive contractions provides a biochemical memory via RLC phosphorylation. Importantly, RLC phosphorylation plays a prominent role in skeletal muscle force potentiation of fast-twitch type IIb but not type I or IIa fibers.

Peptide Binding by a Fragment of Calmodulin Composed of EF-Hands 2 and 3

Calmodulin (CaM) is composed of two EF-hand domains tethered by a flexible linker. Upon Ca2+-binding, a fragment of CaM encompassing EF-hands 2 and 3 (CaM2/3; residues 46-113) folds into a structure remarkably similar to the N- and C-domains of CaM. In this study, we demonstrate that Ca2+-ligated CaM2/3 can also bind to a peptide representing the CaM-recognition sequence of skeletal muscle myosin light chain kinase (M13) with an equimolar stoichiometry and a dissociation constant of 0.40 +/- 0.05 microM. On the basis of an analytical ultracentrifugation measurement, the resulting complex exists as an equilibrium mixture of 2:2 heterotetrameric and 1:1 heterodimeric species. Chemical shift perturbation mapping indicates that, similar to CaM, the peptide associates with a hydrophobic groove crossing both EF-hands in CaM2/3. However, upon binding the M13 peptide, many residues in CaM2/3 yielded two equal intensity NMR signals with the same 15N relaxation properties. Thus, the 2:2 CaM2/3-M13 tetramer, which predominates under the conditions used for these studies, is asymmetric with each component adopting spectroscopically distinguishable conformations within the complex. CaM2/3 also weakly stimulates the phosphatase activity of calcineurin and inhibits stimulation by native CaM. These studies highlight the remarkable plasticity of EF-hand association and expand the diverse repertoire of mechanisms possible for CaM-target protein interactions.

A novel fluorescent probe for protein binding and folding studies: p‐cyano‐phenylalanine

Recently, it is has been shown that the C=N stretching vibration of a non-natural amino acid, p-cyano-phenylalanine (PheCN), could be used as an infrared reporter of local environment. Here, we further showed that the fluorescence emission of PheCN is also sensitive to solvent and, therefore, could be used as a novel optical probe for protein binding and folding studies. Moreover, we found that the fluorescence quantum yield of PheCN is nearly five times larger than that of phenylalanine and, more importantly, can be selectively excited even when other aromatic amino acids are present, thus making it a more versatile fluorophore. To test the feasibility of using PheCN as a practical fluorescent probe, we studied the binding of calmodulin (CaM) to a peptide derived from the CaM-binding domain of skeletal muscle myosin light chain kinase (MLCK). The peptide (MLCK3CN) contains a single PheCN residue and has been shown to bind to CaM with high affinity. As expected, addition of CaM into a MLCK3CN solution resulted in quenching of the PheCN fluorescence. A series of stochiometric titrations further allowed us to determine the binding affinity (Kd) of this peptide to CaM. Taken together, these results indicated that the PheCN fluorescence is sensitive to environment and could be applicable to a wide variety of biological problems.

Effect of myosin light chain kinase overexpression on twitch force potentiation in skeletal muscle

Repetitive low-frequency stimulation results in potentiation of twitch force development in fast-twitch skeletal muscle. Studies in skeletal muscle myosin light chain kinase (skMLCK) knockout mice show that frequency-dependent twitch potentiation is due in part to myosin regulatory light chain (RLC) phosphorylation by the calcium/calmodulin-dependent skMLCK. We generated transgenic mice that express skMLCK under control of the human skeletal muscle actin promoter. Skeletal muscle from transgenic mice exhibited a 23-fold increase in skMLCK protein expression and a 15-fold increase in enzymatic capacity as compared to skeletal muscle from wild-type mice. In extensor digitorum longus (EDL) muscle from wild-type mice, low-frequency (10 Hz) electrical stimulation led to a progressive potentiation of twitch force. Maximal twitch force potentiation was observed in wild-type EDL after 15 sec of stimulation. Electrical stimulation of transgenic EDL muscle resulted in a similar maximal response; however, maximal twitch-force occurred more rapidly. Time to half-maximal potentiation was 6.8±0.4 sec compared to 2.7±0.1 sec (p<0.001) for wild-type and transgenic mice, respectively. The rate and extent of RLC phosphorylation was also greater in transgenic compared to wild-type EDL. Surprisingly, a dramatic increase in skMLCK expression had only a modest positive effect on frequency-dependent twitch potentiation and RLC phosphorylation. Previous studies suggest that calmodulin may be limiting for smooth muscle activation of MLCK, therefore calmodulin may also be limiting for activation of skMLCK. (This work supported by NIH).