A loss-of-function mutation in KCNJ11 causing sulfonylurea-sensitive diabetes in early adult life.

AIMS/HYPOTHESIS: The ATP-sensitive potassium (KATP) channel couples beta cell electrical activity to glucose-stimulated insulin secretion. Loss-of-function mutations in either the pore-forming (inwardly rectifying potassium channel 6.2 [Kir6.2], encoded by KCNJ11) or regulatory (sulfonylurea receptor 1, encoded by ABCC8) subunits result in congenital hyperinsulinism, whereas gain-of-function mutations cause neonatal diabetes. Here, we report a novel loss-of-function mutation (Ser118Leu) in the pore helix of Kir6.2 paradoxically associated with sulfonylurea-sensitive diabetes that presents in early adult life. METHODS: A 31-year-old woman was diagnosed with mild hyperglycaemia during an employee screen. After three pregnancies, during which she was diagnosed with gestational diabetes, the patient continued to show elevated blood glucose and was treated with glibenclamide (known as glyburide in the USA and Canada) and metformin. Genetic testing identified a heterozygous mutation (S118L) in the KCNJ11 gene. Neither parent was known to have diabetes. We investigated the functional properties and membrane trafficking of mutant and wild-type KATP channels in Xenopus oocytes and in HEK-293T cells, using patch-clamp, two-electrode voltage-clamp and surface expression assays. RESULTS: Functional analysis showed no changes in the ATP sensitivity or metabolic regulation of the mutant channel. However, the Kir6.2-S118L mutation impaired surface expression of the KATP channel by 40%, categorising this as a loss-of-function mutation. CONCLUSIONS/INTERPRETATION: Our data support the increasing evidence that individuals with mild loss-of-function KATP channel mutations may develop insulin deficiency in early adulthood and even frank diabetes in middle age. In this case, the patient may have had hyperinsulinism that escaped detection in early life. Our results support the importance of functional analysis of KATP channel mutations in cases of atypical diabetes.

The dynamic interplay of PIP2 and ATP in the regulation of the KATP channel.

ATP-sensitive potassium (KATP ) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol 4,5-bisphosphate (PIP2 ). Here, we use molecular dynamics simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, simulations suggest that K39 shows a greater preference to co-ordinate with PIP2 than with ATP. They also predict that a neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2 , potentially accounting for the reduced ATP inhibition observed in electrophysiological experiments. Our work suggests that PIP2 and ATP interact allosterically to regulate KATP channel activity. KEY POINTS: The KATP channel is activated by the binding of phosphatidylinositol 4,5-bisphosphate (PIP2 ) lipids and inactivated by the binding of ATP. K39 has the potential to bind to both PIP2 and ATP. A mutation to this residue (K39R) results in neonatal diabetes. This study uses patch-clamp fluorometry, electrophysiology and molecular dynamics simulation. We show that PIP2 competes with ATP for K39, and this reduces channel inhibition by ATP. We show that K39R increases channel affinity to PIP2 by increasing the number of hydrogen bonds with PIP2 , when compared with the wild-type K39. This therefore decreases KATP channel inhibition by ATP.

NMDA Receptor Antagonists Increase the Release of GLP-1 From Gut Endocrine Cells.

Type 2 diabetes mellitus (T2DM) remains one of the most pressing health issues facing modern society. Several antidiabetic drugs are currently in clinical use to treat hyperglycaemia, but there is a need for new treatments that effectively restore pancreatic islet function in patients. Recent studies reported that both murine and human pancreatic islets exhibit enhanced insulin release and ß-cell viability in response to N-methyl-D-aspartate (NMDA) receptor antagonists. Furthermore, oral administration of dextromethorphan, an over-the-counter NMDA receptor antagonist, to diabetic patients in a small clinical trial showed improved glucose tolerance and increased insulin release. However, the effects of NMDA receptor antagonists on the secretion of the incretin hormone GLP-1 was not tested, and nothing is known regarding how NMDA receptor antagonists may alter the secretion of gut hormones. This study demonstrates for the first time that, similar to ß-cells, the NMDA receptor antagonist MK-801 increases the release of GLP-1 from a murine L-cell enteroendocrine model cell line, GLUTag cells. Furthermore, we report the 3' mRNA expression profiling of GLUTag cells, with a specific focus on glutamate-activated receptors. We conclude that if NMDA receptor antagonists are to be pursued as an alternative, orally administered treatment for T2DM, it is essential that the effects of these drugs on the release of gut hormones, and specifically the incretin hormones, are fully investigated.

Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding.

The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg2+ was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount.

Role of the C-terminus of SUR in the differential regulation of ß-cell and cardiac KATP channels by MgADP and metabolism.

KEY POINTS: ß-Cell KATP channels are partially open in the absence of metabolic substrates, whereas cardiac KATP channels are closed. Using cloned channels heterologously expressed in Xenopus oocytes we measured the effect of MgADP on the MgATP concentration-inhibition curve immediately after patch excision. MgADP caused a far more striking reduction in ATP inhibition of Kir6.2/SUR1 channels than Kir6.2/SUR2A channels; this effect declined rapidly after patch excision. Exchanging the final 42 amino acids of SUR was sufficient to switch the Mg-nucleotide regulation of Kir6.2/SUR1 and Kir6.2/SUR2A channels, and partially switch their sensitivity to metabolic inhibition. Deletion of the C-terminal 42 residues of SUR abolished MgADP activation of both Kir6.2/SUR1 and Kir6.2/SUR2A channels. We conclude that the different metabolic sensitivity of Kir6.2/SUR1 and Kir6.2/SUR2A channels is at least partially due to their different regulation by Mg-nucleotides, which is determined by the final 42 amino acids. ABSTRACT: ATP-sensitive potassium (KATP ) channels couple the metabolic state of a cell to its electrical activity and play important physiological roles in many tissues. In contrast to ß-cell (Kir6.2/SUR1) channels, which open when extracellular glucose levels fall, cardiac (Kir6.2/SUR2A) channels remain closed. This is due to differences in the SUR subunit rather than cell metabolism. As ATP inhibition and MgADP activation are similar for both types of channels, we investigated channel inhibition by MgATP in the presence of 100 µm MgADP immediately after patch excision [when the channel open probability (PO ) is near maximal]. The results were strikingly different: 100 µm MgADP substantially reduced MgATP inhibition of Kir6.2/SUR1, but had no effect on MgATP inhibition of Kir6.2/SUR2A. Exchanging the final 42 residues of SUR2A with that of SUR1 switched the channel phenotype (and vice versa), and deleting this region abolished Mg-nucleotide activation. This suggests the C-terminal 42 residues are important for the ability of MgADP to influence ATP inhibition at Kir6.2. This region was also necessary, but not sufficient, for activation of the KATP channel in intact cells by metabolic inhibition (azide). We conclude that the ability of MgADP to impair ATP inhibition at Kir6.2 accounts, in part, for the differential metabolic sensitivities of ß-cell and cardiac KATP channels.

Hypothermia-induced dystonia and abnormal cerebellar activity in a mouse model with a single disease-mutation in the sodium-potassium pump.

Mutations in the neuron-specific α3 isoform of the Na+/K+-ATPase are found in patients suffering from Rapid onset Dystonia Parkinsonism and Alternating Hemiplegia of Childhood, two closely related movement disorders. We show that mice harboring a heterozygous hot spot disease mutation, D801Y (α3+/D801Y), suffer abrupt hypothermia-induced dystonia identified by electromyographic recordings. Single-neuron in vivo recordings in awake α3+/D801Y mice revealed irregular firing of Purkinje cells and their synaptic targets, the deep cerebellar nuclei neurons, which was further exacerbated during dystonia and evolved into abnormal high-frequency burst-like firing. Biophysically, we show that the D-to-Y mutation abolished pump-mediated Na+/K+ exchange, but allowed the pumps to bind Na+ and become phosphorylated. These findings implicate aberrant cerebellar activity in α3 isoform-related dystonia and add to the functional understanding of the scarce and severe mutations in the α3 isoform Na+/K+-ATPase.

