Coupled ER to Golgi transport reconstituted with purified cytosolic proteins

A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-alpha-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi transport even in the presence of saturating levels of purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-alpha-factor from docked vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking phase, but are required for efficient fusion of ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.     

Purified cystic fibrosis transmembrane conductance regulator (CFTR) does not function as an ATP channel

The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR). Previously, we provided definitive evidence that CFTR functions as a phosphorylation-regulated chloride channel in our planar lipid bilayer studies of the purified, reconstituted protein. Recent patch-clamp studies have lead to the suggestion that CFTR may also be capable of conducting ATP or inducing this function in neighboring channels. In the present study, we assessed the ATP channel activity of purified CFTR and found that the purified protein does not function as an ATP channel in planar bilayer studies of single channel activity nor in ATP flux measurements in proteoliposomes. Hence, CFTR does not possess intrinsic ATP channel activity and its putative role in cellular ATP transport may be indirect.     

Effect of quercetin on Hoechst 33342 transport by purified and reconstituted P-glycoprotein

Multidrug resistance due to P-glycoprotein is a serious impediment to successful chemotherapy of cancer. Numerous compounds are known that inhibit the drug-exporting function of P-glycoprotein. Understanding the mechanisms of action of these chemosensitizers is made difficult by the complexity of the in vivo cell systems usually employed. To examine the direct effects of chemosensitizers, we have developed a system in which purified and reconstituted P-glycoprotein transports. Hoechst 33342 from the lipid membrane to the aqueous interior of proteoliposomes, requiring ATP hydrolysis (Shapiro AB and Ling V, J Biol Chem 270: 16167-16175, 1995). Here, we use this system to understand the effect on P-glycoprotein of quercetin, one of three flavonoids that have been reported to have the unique property of stimulating drug transport by P-glycoprotein in vivo (Phang et al., Cancer Res 53: 5977-5981, 1993). Since flavonoids are abundant in food, it is important to understand their effects on the function of P-glycoprotein because of the implications for cancer chemotherapy. In our hands, quercetin inhibited P-glycoprotein-mediated Hoechst 33342 efflux and enhanced accumulation, as measured by flow cytometry, by multidrug-resistant CHRC5 cells. In the purified system, quercetin strongly inhibited Hoechst 33342 transport by P-glycoprotein, at least in part by inhibiting the ATPase activity of P-glycoprotein required for transport. We conclude that the previously reported stimulatory effect of quercetin on drug efflux from multidrug-resistant cells is not a direct effect on P-glycoprotein. The ATPase domain of P-glycoprotein may be an attractive target for new chemosensitizing agents.     

Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II. 1. A DNA-dependent burst in ATP hydrolysis

When bound to DNA, topoisomerase II from Saccharomyces cerevisiae exhibits burst kinetics with respect to ATP hydrolysis. Pre-steady-state analysis shows that the enzyme binds and hydrolyzes two ATP per reaction cycle. Our data indicate that at least one of the two ATP is rapidly hydrolyzed prior to the rate-determining step in the reaction mechanism. When DNA is not bound to topoisomerase II, the rate-determining step shifts to become either ATP binding or hydrolysis. Two possible mechanisms are proposed that agree with our observations.     

Molecular determinants of KATP channel inhibition by ATP

ATP-sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore-forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg-nucleotides complicates analysis of nucleotide inhibition of Kir6. 2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP-sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the beta-phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N-terminus preceding, and the C-terminus immediately following, the transmembrane domains. Some mutations in the C-terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single-channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C-terminus in KATP channel gating.     

