Voltage-dependent Na + channels are thought to sense membrane potential with fixed charges located within the membrane's electrical field. Measurement of open probability (Po) as a function of membrane potential gives a quantitative indication of the number of such charges that move through the field in opening the channel. We have used single-channel recording to measure skeletal muscle Na § channel open probability at its most negative extreme, where channels may open as seldom as once per minute. To prevent fast inactivation from masking the voltage dependence of Po, we have generated a clone of the rat skeletal muscle Na + channel that is lacking in fast inactivation (IFM1303QQQ). Using this mutant channel expressed in Xenopus oocytes, and the extra resolution afforded by single-channel analysis, we have extended the resolution of the hyperpolarized tail of the Po curve by four orders of magnitude. We show that previous measurements, which indicated a minimum of six effective gating charges, may have been made in a range of Po values that had not yet arrived at its limiting slope. In our preparation, a minimum of 12 charges must function in the activation gating of the channel. Our results will require reevaluation of kinetic models based on six charges, and they have major implications for the interpretation of $4 mutagenesis studies and structure/function models of the Na § channel.

The R403Q mutation in the ␤-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly ␣-MHC as opposed to the ␤-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q ␣-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous ␣-MHC was largely replaced with transgenically encoded ␤-MHC. A His 6 tag was cloned at the N terminus of the ␣-and ␤-MHC to facilitate protein isolation by Ni 2؉ -chelating chromatography. Characterization of the R403Q ␣-MHC by the in vitro motility assay showed a 30 -40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a ␤-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q ␣-S1 is ϳ30% higher than for wild type, whereas the enzymatic activity for R403Q ␤-S1 is reduced by ϳ10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.The arginine to glutamine mutation at amino acid 403 (R403Q) in the ␤-myosin heavy chain (MHC) 2 was the first MHC missense mutation linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle (1). Since that time several hundred mutations in genes encoding sarcomeric proteins have been identified, leading to the designation of FHC as primarily a "disease of the sarcomere" (2, 3). However, the pathways linking the various genetic defects to the characteristic human disease phenotype remain largely unknown. Even the fundamental question of whether these mutations cause a gain or a loss of function in myosin remains unresolved. We have confined our study to the R403Q mutation, because it is one of the most extensively studied FHC mutations and because it has a poor clinical prognosis (2, 4). The majority of the earlier biochemical/biophysical results, derived from expression systems and biopsies, supported the hypothesis that the R403Q mutation leads to a large decrease in motor function, which ultimately results in a compensatory hypertrophic response (for review, see Ref. 5).The first murine model for FHC replaced one allele of the endogenous cardiac mouse ␣-MHC with the mutant ␣-MHC (R403Q) gene by homologous recombination (6); this heterozygous mouse (R403Q/ϩ) resembled the human cardiac phenotype in many ways, except cardiac hypertrophy was notably milder. Subsequent studies on myosin extracted from a Ͻ1-week-o...

Blebbistatin is reported to be a selective and specific small molecule inhibitor of the myosin II isoforms expressed by striated muscles and nonmuscle (IC 50 ϭ 0.5-5 M) but is a poor inhibitor of purified turkey smooth muscle myosin II (IC 50 ϳ80 M). We found that blebbistatin potently (IC 50 ϳ3 M) inhibited the actomyosin ATPase activities of expressed "slow" [smooth muscle myosin IIA (SMA)] and "fast" [smooth muscle myosin IIB (SMB)] smooth muscle myosin II heavy-chain isoforms. Blebbistatin also inhibited the KCl-induced tonic contractions produced by rabbit femoral and renal arteries that express primarily SMA and the weaker tonic contraction produced by the saphenous artery that expresses primarily SMB, with an equivalent potency comparable with that identified for nonmuscle myosin IIA (IC 50 ϳ5 M). In femoral and saphenous arteries, blebbistatin had no effect on unloaded shortening velocity or the tonic increase in myosin light-chain phosphorylation produced by KCl but potently inhibited ␤-escin permeabilized artery contracted with calcium at pCa 5, suggesting that cell signaling events upstream from KCl-induced activation of cross-bridges were unaffected by blebbistatin. It is noteworthy that KCl-induced contractions of chicken gizzard were less potently inhibited (IC 50 ϳ20 M). Adult femoral, renal, and saphenous arteries did not express significant levels of nonmuscle myosin. These data together indicate that blebbistatin is a potent inhibitor of smooth muscle myosin II, supporting the hypothesis that the force-bearing structure responsible for tonic force maintenance in adult mammalian vascular smooth muscle is the cross-bridge formed from the blebbistatin-dependent interaction between actin and smooth muscle myosin II.