Neonatal Diabetes and the KATP Channel: From Mutation to Therapy.

Activating mutations in one of the two subunits of the ATP-sensitive potassium (KATP) channel cause neonatal diabetes (ND). This may be either transient or permanent and, in approximately 20% of patients, is associated with neurodevelopmental delay. In most patients, switching from insulin to oral sulfonylurea therapy improves glycemic control and ameliorates some of the neurological disabilities. Here, we review how KATP channel mutations lead to the varied clinical phenotype, how sulfonylureas exert their therapeutic effects, and why their efficacy varies with individual mutations.

Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels.

KATP channels act as key regulators of electrical excitability by coupling metabolic cues-mainly intracellular adenine nucleotide concentrations-to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.

Successful transfer to sulfonylureas in KCNJ11 neonatal diabetes is determined by the mutation and duration of diabetes.

AIMS/HYPOTHESIS: The finding that patients with diabetes due to potassium channel mutations can transfer from insulin to sulfonylureas has revolutionised the management of patients with permanent neonatal diabetes. The extent to which the in vitro characteristics of the mutation can predict a successful transfer is not known. Our aim was to identify factors associated with successful transfer from insulin to sulfonylureas in patients with permanent neonatal diabetes due to mutations in KCNJ11 (which encodes the inwardly rectifying potassium channel Kir6.2). METHODS: We retrospectively analysed clinical data on 127 patients with neonatal diabetes due to KCNJ11 mutations who attempted to transfer to sulfonylureas. We considered transfer successful when patients completely discontinued insulin whilst on sulfonylureas. All unsuccessful transfers received ≥0.8 mg kg(-1) day(-1) glibenclamide (or the equivalent) for >4 weeks. The in vitro response of mutant Kir6.2/SUR1 channels to tolbutamide was assessed in Xenopus oocytes. For some specific mutations, not all individuals carrying the mutation were able to transfer successfully; we therefore investigated which clinical features could predict a successful transfer. RESULTS: In all, 112 out of 127 (88%) patients successfully transferred to sulfonylureas from insulin with an improvement in HbA1c from 8.2% (66 mmol/mol) on insulin, to 5.9% (41 mmol/mol) on sulphonylureas (p = 0.001). The in vitro response of the mutation to tolbutamide determined the likelihood of transfer: the extent of tolbutamide block was <63% for the p.C166Y, p.I296L, p.L164P or p.T293N mutations, and no patients with these mutations successfully transferred. However, most individuals with mutations for which tolbutamide block was >73% did transfer successfully. The few patients with these mutations who could not transfer had a longer duration of diabetes than those who transferred successfully (18.2 vs 3.4 years, p = 0.032). There was no difference in pre-transfer HbA1c (p = 0.87), weight-for-age z scores (SD score; p = 0.12) or sex (p = 0.17). CONCLUSIONS/INTERPRETATION: Transfer from insulin is successful for most KCNJ11 patients and is best predicted by the in vitro response of the specific mutation and the duration of diabetes. Knowledge of the specific mutation and of diabetes duration can help predict whether successful transfer to sulfonylureas is likely. This result supports the early genetic testing and early treatment of patients with neonatal diabetes aged under 6 months.

Neonatal diabetes caused by a homozygous KCNJ11 mutation demonstrates that tiny changes in ATP sensitivity markedly affect diabetes risk.

