Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels

Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1–KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1–KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1)₄(KCNE1)_n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or I_Ks, is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether I_Ks is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments I_Ks current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1)₄(KCNE1)_n channels are present as components of I_Ks and are pharmacologically distinct from KCNE-saturated KCNQ1–KCNE1 channels.     

Functionally Aberrant Mutant KCNQ1 With Intermediate Heterozygous and Homozygous Phenotypes

Background

Deleterious mutations in KCNQ1 may lead to an autosomal dominant form of long QT syndrome (LQTS) (Romano-Ward) or autosomal recessive form (Jervell and Lange-Nielsen). Both are associated with severe ventricular tachyarrhythmias due to the reduction of the slowly activating delayed rectifier K⁺ current (I_Ks). Our objective was to investigate the functional consequences of KCNQ1-R562S mutation in an atypical form of KCNQ1-linked LQTS.

BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs)

KCNQ1 (Kv7.1) proteins form a homotetrameric channel, which produces a voltage-dependent K⁺ current. Co-assembly of KCNQ1 with the auxiliary β-subunit KCNE1 strongly up-regulates this current. In cardiac myocytes, KCNQ1/E1 complexes are thought to give rise to the delayed rectifier current I_Ks, which contributes to cardiac action potential repolarization. We report here that the type I membrane protein BACE1 (β-site APP-cleaving enzyme 1), which is best known for its detrimental role in Alzheimer's disease, but is also, as reported here, present in cardiac myocytes, serves as a novel interaction partner of KCNQ1. Using HEK293T cells as heterologous expression system to study the electrophysiological effects of BACE1 and KCNE1 on KCNQ1 in different combinations, our main findings were the following: (1) BACE1 slowed the inactivation of KCNQ1 current producing an increased initial response to depolarizing voltage steps. (2) Activation kinetics of KCNQ1/E1 currents were significantly slowed in the presence of co-expressed BACE1. (3) BACE1 impaired reconstituted cardiac I_Ks when cardiac action potentials were used as voltage commands, but interestingly augmented the I_Ks of ATP-deprived cells, suggesting that the effect of BACE1 depends on the metabolic state of the cell. (4) The electrophysiological effects of BACE1 on KCNQ1 reported here were independent of its enzymatic activity, as they were preserved when the proteolytically inactive variant BACE1 D289N was co-transfected in lieu of BACE1 or when BACE1-expressing cells were treated with the BACE1-inhibiting compound C3. (5) Co-immunoprecipitation and fluorescence recovery after photobleaching (FRAP) supported our hypothesis that BACE1 modifies the biophysical properties of I_Ks by physically interacting with KCNQ1 in a β-subunit-like fashion. Strongly underscoring the functional significance of this interaction, we detected BACE1 in human iPSC-derived cardiomyocytes and murine cardiac tissue and observed decreased I_Ks in atrial cardiomyocytes of BACE1-deficient mice.     

Augmented potassium current is a shared phenotype for two genetic defects associated with familial atrial fibrillation

Mutations in multiple genes have been implicated in familial atrial fibrillation (AF), but the underlying mechanisms, and thus implications for therapy, remain ill-defined. Among 231 participants in the Vanderbilt AF Registry, we found a mutation in KCNQ1 (encoding the α-subunit of slow delayed rectifier potassium current [I_Ks]) and separately a mutation in natriuretic peptide precursor A (NPPA) gene (encoding atrial natriuretic peptide, ANP), both segregating with early onset lone AF in different kindreds. The functional effects of these mutations yielded strikingly similar I_Ks “gain-of-function.” In Chinese Hamster Ovary (CHO) cells, coexpression of mutant KCNQ1 with its ancillary subunit KCNE1 generated ∼ 3-fold larger currents that activated much faster than wild-type (WT)-I_Ks. Application of the WT NPPA peptide fragment produced similar changes in WT-I_Ks, and these were exaggerated with the mutant NPPA S64R peptide fragment. Anantin, a competitive ANP receptor antagonist, completely inhibited the changes in I_Ks gating observed with NPPA S64R. Computational simulations identified accelerated transitions into open states as the mechanism for variant I_Ks gating. Incorporating these I_Ks changes into computed human atrial action potentials (AP) resulted in 37% shortening (120 vs. 192 ms at 300 ms cycle length), reflecting loss of the phase II dome which is dependent on L-type calcium channel current. We found striking functional similarities due to mutations in KCNQ1 and NPPA genes which led to I_Ks “gain-of-function”, atrial AP shortening, and consequently altered calcium current as a common mechanism between diverse familial AF syndromes.     

