The tethered motor domain of a kinesin-microtubule complex catalyzes reversible synthesis of bound ATP

Although the steps for the forward reaction of ATP hydrolysis by the motor protein kinesin have been studied extensively, the rates for the reverse reactions and thus the energy changes at each step are not as well defined. Oxygen isotopic exchange between water and P(i) was used to evaluate the reverse rates. The fraction of the kinesin x ADP x P(i) complex that reverts to ATP before release of P(i) during net hydrolysis was approximately 0 and approximately 2.6% in the absence and presence of microtubules (MTs), respectively. The rate of synthesis of bound ATP from free P(i) and the MT x kinesin x ADP complex was approximately 1.7 M(-1) x s(-1) (K0.5 ADP = 70 microM) with monomeric kinesin in the absence of net hydrolysis. Synthesis of bound ATP from the ADP of the tethered head of a dimer-MT complex was 20-fold faster than for the monomer-MT complex. This MT-activated ATP synthesis at the tethered head is in marked contrast to the lack of MT stimulation of ADP release from the same site. The more rapid ATP synthesis with dimers suggests that the tethered head binds behind the strongly attached head, because this positions the neck linker of the tethered head toward the plus end of the MT and would thus facilitate its docking on synthesis of ATP. The observed rate of ATP synthesis also puts limits on the overall energetics that suggest that a significant fraction of the free energy of ATP hydrolysis is available to drive the docking of the neck linker on binding of ATP.     

Kinesin's processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains

Kinesin is a processive motor protein: A single molecule can walk continuously along a microtubule for several micrometers, taking hundreds of 8-nm steps without dissociating. To elucidate the biochemical and structural basis for processivity, we have engineered a heterodimeric one-headed kinesin and compared its biochemical properties to those of the wild-type two-headed molecule. Our construct retains the functionally important neck and tail domains and supports motility in high-density microtubule gliding assays, though it fails to move at the single-molecule level. We find that the ATPase rate of one-headed kinesin is 3-6 s(-1) and that detachment from the microtubule occurs at a similar rate (3 s(-1)). This establishes that one-headed kinesin usually detaches once per ATP hydrolysis cycle. Furthermore, we identify the rate-limiting step in the one-headed hydrolysis cycle as detachment from the microtubule in the ADP.P(i) state. Because the ATPase and detachment rates are roughly an order of magnitude lower than the corresponding rates for two-headed kinesin, the detachment of one head in the homodimer (in the ADP.P(i) state) must be accelerated by the other head. We hypothesize that this results from internal strain generated when the second head binds. This idea accords with a hand-over-hand model for processivity in which the release of the trailing head is contingent on the binding of the forward head. These new results, together with previously published ones, allow us to propose a pathway that defines the chemical and mechanical cycle for two-headed kinesin.     

Probing the kinesin reaction cycle with a 2D optical force clamp

With every step it takes, the kinesin motor undergoes a mechanochemical reaction cycle that includes the hydrolysis of one ATP molecule, ADPP(i) release, plus an unknown number of additional transitions. Kinesin velocity depends on both the magnitude and the direction of the applied load. Using specialized apparatus, we subjected single kinesin molecules to forces in differing directions. Sideways and forward loads up to 8 pN exert only a weak effect, whereas comparable forces applied in the backward direction lead to stall. This strong directional bias suggests that the primary working stroke is closely aligned with the microtubule axis. Sideways loads slow the motor asymmetrically, but only at higher ATP levels, revealing the presence of additional, load-dependent transitions late in the cycle. Fluctuation analysis shows that the cycle contains at least four transitions, and confirms that hydrolysis remains tightly coupled to stepping. Together, our findings pose challenges for models of kinesin motion.     