AIMS/HYPOTHESIS: The pancreatic ATP-sensitive potassium (KATP) channel plays a pivotal role in linking beta cell metabolism to insulin secretion. Mutations in KATP channel genes can result in hypo- or hypersecretion of insulin, as in neonatal diabetes mellitus and congenital hyperinsulinism, respectively. To date, all patients affected by neonatal diabetes due to a mutation in the pore-forming subunit of the channel (Kir6.2, KCNJ11) are heterozygous for the mutation. Here, we report the first clinical case of neonatal diabetes caused by a homozygous KCNJ11 mutation. METHODS: A male patient was diagnosed with diabetes shortly after birth. At 5 months of age, genetic testing revealed he carried a homozygous KCNJ11 mutation, G324R, (Kir6.2-G324R) and he was successfully transferred to sulfonylurea therapy (0.2 mg kg(-1) day(-1)). Neither heterozygous parent was affected. Functional properties of wild-type, heterozygous and homozygous mutant KATP channels were examined after heterologous expression in Xenopus oocytes. RESULTS: Functional studies indicated that the Kir6.2-G324R mutation reduces the channel ATP sensitivity but that the difference in ATP inhibition between homozygous and heterozygous channels is remarkably small. Nevertheless, the homozygous patient developed neonatal diabetes, whereas the heterozygous parents were, and remain, unaffected. Kir6.2-G324R channels were fully shut by the sulfonylurea tolbutamide, which explains why the patient's diabetes was well controlled by sulfonylurea therapy. CONCLUSIONS/INTERPRETATION: The data demonstrate that tiny changes in KATP channel activity can alter beta cell electrical activity and insulin secretion sufficiently to cause diabetes. They also aid our understanding of how the Kir6.2-E23K variant predisposes to type 2 diabetes.

The Nucleotide-Binding Sites of SUR1: A Mechanistic Model.

ATP-sensitive potassium (KATP) channels comprise four pore-forming Kir6.2 subunits and four modulatory sulfonylurea receptor (SUR) subunits. The latter belong to the ATP-binding cassette family of transporters. KATP channels are inhibited by ATP (or ADP) binding to Kir6.2 and activated by Mg-nucleotide interactions with SUR. This dual regulation enables the KATP channel to couple the metabolic state of a cell to its electrical excitability and is crucial for the KATP channel's role in regulating insulin secretion, cardiac and neuronal excitability, and vascular tone. Here, we review the regulation of the KATP channel by adenine nucleotides and present an equilibrium allosteric model for nucleotide activation and inhibition. The model can account for many experimental observations in the literature and provides testable predictions for future experiments.

Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps.

A single Na(+)/K(+)-ATPase pumps three Na(+) outwards and two K(+) inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na(+) than K(+) generates outward current across the cell membrane. Less well understood is the ability of Na(+)/K(+) pumps to generate an inward current of protons. Originally noted in pumps deprived of external K(+) and Na(+) ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K(+) and Na(+) concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na(+) release from phosphorylated Na(+)/K(+) pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na(+)/K(+) pumps that enables proton import is not required for completion of the 3 Na(+)/2 K(+) transport cycle. However, the back-step occurs readily during Na(+)/K(+) transport when external K(+) ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na(+)-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na(+) and K(+) ions that passes through binding site II. The inferred occurrence of Na(+)/K(+) exchange and H(+) import during the same conformational cycle of a single molecule identifies the Na(+)/K(+) pump as a hybrid transporter. Whether Na(+)/K(+) pump-mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.

The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions.

Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K-adenosine triphosphatase (ATPase) alpha subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane's electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis alpha1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain-sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 microM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump-induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the binding pocket. Gating measurements of palytoxin-opened Na/K pump channels additionally imply that the C-terminal contacts also help stabilize pump conformations with occluded K ions.

Plasma membrane-porating domain in poliovirus 2B protein. A short peptide mimics viroporin activity.

Picornavirus 2B, a non-structural protein required for effective viral replication, has been implicated in cell membrane permeabilization during the late phases of infection. Here, we have approached the molecular mechanism of this process by assessing the pore-forming activity of an overlapping peptide library that spanned the complete 2B sequence. At non-cytopathic concentrations, only the P3 peptide, spanning 2B residues 35-55, effectively assembled hydrophilic pores that allowed diffusion of low molecular mass solutes across the cell plasma membrane (IC(50) approximately 4x10(-7) M) and boundary liposome bilayers (starting at peptide to lipid molar ratios>1:10(4)). Circular dichroism data were consistent with its capacity to fold as a helix in a membrane-like environment. Furthermore, addition of this peptide to a sealed plasma-membrane model, consisting of retinal rod outer segments patch-clamped in a whole-cell configuration, induced ion channel activity within seconds at concentrations as low as 10(-8) M. Thus, we have established a "one-helix" 2B version that possesses the intrinsic pore-forming activity required to directly and effectively permeabilize the cell plasma membrane. We conclude that 2B viroporin can be classified as a genuine pore-forming toxin of viral origin, which is produced intracellularly at certain times post infection.