Selective alteration of sodium channel gating by Australian funnel-web spider toxins

The actions of potent mammalian neurotoxins isolated from the venom of two Australian funnel-web spiders were investigated using both electrophysiological and neurochemical techniques. Whole-cell patch clamp recording of sodium currents in rat dorsal root ganglion neurons revealed that versutoxin (VTX), isolated from the venom of Hadronyche versuta, produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation and a reduction in peak TTX-S sodium current. In contrast, VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. VTX also shifted the voltage dependence of sodium channel activation in the hyperpolarizing direction and increased the rate of recovery from inactivation. Ion flux studies performed in rat brain synaptosomes also revealed that robustoxin (RTX), from the venom of Atrax robustus, and VTX both produced a partial activation of 22Na+ flux and an inhibition of batrachotoxin-activated 22Na+ flux. This inhibition of flux through batrachotoxin-activated channels was not due to an interaction with neurotoxin receptor site 1 since [3H]saxitoxin binding was unaffected. In addition, the partial activation of 22Na+ flux was not enhanced in the presence of alpha-scorpion toxin and further experiments suggest that VTX also enhances [3H]batrachotoxin binding. These selective actions of funnel-web spider toxins on sodium channel function are comparable to those of alpha-scorpion and sea anemone toxins which bind to neurotoxin receptor site 3 on the channel to slow channel inactivation profoundly. Also, these modifications of sodium channel gating and kinetics are consistent with actions of the spider toxins to produce repetitive firing of action potentials.     

Nonequilibrium response spectroscopy of voltage-sensitive ion channel gating

We describe a new electrophysiological technique called nonequilibrium response spectroscopy, which involves application of rapidly fluctuating (as high as 14 kHz) large-amplitude voltage clamp waveforms to ion channels. As a consequence of the irreversible (in the sense of Carnot) exchange of energy between the fluctuating field and the channel protein, the gating response is exquisitely sensitive to features of the kinetics that are difficult or impossible to adequately resolve by means of traditional stepped potential protocols. Here we focus on the application of dichotomous (telegraph) noise voltage fluctuations, a broadband Markovian colored noise that fluctuates between two values. Because Markov kinetic models of channel gating can be embedded within higher-dimensional Markov models that take into account the effects of the voltage fluctuations, many features of the response of the channels can be calculated algebraically. This makes dichotomous noise and its generalizations uniquely suitable for model selection and kinetic analysis. Although we describe its application to macroscopic ionic current measurements, the nonequilibrium response method can also be applied to gating and single channel current recording techniques. We show how data from the human cardiac isoform (hH1a) of the Na+ channel expressed in mammalian cells can be acquired and analyzed, and how these data reveal hidden aspects of the molecular kinetics that are not revealed by conventional methods.     

PinA inhibits ATP hydrolysis and energy-dependent protein degradation by Lon protease

The bacteriophage T4 PinA protein inhibited degradation of [3H]alpha-methyl casein by purified Lon protease from Escherichia coli, but inhibition was noncompetitive with respect to casein. PinA did not inhibit cleavage of the fluorogenic peptide, N-glutaryl-alanylalanylphenylalanyl-3-methoxynaphthylamide and, moreover, did not block the ability of protein substrates, such as casein, to activate cleavage of fluorogenic peptides by Lon. Thus, PinA does not block the proteolytic active site or the allosteric protein-binding site on Lon. Inhibition of basal ATPase activity was variable (50-90%), whereas inhibition of protein-activated ATPase activity was usually 80-95%. Inhibition was noncompetitive with respect to ATP. PinA did not block activation of peptide cleavage by nonhydrolyzable analogs of ATP. These data suggest that PinA does not bind at the ATPase active site of Lon and does not interfere with nucleotide binding to the enzyme. PinA inhibited cleavage of the 72-amino acid protein, CcdA, degradation of which requires ATP hydrolysis, but did not inhibit cleavage of the carboxyl-terminal 41-amino acid fragment of CcdA, degradation of which does not require ATP hydrolysis. PinA thus appears to interact at a novel regulatory or enzymatic site involved in the coupling between ATP hydrolysis and proteolysis, possibly blocking the protein unfolding or remodeling step essential for degradation of high molecular weight protein substrates by Lon.     

ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin. 2. Relationships between proton motive force and ATP synthesis

The correlation between the rate of ATP synthesis and light-induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from thermophilic Bacillus PS3. By variation of the actinic light intensity it was found that ATP synthase activity depended in a sigmoidal manner on the amplitude of the transmembrane light-induced pH gradient. Maximal rates of ATP synthesis (up to to 200 nmol ATP x min(-1) x mg protein (-1) were obtained at saturating light intensities under a steady-state pH gradient of about pH 1.25. It was demonstrated that this was the maximal deltapH attainable at 40 degrees C in reconstituted proteoliposomes, due to the feedback inhibition of bacteriorhodopsin by the proton gradient it generates. In the absence of valinomycin, a small but significant transmembrane electrical potential could develop at 40 degrees C, contributing to an increase in the rate of ATP synthesis. The H+/ATP stoichiometry was measured at the static-head (equilibrium) conditions from the ratio of the phosphate potential to the size of the light-induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gradient. Increasing the amount of bacteriorhodopsin in the proteoliposomes at a constant F0F1 concentration led to a large increase in the rate of ATP synthesis whereas the magnitude of delta pH remained the same or, at very high bacteriorhodopsin levels, decreased. Consequently the H+/ATP stoichiometry was found to increase significantly with increasing bacteriorhodopsin content. Reconstitutions with mixtures of native and impaired bacteriorhodopsin (Asp96-->Asn mutated bacteriorhodopsin) further demonstrated that this increase in the coupling efficiency could not be related to protein-protein interactions but rather to bacteriorhodopsin donating H+ to the ATP synthase. Increasing the amount of negatively charged phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transmembrane pH gradient. Similar results were obtained with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by delta pH/delta psi transitions were independent of bacteriorhodopsin or anionic lipid levels. These observations were interpreted as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposomes, a localized pathway for coupling light-driven H+ transport by bacteriorhodopsin to ATP synthesis by F0F1 might exist under specific experimental conditions.     

ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits

Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase delta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report, we introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC. These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits.     

Three-dimensional architecture and gating mechanism of a K+ channel studied by EPR spectroscopy

The transmembrane organization of a potassium channel from Streptomyces lividans has been studied using site-directed spin labeling techniques and electron paramagnetic resonance spectroscopy. In the tetrameric channel complex, two alpha-helices were identified per monomer and assigned to the amino acid sequence. Probe mobility and accessibility data clearly establish that the first helix (TM1) is located in the perimeter of the channel, showing extensive protein-lipid contacts, while the second helix (TM2) is closer to the four-fold symmetric axis of the channel, lining the intracellular vestibule. A large conformational change in the C-terminal end of TM2 was measured when comparing conditions that favor either the open or closed states. The present data suggest that the diameter of the internal vestibule increases with channel opening.     

Minireview: on the structure and gating mechanism of the mitochondrial channel, VDAC

The peripheral artery occlusive disease is a widely spread disease and its diagnosis, treatment options and consequences are frequently underestimated. Especially for the old patient, preservation of an extremity may mean mobility and quality of life. The increasing life expectancy and behaviour of prosperity including a lack of mobility are causing a rise in the frequence of atherosclerotic diseases. The prevalence of occlusive vascular diseases in patients between 55 to 64 years of age is currently 11% and is, therefore, a wide-spread disease. However, the socio-economic relevance of the occlusive vascular diseases is frequently underestimated. It causes both very high direct costs (treatment procedures, prostheses etc) as well as high indirect costs (permanent disability). Therefore, early diagnosis and treatment plays an important role in the avoidance of a progression of the disease. For an early diagnosis of the stage I of occlusive vascular diseases it makes sense to examine the vessels of patients at risk (i.e. diabetes mellitus, hypertension, hyperlipidemia, nicotine abuse, and overweight). Dopplerultra-sound and oscillometry are highly sensitive and specific diagnostic measures. The eradication of risk factor and the treatment of the secondary diseases plays the most important role in this disease stage without symptoms. A specific vessel training is indicated during stage II to encourage the development of collateral blood flow. Additionally, an interventional diagnostic and therapy should be considered in this stage with limitations in the daily activities. The administration of vasoactive drugs is controversly discussed. The acetylsalicylic acid (ASA) is remaining the most investigated substance for reducing the progress of the arteriosclerotic process. The administration of ticlopidine is justified in cases of ASA-allergies. The stages III and IV are characterized by pain at rest and necrosis. Firstly, the indication for a transcutaneous transluminal angioplasty, thrombolysis or bypass-surgery should be proofed. If procedures of revascularization are not possible, prostaglandines may improve the pain at rest and wound healing. Beside the stage of the occlusive vascular disease, the presence of risk factors, the physical status of the patient, and the location of the occlusion are of great importance for the decision about the treatment procedure.     