Ecto-nucleotide pyrophosphatase/phosphodiesterase as part of a multiple system for nucleotide hydrolysis by platelets from rats: kinetic characterization and biochemical properties

In this study, we describe an ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activity in rat platelets. Using p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) as a substrate for E-NPP, we demonstrate an enzyme activity that shares the major biochemical properties described for E-NPPs: alkaline pH dependence, divalent cation dependence and blockade of activity by metal ion chelator. K(m) and V(max) values for p-Nph-5'-TMP hydrolysis were found to be 106 +/- 18 microM and 3.44 +/- 0.18 nmol p-nitrophenol/min/mg (mean +/- SD, n = 5). We hypothesize that an E-NPP is co-localized with an ecto-nucleoside triphosphate diphosphohydrolase and an ecto-5'-nucleotidase on the platelet surface, as part of a multiple system for nucleotide hydrolysis, since they can act under distinct physiological conditions and can be differently regulated. Thus, 0.25 mM suramin inhibited p-Nph-5'-TMP, ATP and ADP hydrolysis, while 0.5 mM AMP decreased only p-Nph-5'-TMP hydrolysis. Besides, 5.0, 10 and 20 mM sodium azide just inhibited ATP and ADP hydrolysis. Angiotensin II (5.0 and 10 nM) affected only ADP hydrolysis. Gadolinium chloride (0.2 and 0.5 mM) strongly inhibited the ATP and ADP hydrolysis. The E-NPP described here represents a novel insight into the control of platelet purinergic signaling.     

Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein

P-glycoprotein (Pgp) is an ATP-dependent hydrophobic natural product anticancer drug efflux pump whose overexpression confers multidrug resistance to tumor cells. The work reported here deals with the elucidation of the energy requirement for substrate interaction with Pgp during the catalytic cycle. We show that the K(d) (412 nM) of the substrate analogue [(125)I]iodoarylazidoprazoin for Pgp is not altered by the presence of the nonhydrolyzable nucleotide 5'-adenylylimididiphosphate and vanadate (K(d) = 403 nM). Though binding of nucleotide per se does not affect interactions with the substrate, ATP hydrolysis results in a dramatic conformational change where the affinity of [(125)I]iodoarylazidoprazoin for Pgp trapped in transition-state conformation (Pgp x ADP x vanadate) is reduced >30-fold. To transform Pgp from this intermediate state of low affinity for substrate to the next catalytic cycle, i.e., a conformation that binds substrate with high affinity, requires conditions that permit ATP hydrolysis. Additionally, there is an inverse correlation (R(2) = 0.96) between 8AzidoADP (or ADP) release and the recovery of substrate binding. These results suggest that the release of nucleotide is necessary for reactivation but not sufficient. The hydrolysis of additional molecule(s) of ATP (or 8AzidoATP) is obligatory for the catalytic cycle to advance to completion. These data are consistent with the observed stoichiometry of two ATP molecules hydrolyzed for the transport of every substrate molecule. Our data demonstrate two distinct roles for ATP hydrolysis in a single turnover of the catalytic cycle of Pgp, one in the transport of substrate and the other in effecting conformational changes to reset the pump for the next catalytic cycle.     

Amiodarone inhibits cardiac ATP-sensitive potassium channels

INTRODUCTION: ATP-sensitive K+ channels (K(ATP)) are expressed abundantly in cardiovascular tissues. Blocking this channel in experimental models of ischemia can reduce arrhythmias. We investigated the acute effects of amiodarone on the activity of cardiac sarcolemmal K(ATP) channels and their sensitivity to ATP. METHODS AND RESULTS: Single K(ATP) channel activity was recorded using inside-out patches from rat ventricular myocytes (symmetric 140 mM K+ solutions and a pipette potential of +40 mV). Amiodarone inhibited K(ATP) channel activity in a concentration-dependent manner. After 60 seconds of exposure to amiodarone, the fraction of mean patch current relative to baseline current was 1.0 +/- 0.05 (n = 4), 0.8 +/- 0.07 (n = 4), 0.6 +/- 0.07 (n = 5), and 0.2 +/- 0.05 (n = 7) with 0, 0.1, 1.0, or 10 microM amiodarone, respectively (IC50 = 2.3 microM). ATP sensitivity was greater in the presence of amiodarone (EC50 = 13 +/- 0.2 microM in the presence of 10 microM amiodarone vs 43 +/- 0.1 microM in controls, n = 5; P < 0.05). Kinetic analysis showed that open and short closed intervals (bursting activity) were unchanged by 1 microM amiodarone, whereas interburst closed intervals were prolonged. Amiodarone also inhibited whole cell K(ATP) channel current (activated by 100 microM bimakalim). After a 10-minute application of amiodarone (10 microM), relative current was 0.71 +/- 0.03 vs 0.92 +/- 0.09 in control (P < 0.03). CONCLUSION: Amiodarone rapidly inhibited K(ATP) channel activity by both promoting channel closure and increasing ATP sensitivity. These actions may contribute to the antiarrhythmic properties of amiodarone.     