Regulatory light chain (RLC) phosphorylation is necessary to activate smooth muscle myosin, unlike constitutively active striated muscle myosins. Here we show that an actin-binding surface loop located at the 50/20-kDa junction contributes to this fundamental difference between myosins. Substitution of the native actin-binding loop of smooth muscle heavy meromyosin (HMM) with that from either skeletal or ␤-cardiac myosin caused the chimeric HMMs to become unregulated like the myosin from which the loop was derived. Dephosphorylated chimeric HMMs gained the ability to move actin in a motility assay and had 50 -70% of the actinactivated ATPase activity of phosphorylated wild-type HMM. Direct binding measurements showed that the affinity of HMM for actin in the presence of MgATP was unaffected by loop substitution; thus the rate of a step other than binding is increased. Phosphorylation of the chimeras did not lead to a higher V max than obtained for wild-type HMM. In the absence of actin, a foreign loop did not affect nucleotide trapping. Native regulated molecules have thus evolved a loop sequence which prevents rapid product release by actin when the RLC is dephosphorylated, thereby allowing activity to be controlled by RLC phosphorylation.Myosins differ from each other in how fast they can move actin, the rates of their actin-activated ATPases, and whether or not these activities are regulated. It has been recently suggested that a divergent surface loop at the actin-binding interface "tunes" the rate of phosphate release and thus sets the maximum velocity (V max ) for ATPase activity. A second loop, at the 25/50-kDa interface near the ATP-binding site, was proposed to control the rate of ADP release and thus be responsible for determining the velocity at which different myosins move actin (Spudich, 1994). The actin-activated ATPase activity of a chimeric Dictyostelium myosin containing the actin-binding loop from skeletal muscle myosin was 5-fold higher than wildtype Dictyostelium myosin, providing experimental evidence in support of the first part of this hypothesis (Uyeda et al., 1994). This study did not show if such a generalization would hold true for other myosin motors nor was the question of heavy chain sequences involved in regulation of activity addressed.The molecular step that is controlled by light chain phosphorylation in smooth muscle myosin is phosphate release from the active site (Sellers, 1985). It has been recently proposed that myosin is a "back door" enzyme, whereby the cleaved phosphate leaves via a cleft in the 50-kDa domain, instead of through the nucleotide-binding pocket from which it entered. Binding of actin is suggested to promote movement of the highly conserved P-loop near the active site so that phosphate can leave (Yount et al., 1995). The interaction of actin with myosin probably involves several steps; the first is thought to be a weak interaction between the N terminus of actin and the 50/20-kDa junction of myosin, followed by stronger interactions involving hydrophobic res...

It has been well established that muscle contraction is driven by structural rearrangements within myosin during its ATPase cycle and interaction with actin. The crystal structures of myosin (Fig. 1) solved in different nucleotide states have provided a framework for examining the structural basis of the ATPase cycle of myosin (1-6). In addition, solution techniques that include monitoring the intrinsic fluorescence of myosin have proven extremely valuable for examining the enzymatic and kinetic properties of the molecule (7-13). The following kinetic scheme of the myosin MgATPase cycle was developed based on observed changes in intrinsic fluorescence corresponding to structural changes within myosin, where M represents myosin and an asterisk represents enhanced protein fluorescence (14).In this reaction myosin first forms a collision complex with ATP followed by an isomerization to the M*⅐ATP complex, which results in the first level of fluorescence enhancement (*). During the rapid reversible process of ATP hydrolysis, a structural change induces an additional fluorescence enhancement (**). Then, following hydrolysis of ATP the fluorescence decreases back to the first level of enhancement (*), which is thought to correspond to the rate-limiting structural change resulting in phosphate release. The phosphate release step then shifts myosin from a weak to strong binding conformation and is the step believed to be associated with force generation during muscle contraction. The release of ADP is a much faster two-step process in which the second step results in a decrease in fluorescence to basal levels. Thus, examining the conformational changes that result in alterations in intrinsic tryptophan fluorescence may lead to important insights about the structural properties of the myosin MgATPase cycle.Dynamic structural information about domain motions within myosin during its MgATPase cycle has been pursued extensively (reviewed in Ref. 15). Indeed, several studies have demonstrated key rearrangements in the light chain-binding region (residues 781-820 highlighted in pink in Fig. 1), also referred to as the lever arm, providing a structural mechanism for force generation (reviewed in Ref. 16). Electron paramagnetic resonance studies, utilizing a probe on the regulatory light chain have shown a 30°rotation of this region (17), suggesting that the lever arm region rotates relative to the catalytic domain during muscle contraction. In addition, fluorescence studies utilizing a fluorescent probe at the regulatory light chain demonstrated rotation of the lever arm in contracting muscle fibers (15). Cryo-electron microscopy studies have revealed that smooth muscle myosin decorated on actin filaments undergoes a large structural change (30 -35 Å) of the lever arm upon ADP release (18). Furthermore, nucleotide analogs, which trap myosin in specific nucleotide states, have been useful in elucidating the structural properties of the normally short-lived stages of the MgATPase cycle. Structural studies suggest that MgADP-BeF ...