Allosteric gating of a large conductance Ca-activated K+ channel

Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca2+. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca2+ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca2+ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca2+ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca2+ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations.     

Musing of the feline hepatic voltage-dependent delayed-rectifier potassium channel gating

SETTING: Measures known to improve adherence such as short course chemoprophylaxis and directly observed therapy can be enhanced to a significant extent/by the use of incentives. Adherence to tuberculosis therapy is influenced by several factors, including the health care system, complexity of therapeutic regimens and patient's characteristics. Individual factors that negatively influence patient's adherence are the most difficult to counter. Preventive tuberculosis therapy is doubly challenging because the benefit of treatment is not felt, while toxicity from the medication, when it occurs, is experienced immediately. Ingenious incentives therefore have to make it worth the patient's while. During a study on preventive regimens, a request for an incentive, Sustacal, was observed to help completion of preventive regimens. Components of individual TB programs may help in patient adherence; it is important for health care staff to identify these aspects and, if they are successful, utilize these as an incentive to complete treatment.     

Open channel block and alteration of N-methyl-D-aspartic acid receptor gating by an analog of phencyclidine

We investigated inhibition of the N-methyl-D-aspartic acid (NMDA) receptor-channel complex by N-ethyl-1,4,9, 9alpha-tetrahydro-4alphaR-cis-4alphaH-fluoren-++ +4alpha-amine (NEFA), a structural analog of phencyclidine (PCP). Using the whole-cell recording technique, we demonstrated that NEFA inhibits NMDA responses with an IC50 of 0.51 microM at -66 mV. We determined that NEFA binds to the open channel, and subsequently the channel can close and trap the blocker. Once the channel has closed, NEFA is unable to dissociate until the channel reopens. Single-channel recordings revealed that NEFA reduces the mean open time of single NMDA-activated channels in a concentration-dependent manner with a forward blocking rate (k+) of 39.9 microM-1 s-1. A computational model of antagonism by NEFA was developed and constrained using kinetic measurements of single-channel data. By multiple criteria, only models in which blocker binding in the channel causes a change in receptor operation adequately fit or predicted whole-cell data. By comparing model predictions and experimental measurements of NEFA action at a high NMDA concentration, we determined that NEFA affects receptor operation through an influence on channel gating. We conclude that inhibition of NMDA receptors by PCP-like blockers involves a modification of channel gating as well as block of current flow through the open channel.     

Depolarization shifts the voltage dependence of cardiac sodium channel and calcium channel gating charge movements

In cardiac ventricular myocytes, membrane depolarization leads to the inactivation of the Na channel and Ca channel ionic currents. The inactivation of the ionic currents has been associated with a reduction of the gating charge movement ("immobilization") which governs the activation of Na channels and Ca channels. The nature of the apparent "immobilization" of the charge movement following depolarization was explored in embryonic chick ventricular myocytes using voltage protocols applied from depolarized holding potentials. It was found that although all of the charge was mobile following inactivation, the voltage dependence of its movement was shifted to more negative potentials. In addition, the shift in the distribution of the Na channel charge could be differentiated from that of the Ca channel charge on the basis of kinetic as well as steady-state criteria. These results suggest that the voltage-dependent activation of Na channel and Ca channel charge movements leads to conformational changes and charge rearrangements that differentially bias the movements of these voltage sensors, and concomitantly produce channel inactivation.     

Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator

CFTR is a cyclic AMP (cAMP)-activated chloride (Cl-) channel and a regulator of outwardly rectifying Cl- channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl- channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl- efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl- channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted alpha-helices 5 and 6, forms an essential part of the Cl- channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl- channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.     

Insertion scanning mutagenesis of subunit a of the F1F0 ATP synthase near His245 and implications on gating of the proton channel

Subunit a of the E. coli F1F0 ATP synthase was probed by insertion scanning mutagenesis in a region between residues Glu219 and His245. A series of single amino acid insertions, of both alanine and aspartic acid, were constructed after the following residues: 225, 229, 233, 238, 243, and 245. The mutants were tested for growth yield, binding of F1 to membranes, dicyclohexylcarbodiimide sensitivity of ATPase activity, ATP-driven proton translocation, and passive proton permeability of membranes stripped of F1. Significant loss of function was seen only with insertions after positions 238 and 243. In contrast, both insertions after residue 225 and the alanine insertion after residue 245 were nearly identical in function to the wild type. The other insertions showed an intermediate loss of function. Missense mutations of His245 to serine and cysteine were nonfunctional, while the W241C mutant showed nearly normal ATPase function. Replacement of Leu162 by histidine failed to suppress the 245 mutants, but chemical rescue of H245S was partially successful using acetate. An interaction between Trp241 and His245 may be involved in gating a "half-channel" from the periplasmic surface of F0 to Asp61 of subunit a.     

Recovery of free ADP, Pi, and free energy of ATP hydrolysis in human skeletal muscle

BACKGROUND: Recent studies have demonstrated that a high concentration of phosphate directly stimulates parathyroid hormone (PTH) secretion. High serum levels of phosphate are usually observed in patients with end-stage renal disease. The aim of the present study was to evaluate whether serum phosphate concentration had an acute effect on PTH secretion in hemodialysis patients. The levels of serum phosphate were manipulated during the hemodialysis session by using a phosphate free dialysate or a dialysate with a high content of phosphate. METHODS: Ten stable hemodialysis patients with PTH values above 300 pg/ml were included in the study. A PTH-calcium curve was obtained during both high phosphate and phosphate free hemodialysis. RESULTS: The serum phosphate concentration remained high (2.17 +/- 0.18 mM) throughout the high phosphate hemodialysis and decreased progressively to normal levels (1.02 +/- 0.06 mM) during the phosphate free hemodialysis. The serum PTH levels at maximal inhibition by hypercalcemia (minimal PTH) were greater during the high phosphate than the phosphate free hemodialysis (413 +/- 79 vs. 318 +/- 76 pg/ml, P < 0.003). In all patients the values of minimum PTH were greater during the high phosphorus than the phosphorus free hemodialysis. The values of maximally stimulated PTH during hypocalcemia and the set point of the PTH-calcium curve were similar during the high phosphate and the phosphate free hemodialysis. CONCLUSION: The maintenance of high serum phosphorus levels during hemodialysis prevented, in part, the inhibition of PTH secretion by calcium, which strongly suggests that in hemodialysis patients high serum phosphate contributes directly to the elevation of PTH levels despite normal or high serum calcium concentration.     

ATP flux is controlled by a voltage-gated channel from the mitochondrial outer membrane

A voltage-gated channel, called VDAC (mitochondrial porin) is known to be responsible for most of the metabolite flux across the mitochondrial outer membrane. Here, direct measurements of ATP flux through VDAC channels reconstituted into planar phospholipid membranes establish that VDAC is sufficient to provide passage for ATP efflux from mitochondria. Further, the gating of the channel can shut down ATP flux completely while, simultaneously, allowing the flow of small ions. Thus, these channels are ideally suited to control ATP flux through the mitochondrial outer membrane and, consequently, mitochondrial function. The block to ATP flow through the closed state is likely to be not steric but electrostatic.