Local regulation of vasopressin and oxytocin secretion by extracellular ATP in the isolated posterior lobe of the rat hypophysis

It is now widely accepted that ATP functions as a signalling substance in the nervous system. The presence of P2 receptors mediating the action of extracellular ATP in brain regions involved in hormonal regulation raises the possibility that a similar role for ATP might also exist in the neuroendocrine system. In this study, the release from the rat isolated neurohypophysis preparation of endogenous ATP, oxytocin and vasopressin (AVP) were measured simultaneously using luciferin-luciferase and RIA techniques. After 70 min preperfusion, electrical field stimulation caused a rapid increase in the amount of ATP in the effluent and the release of AVP and oxytocin also increased stimulation-dependently. Inhibition of voltage-dependent Na+ channels by tetrodotoxin (1 microM) reduced the stimulation-evoked release of AVP and oxytocin; however, the evoked release of ATP remained unaffected. The effect of endogenous ATP on the hormone secretion was tested by suramin (300 microM), the P2 receptor antagonist. Suramin significantly increased the release of AVP, and the release of oxytocin was also enhanced. ATP, when applied to the superfusing medium, decreased the release of AVP, but not that of oxytocin, and its effect was prevented by suramin. ATP (60 nmol), added to the tissues, was readily decomposed to ADP, AMP and adenosine measured by HPLC combined with ultraviolet light detection, and the kinetic parameters of the enzymes responsible for inactivation of ATP (ectoATPase and ecto5'-nucleotidase) were also determined (Km=264+/-2.7 and 334+/-165 microM and vmax=6.7+/-1.1 and 2.54+/-0.24 nmol/min per preparation (n=3) for ectoATPase and ecto5'-nucleotidase respectively). Taken together, our data demonstrate the stimulation-dependent release, P2 receptor-mediated action and extracellular metabolism of endogenous ATP in the posterior lobe of the hypophysis and indicate its role, as a paracrine regulator, in the local control of hormone secretion.     

The in-vitro and ex-vivo effects of chloroquine sulphate on platelet function: implications for malaria prophylaxis in patients with impaired haemostasis

Platelet aggregation responses were studied in platelet-rich plasma from six healthy volunteers before and 2 and 6 h after ingestion of 600 mg chloroquine sulphate. Apart from a mild reduction in height of aggregation response to 1 microgram ml-1 collagen 2 h post-drug ingestion (mean percentage of pre-drug values +/- s.e.m. = 87.8% +/- 4.0%; P = 0.04), no significant differences were observed in platelet responses to ADP (1 and 5 microM) or collagen (1 and 4 micrograms ml-1) at 2 or 6 h post-chloroquine compared to the pre-drug values. In vitro, drug concentrations approximately 1000 times greater than those used therapeutically were required for 50% inhibition of platelet aggregation and ATP release in response to 5 microM ADP, 1 microgram ml-1 collagen and 4 micrograms ml-1 collagen (IC50 concentrations +/- s.e.m. for inhibition of aggregation = 98.5 +/- 3.7, 53.5 +/- 56.4 and 113.0 +/- 6.2 mg l-1 respectively; IC50s +/- s.e.m. for inhibition of ATP release = 0.9 +/- 0.2, 14.7 +/- 4.0 and 23.0 +/- 5.3 mg l-1 respectively). These data provide no cause for concern in using chloroquine for malaria prophylaxis in patients with impaired haemostasis.     