In silico investigation of a KCNQ1 mutation associated with familial atrial fibrillation

Mutations in transmembrane domains of the KCNQ1 subunit of the I_Ks potassium channel have been associated with familial atrial fibrillation. We have investigated mechanisms by which the S1 domain S140G KCNQ1 mutation influences atrial arrhythmia risk and, additionally, whether it can affect ventricular electrophysiology. In perforated-patch recordings, S140G-KCNQ1 + KCNE1 exhibited leftward-shifted activation, slowed deactivation and marked residual current. In human atrial action potential (AP) simulations, AP duration and refractoriness were shortened and rate-dependence flattened. Simulated I_Ks but not I_Kr block offset AP shortening produced by the mutation. In atrial tissue simulations, temporal vulnerability to re-entry was little affected by the S140G mutation. Spatial vulnerability was markedly increased, leading to more stable and stationary spiral wave re-entry in 2D stimulations, which was offset by I_Ks block, and to scroll waves in 3D simulations. These changes account for vulnerability to AF with this mutation. Ventricular AP clamp experiments indicate a propensity for increased ventricular I_Ks with the S140G KCNQ1 mutation and ventricular AP simulations showed model-dependent ventricular AP abbreviation.     

Intracellular ATP binding is required to activate the slowly activating K+ channel IKs

Gating of ion channels by ligands is fundamental to cellular function, and ATP serves as both an energy source and a signaling molecule that modulates ion channel and transporter functions. The slowly activating K⁺ channel I_Ks in cardiac myocytes is formed by KCNQ1 and KCNE1 subunits that conduct K⁺ to repolarize the action potential. Here we show that intracellular ATP activates heterologously coexpressed KCNQ1 and KCNE1 as well as I_Ks in cardiac myocytes by directly binding to the C terminus of KCNQ1 to allow the pore to open. The channel is most sensitive to ATP near its physiological concentration, and lowering ATP concentration in cardiac myocytes results in I_Ks reduction and action potential prolongation. Multiple mutations that suppress I_Ks by decreasing the ATP sensitivity of the channel are associated with the long QT (interval between the Q and T waves in electrocardiogram) syndrome that predisposes afflicted individuals to cardiac arrhythmia and sudden death. A cluster of basic and aromatic residues that may form a unique ATP binding site are identified; ATP activation of the wild-type channel and the effects of the mutations on ATP sensitivity are consistent with an allosteric mechanism. These results demonstrate the activation of an ion channel by intracellular ATP binding, and ATP-dependent gating allows I_Ks to couple myocyte energy state to its electrophysiology in physiologic and pathologic conditions.Significant energy is required to sustain both the electrical and contractile events that accompany each heart beat, suggesting that the level of ATP is one key to normal cardiac functions. Not surprisingly, a reduction in ATP concentration ([ATP]) plays a key role in the pathogenesis and progression of ischemic heart diseases, including heart failure. Intracellular ATP is important not only in providing energy (1) and as a substrate for protein kinases (2), but also as signaling molecules to bind and modulate proteins. Only a handful of results have shown that intracellular ATP serves as a signal for membrane channels and transporters (3). The best-studied example is the K_ATP channel (4, 5). This channel is inhibited in physiologic conditions by [ATP]s of 5–10 mM (6), but when the ATP levels drop to submillimolar concentrations, as in cardiac ischemia, the K_ATP channels open, shortening the action potential duration and providing metabolic protection against the insult of ischemia (7). However, at normal physiologic conditions, whether and how ATP serves as a signal connecting the energetic state of the cell to membrane excitability is still unknown.The slowly activating K⁺ channel I_Ks plays an important role in controlling the action potential duration (APD) in cardiac myocytes; it opens in response to depolarization to conduct potassium ions out of the cell, which contributes to repolarization of the membrane, terminating the cardiac action potential and thereby the myocyte contraction. The I_Ks channel consists of pore-forming KCNQ1 subunits and the single-transmembrane auxiliary subunits KCNE1 (8, 9). Loss-of-function mutations in either KCNQ1 or KCNE1 lead to prolongation of ventricular action potentials and long QT syndrome (LQTS) that manifests as QT (interval between the Q and T waves in electrocardiogram) prolongation in the electrocardiogram. LQTS predisposes patients to cardiac arrhythmias that lead to syncope and sudden death (10). Previous studies show that ATP is required for activation of I_Ks channels (11), but the molecular mechanism and the physiologic function of this ATP modulation is not known. It was the purpose of this study to investigate the mechanism by which ATP regulated I_Ks. Our results show that ATP directly binds to the KCNQ1 protein to regulate channel function at concentrations ([ATP]) close to the physiologic intracellular [ATP] in cardiac myocytes. Our studies also reveal a unique ATP binding site and mechanism for modulation of ion channel function. Further, we found that several LQT-associated mutations alter I_Ks function by reducing ATP sensitivity. These results demonstrate that ATP regulation is vital for I_Ks channel function; a disruption of these regulations predisposes to life-threatening cardiac arrhythmias.     