A novel technique to study pore-forming peptides in a natural membrane.

The biophysical characteristics and the pore formation dynamics of synthetic or naturally occurring peptides forming membrane-spanning channels were investigated by using isolated photoreceptor rod outer segments (OS) recorded in whole-cell configuration. Once blocking the two OS endogenous conductances (the cGMP channels by light and the Na(+):Ca(2+),K(+) exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K(+), Na(+) or Ca(2+)), the OS membrane resistance (R ( m )) was typically larger than 1 GOmega in the presence of 1 mM external Ca(2+). Therefore, any exogenous current could be studied down to the single channel level. The peptides were applied to (and removed from) the extracellular OS side in approximately 50 ms with a computer-controlled microperfusion system, in which every perfusion parameter, as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles were controlled by a user-friendly interface. This technique was then used to determine the biophysical properties and the pore formation dynamics of antibiotic peptaibols, as the native alamethicin mixture, the synthesized major component of the neutral fraction (F50/5) of alamethicin, and the synthetic trichogin GA IV.

Pore-forming properties of alamethicin F50/5 inserted in a biological membrane.

The pore-forming properties of native and synthetic alamethicins were investigated in photoreceptor rod outer segments (OS) isolated from frog retina, and recorded in whole-cell configuration. The peptaibols were applied (and removed) to (from) the OS within less than 50 ms by means of a computer-controlled micro-perfusion system. Once blocked with light, the main OS endogenous conductance, the OS membrane resistance was >1 GOmega, allowing low-noise and high-resolution recordings. Currents of ca. 700 pA were recorded in symmetric K(+) (100 mM) and Ca(2+) (1 mM), upon applying 1 microM of alamethicin F50/5 or its [L-Glu(OMe)(7,18,19)] analogue to the OS membrane (clamped at -20 mV). In the latter peptide, the Gln residues at positions 7, 18, and 19 were substituted with side-chain esterified Glu residues. For both peptides, the current activated exponentially, with a delay from peptide application, and exponentially returned to zero without any delay, upon removing the peptide from the external solution. The delay as well as the activation (tau(a)) and deactivation (tau(d)) time constants of the current produced by the modified alamethicin were much slower, and the current noise was much larger, with respect to the corresponding values for alamethicin F50/5. Therefore, the above three Gln residues are not a key factor for pore formation, but the [L-Glu(OMe)(7,18,19)] analogue produces larger pores with a lower probability of formation.

Modulation of the reaction cycle of the Na+:Ca2+, K+ exchanger.

Ca(2+) concentration in retinal photoreceptor rod outer segment (OS) strongly affects the generator potential kinetics and the receptor light adaptation. The response to intense light stimuli delivered in the dark produce potential changes exceeding 40 mV: since the Ca(2+) extrusion in the OS is entirely controlled by the Na(+):Ca(2+), K(+) exchanger, it is important to assess how the exchanger ion transport rate is affected by the voltage and, in general, by intracellular factors. It is indeed known that the cardiac Na(+):Ca(2+) exchanger is regulated by Mg-ATP via a still unknown metabolic pathway. In the present work, the Na(+):Ca(2+), K(+) exchanger regulation was investigated in isolated OS, recorded in whole-cell configuration, using ionic conditions that activated maximally the exchanger in both forward and reverse mode. In all species examined (amphibia: Rana esculenta and Ambystoma mexicanum; reptilia: Gecko gecko), the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. Since hyperpolarisation increases Ca(2+) extrusion rate, the recovery of the dark level of Ca(2+) (and, in turn, of the generator potential) after intense light stimuli results accelerated. Mg-ATP increased the size of forward and reverse exchange current by a factor of approximately 2.3 and approximately 2.6, respectively, without modifying their voltage dependence. This indicates that Mg-ATP regulates the number of active exchanger sites and/or the exchanger turnover number, although via an unknown mechanism.