Influence of cellular energy metabolism on contractions of porcine carotid artery smooth muscle

A number of cellular metabolites, including inorganic phosphate and ADP, have been proposed to regulate the contractions of smooth muscle. Hypothesizing that one of these would have a greater influence than the others, parallel experiments using tissue mechanics and (31)P-NMR allowed comparison of several metabolic components with the generation of force in porcine carotid artery smooth muscle during long-term contractions. P(i), ADP, ATP, PCr, free energy, pH, and free Mg(2+) were determined from phosphate spectra during a control-hypoxia-postcontrol sequence generated during K(+) stimulation by replacement of oxygen with nitrogen using either pyruvate or glucose as substrate. Both pH and free Mg(2+) were significantly lower in control pyruvate-supplied tissues than in glucose-supplied tissues. Mechanical experiments following the same protocol produced variations in force. The pyruvate series produced the greater range of mechanical and metabolic changes. Linear and logarithmic regression analysis found the order of correlation with force to be highest for P(i), followed by pH, free energy, PCr, ATP, ADP, and free Mg(2+). The results are consistent with models for the regulation of myosin ATPase by free phosphate inhibition. The results are inconsistent with models of ADP as a regulator of smooth muscle force. Perturbations which alter intracellular phosphate, such as creatine loading, may produce side effects on the contractions of vascular smooth muscle.     

Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain

The two-headed kinesin motor harnesses the energy of ATP hydrolysis to take 8-nm steps, walking processively along a microtubule, alternately stepping with each of its catalytic heads in a hand-over-hand fashion. Two persistent challenges for models of kinesin motility are to explain how the two heads are coordinated ("gated") and when the translocation step occurs relative to other events in the mechanochemical reaction cycle. To investigate these questions, we used a precision optical trap to measure the single-molecule kinetics of kinesin in the presence of substrate analogs beryllium fluoride or adenylyl-imidodiphosphate. We found that normal stepping patterns were interspersed with long pauses induced by analog binding, and that these pauses were interrupted by short-lived backsteps. After a pause, processive stepping could only resume once the kinesin molecule took an obligatory, terminal backstep, exchanging the positions of its front and rear heads, presumably to allow release of the bound analog from the new front head. Preferential release from the front head implies that the kinetics of the two heads are differentially affected when both are bound to the microtubule, presumably by internal strain that is responsible for the gating. Furthermore, we found that ATP binding was required to reinitiate processive stepping after the terminal backstep. Together, our results support stepping models in which ATP binding triggers the mechanical step and the front head is gated by strain.     

On the hand-over-hand mechanism of kinesin

We present here a simple theoretical model for conventional kinesin. The model reproduces the hand-over-hand mechanism for kinesin walking to the plus end of a microtubule. A large hindering force induces kinesin to walk slowly to the minus end, again by a hand-over-hand mechanism. Good agreement is obtained between the calculated and experimental results on the external force dependence of the walking speed, the forward/backward step ratio, and dwell times for both forward and backward steps. The model predicts that both forward and backward motions of kinesin take place at the same chemical state of the motor heads, with the front head being occupied by an ATP (or ADP,Pi) and the rear being occupied by an ADP. The direction of motion is a result of the competition between the power stroke produced by the front head and the external load. The other predictions include the external force dependence of the chemomechanical coupling ratio (e.g., the stepping distance/ATP ratio) and the walking speed of kinesin at force ranges that have not been tested by experiments. The model predicts that the chemomechanical coupling remains tight in a large force range. However, when the external force is very large (e.g., approximately 18 pN), kinesin slides in an inchworm fashion, and the translocation of kinesin becomes loosely coupled to ATP turnovers.     

Kinesin ATPase: rate-limiting ADP release.

The ATPase rate of kinesin isolated from bovine brain by the method of S.A. Kuznetsov and V.I. Gelfand [(1986) Proc. Natl. Acad. Sci. USA 83, 8530-8534)] is stimulated 1000-fold by interaction with tubulin (turnover rate per 120-kDa peptide increases from approximately equal to 0.009 sec-1 to 9 sec-1). The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that Pi release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (Ki less than 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound ADP by [14C]ATP at the steady-state ATPase rate as shown by centrifuge gel filtration and inaccessibility to pyruvate kinase. Tubulin accelerates the release of the bound ADP consistent with its activation of the net ATPase reaction. The detailed kinetics of ADP release in the presence of tubulin are biphasic indicating apparent heterogeneity with a fraction of the kinesin active sites being unaffected by tubulin.     

Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis.

The N-terminal 392 amino acids of the Drosophila kinesin alpha subunit (designated DKH392) form a dimer in solution that releases only one of its two tightly bound ADP molecules on association with a microtubule, whereas a shorter monomeric construct (designated DKH340) releases > or = 95% of its one bound ADP on association with a microtubule. This half-site reactivity of dimeric DKH392 is observed over a wide range of ratios of DKH392 to microtubules and steady-state ATPase rates, indicating that it is characteristic of the mechanism of microtubule-stimulated ATP hydrolysis and not the result of a fortuitous balance of rate constants. When [alpha-32P]ATP is included in the medium, incorporation of 32P label into the pool of ADP that is bound to the complex of DKH392 and microtubules occurs rapidly enough for the bound ADP to be an intermediate on the main pathway of ATP hydrolysis. These and other results are consistent with the half-site reactivity being a consequence of the tethering of dimeric DKH392 to the microtubule through one head domain, which is attached in a rigor-like manner without bound nucleotide, whereas the other head is not attached to the microtubule and still contains a tightly bound ADP. An intermediate of this nature and the tight binding of DKH392 to microtubules in the presence of ATP suggest a mechanism for directed motility in which the head domains of dimeric DKH392 alternate in a sequential manner.     

P2-purinoceptor activation stimulates phosphoinositide hydrolysis and inhibits accumulation of cAMP in cultured ventricular myocytes.

Extracellular ATP modulates cardiac contraction through P2-purinoceptors on cardiac myocytes. To elucidate the molecular mechanism of this response, we examined the effects of P2-purinoceptor activation on phosphoinositide (PI) hydrolysis and the cAMP system in cultured ventricular myocytes of fetal mice. In a concentration-dependent manner, ATP stimulated accumulations of [3H]inositol monophosphate, bisphosphate, and trisphosphate with the half-maximum effective concentration of approximately 1 microM in the myocytes labeled with [3H]inositol. The order of efficacy of a series of adenyl compounds for stimulation of PI hydrolysis was adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ATP greater than ADP, 5'-adenylylimidodiphosphate (APPNP) greater than alpha,beta-methyleneadenosine 5'-triphosphate (APCPP) greater than beta,gamma-methyleneadenosine 5'-triphosphate, AMP greater than adenosine. On the other hand, 100 microM ATP gamma S inhibited isoproterenol-induced accumulation of cAMP by approximately 70% without decreasing the time to maximal cAMP levels, as measured by radioimmunoassay. This response was also concentration dependent, with a half-maximum inhibitory concentration (IC50) of approximately 1 microM. All of the tested ATP, ADP, and ATP analogues inhibited the cAMP system, and the responses to ATP gamma S, APPNP, and APCPP were insensitive to an A1-purinoceptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. Pertussis toxin attenuated the ATP-induced PI hydrolysis by no more than 25% at 100 ng/ml but completely suppressed the ATP gamma S-induced inhibition of the cAMP system.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution tracking of microtubule motility driven by a single kinesin motor.

Kinesin is a microtubule-based motor protein that contains two identical force-generating subunits. The kinesin binding sites along the microtubule lie 8 nm apart (the dimension of the tubulin dimer), which implies that kinesin must translocate a minimum distance of 8 nm per hydrolysis cycle. Measurements of kinesin's microtubule-stimulated ATPase activity (approximately 20 ATP per sec) and velocity of transport (approximately 0.6 micron/sec), however, suggest that the net distance moved per ATP (approximately 30 nm) may be greater than one tubulin dimer under zero load conditions. To explore how kinesin translocates during its ATPase cycle, we constructed a microscope capable of tracking movement with 1-nm resolution at a bandwidth of 200 Hz and used this device to examine microtubule movement driven by a single kinesin motor. Regular stepwise movements were not observed in displacement traces of moving microtubules, although Brownian forces acting on elastic elements within the kinesin motor precluded detection of steps that were < 12 nm. Though individual steps of approximately 16 nm were occasionally observed, their infrequent occurrence suggests that kinesin rarely moves abruptly by distances of two or more tubulin subunits during its ATP hydrolysis cycle. Instead it is more likely that kinesin moves forward by the distance of only a single tubulin subunit under zero load conditions.     

Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution

In order to operate in a coordinated fashion, multisubunit enzymes use cooperative interactions intrinsic to their enzymatic cycle, but this process remains poorly understood. Accordingly, ATP number distributions in various hydrolyzed states have been obtained for single copies of the mammalian double-ring multisubunit chaperonin TRiC/CCT in free solution using the emission from chaperonin-bound fluorescent nucleotides and closed-loop feedback trapping provided by an Anti-Brownian ELectrokinetic trap. Observations of the 16-subunit complexes as ADP molecules are dissociating shows a peak in the bound ADP number distribution at 8 ADP, whose height falls over time with little shift in the position of the peak, indicating a highly cooperative ADP release process which would be difficult to observe by ensemble-averaged methods. When AlFx is added to produce ATP hydrolysis transition state mimics (ADP·AlFx) locked to the complex, the peak at 8 nucleotides dominates for all but the lowest incubation concentrations. Although ensemble averages of the single-molecule data show agreement with standard cooperativity models, surprisingly, the observed number distributions depart from standard models, illustrating the value of these single-molecule observations in constraining the mechanism of cooperativity. While a complete alternative microscopic model cannot be defined at present, the addition of subunit-occupancy-dependent cooperativity in hydrolysis yields distributions consistent with the data.     

Kinesin-microtubule binding depends on both nucleotide state and loading direction

Kinesin is a motor protein that transports organelles along a microtubule toward its plus end by using the energy of ATP hydrolysis. To clarify the nucleotide-dependent binding mode, we measured the unbinding force for one-headed kinesin heterodimers in addition to conventional two-headed kinesin homodimers under several nucleotide states. We found that both a weak and a strong binding state exist in each head of kinesin corresponding to a small and a large unbinding force, respectively; that is, weak for the ADP state and strong for the nucleotide-free and adenosine 5'-[beta,gamma-imido]triphosphate states. Model analysis showed that (i) the two binding modes in each head could be explained by a difference in the binding energy and (ii) the directional instability of binding, i.e., dependence of unbinding force on loading direction, could be explained by a difference in the characteristic distance for the kinesin-microtubule interaction during plus- and minus-end-directed loading. Both these factors must play an important role in the molecular mechanism of kinesin motility.     

Mechanism of the myosin catalyzed hydrolysis of ATP as rationalized by molecular modeling

The intrinsic chemical reaction of adenosine triphosphate (ATP) hydrolysis catalyzed by myosin is modeled by using a combined quantum mechanics and molecular mechanics (QM/MM) methodology that achieves a near ab initio representation of the entire model. Starting with coordinates derived from the heavy atoms of the crystal structure (Protein Data Bank ID code 1VOM) in which myosin is bound to the ATP analog ADP.VO(4)(-), a minimum-energy path is found for the transformation ATP + H(2)O --> ADP + P(i) that is characterized by two distinct events: (i) a low activation-energy cleavage of the P(gamma) O(betagamma) bond and separation of the gamma-phosphate from ADP and (ii) the formation of the inorganic phosphate as a consequence of proton transfers mediated by two water molecules and assisted by the Glu-459-Arg-238 salt bridge of the protein. The minimum-energy model of the enzyme-substrate complex features a stable hydrogen-bonding network in which the lytic water is positioned favorably for a nucleophilic attack of the ATP gamma-phosphate and for the transfer of a proton to stably bound second water. In addition, the P(gamma) O(betagamma) bond has become significantly longer than in the unbound state of the ATP and thus is predisposed to cleavage. The modeled transformation is viewed as the part of the overall hydrolysis reaction occurring in the closed enzyme pocket after ATP is bound tightly to myosin and before conformational changes preceding release of inorganic phosphate.     

P1075 opens mitochondrial K(ATP) channels and generates reactive oxygen species resulting in cardioprotection of rabbit hearts

We have recently proposed that opening of mitochondrial K(ATP) channels (mitoK(ATP)) acts as a trigger for preconditioning (PC) by causing mitochondria to produce reactive oxygen species (ROS). Controversy exists as to whether the putative sarcolemma-selective K(ATP) channel opener P1075 also opens mitoK(ATP) channels and may be cardioprotective. We purified mitoK(ATP) channels from either rabbit heart, rat heart or rat brain and reconstituted the proteins into liposomes. mitoK(ATP) channels from each of these tissues were opened by P1075 with EC(50) values of 60-90 nM. We next tested whether P1075 causes rabbit cardiomyocytes to produce ROS in a K(ATP)-dependent fashion. Mitochondrial ROS production was monitored by the appearance of fluorescence as reduced MitoTracker Red was oxidized. P1075 (100 microM) led to a 44 +/- 9% increase in ROS generation (P < 0.001 vs. untreated cells), which was similar to the increase seen with 50 microM diazoxide, a selective mitoK(ATP) channel opener (49 +/- 9%, P < 0.001 vs. untreated cells). The effect of P1075 was equally potent at a concentration of 150 nM. The P1075-induced increase in ROS production was blocked by 50 microM glibenclamide (GLI), a non-selective K(ATP) blocker, and by 5-hydroxydecanoate (1 mM), a highly selective mitoK(ATP) blocker (-6 +/- 14% and +4 +/- 12%, respectively; P = n.s). In isolated rabbit hearts, P1075 (150 nM) markedly reduced infarct size compared to control animals (10.6 +/- 8.1% of the area at risk vs. 31.5 +/- 5.6%, P < 0.05). GLI (5 microM) as well as 5-hydroxydecanoate (200 microM) completely blocked P1075's anti-infarct effect (31.7 +/- 9.5% and 27.7 +/- 4.6% infarction, respectively; P = n.s. vs. untreated hearts). These data provide strong evidence that P1075 does open mitoK(ATP) channels and protects the ischemic rabbit heart in a mitoK(ATP)-dependent manner.     

Activation of pausing F1 motor by external force

A rotary motor F(1), a catalytic part of ATP synthase, makes a 120 degrees step rotation driven by hydrolysis of one ATP, which consists of 80 degrees and 40 degrees substeps initiated by ATP binding and probably by ADP and/or P(i) dissociation, respectively. During active rotations, F(1) spontaneously fails in ADP release and pauses after a 80 degrees substep, which is called the ADP-inhibited form. In the present work, we found that, when pushed >+40 degrees with magnetic tweezers, the pausing F(1) resumes its active rotation after releasing inhibitory ADP. The rate constant of the mechanical activation exponentially increased with the pushed angle, implying that F(1) weakens the affinity of its catalytic site for ADP as the angle goes forward. This finding explains not only its unidirectional nature of rotation, but also its physiological function in ATP synthesis; it would readily bind ADP from solution when rotated backward by an F(o) motor in the ATP synthase. Furthermore, the mechanical work for the forced rotation was efficiently converted into work for expelling ADP from the catalytic site, supporting the tight coupling between the rotation and catalytic event.