Polyunsaturated fatty acid analogs act antiarrhythmically on the cardiac IKs channel

Polyunsaturated fatty acids (PUFAs) affect cardiac excitability. Kv7.1 and the β-subunit KCNE1 form the cardiac I_Ks channel that is central for cardiac repolarization. In this study, we explore the prospects of PUFAs as I_Ks channel modulators. We report that PUFAs open Kv7.1 via an electrostatic mechanism. Both the polyunsaturated acyl tail and the negatively charged carboxyl head group are required for PUFAs to open Kv7.1. We further show that KCNE1 coexpression abolishes the PUFA effect on Kv7.1 by promoting PUFA protonation. PUFA analogs with a decreased pK_a value, to preserve their negative charge at neutral pH, restore the sensitivity to open I_Ks channels. PUFA analogs with a positively charged head group inhibit I_Ks channels. These different PUFA analogs could be developed into drugs to treat cardiac arrhythmias. In support of this possibility, we show that PUFA analogs act antiarrhythmically in embryonic rat cardiomyocytes and in isolated perfused hearts from guinea pig.The cardiac action potential is initiated and maintained by inward sodium and calcium currents and terminated by outward potassium currents (1). The I_Ks channel, formed by four α subunits (voltage-gated potassium channel subunit Kv7.1, originally called KCNQ1 or KvLQT1) and two to four auxiliary β subunits (Kv channel beta subunit KCNE1, originally called minK) (1, 2), contributes a major component of the repolarizing potassium current. More than 300 mutations in the genes encoding Kv7.1 and KCNE1 have been identified in patients with cardiac arrhythmia (1). Loss-of-function mutations of the I_Ks channel prolong the QT interval as observed in long QT syndrome, leading to ventricular arrhythmias, ventricular fibrillation, and sudden death (1). Gain-of-function mutations of the I_Ks channel shorten the QT interval, possibly leading to arrhythmia such as short QT syndrome or atrial fibrillation (1). Pharmacological augmentation (in the case of long QT syndrome) or inhibition (in the case of short QT syndrome) of I_Ks channel activity is a logical pharmacological strategy to treat these forms of cardiac arrhythmias.Kv7.1 is a tetrameric voltage-gated K (Kv) channel with six transmembrane segments (called S1–S6) per subunit (3). S5 and S6 from all four subunits together form the pore domain with the central ion-conducting pore. In Kv channels, S6 has been shown to function as the activation gate, shutting off the intracellular access to the pore for K⁺ ions in the closed state of the channel (3–5). Most reported activators or inhibitors of Kv7.1 channels target the ion-conducting pore domain of the channel, opening or blocking the ionic pathway (6–10). S1–S4 of each subunit form a voltage-sensor domain (VSD). In Kv channels, each S4 segment has several positively charged residues and has been shown to move in response to changes in the transmembrane voltage (3, 11). In response to membrane depolarization, the S4 segments move outward with respect to the membrane, which causes channel opening. Although four Kv7.1 subunits per se form a functional channel, Kv7.1 needs to coassemble with the auxiliary β-subunit KCNE1 to recapitulate the biophysical properties of the native cardiac I_Ks channel (12, 13). KCNE1, a single transmembrane helix protein, has been proposed to associate with Kv7.1 in the lipid cleft between adjacent VSDs, making contact with VSD transmembrane segments S1 and S4 and pore transmembrane segment S6 (14–16).In this study, we explore the prospects of polyunsaturated fatty acids (PUFAs) and PUFA analogs as small molecules enhancing or inhibiting the activity of the cardiac I_Ks channel by changing I_Ks channel voltage dependence. We previously suggested that PUFAs facilitate opening of the related Shaker Kv channel via electrostatic attraction of S4 (17–20). The pharmacological sensitivity of I_Ks to small-molecule activators has been shown to depend on the Kv7.1:KCNE1 stoichiometry (21–23). We therefore also determine the impact of Kv7.1:KCNE1 stoichiometry on PUFA sensitivity.Below we show that PUFAs affect the Kv7.1 channel by an electrostatic effect on the voltage sensor movement. We also show that KCNE1 abolishes the PUFA sensitivity of the Kv7.1 channel at physiological pH, suggesting that physiologically occurring PUFAs do not act on I_Ks channels in vivo. Furthermore, we identify PUFA analogs that have effects on the I_Ks channel at physiological pH, increase I_Ks in cardiomyocytes, restore rhythmic firing in arrhythmic cardiomyocytes, and shorten the QT interval in isolated perfused guinea pig hearts. These results may form the basis for development of pharmacological drugs that target the I_Ks channel to prevent cardiac arrhythmias.     

Single-channel basis for the slow activation of the repolarizing cardiac potassium current,IKs

Coassembly of potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) with potassium voltage-gated channel, Isk-related family, member 1 (KCNE1) the delayed rectifier potassium channel I_Ks. Its slow activation is critically important for membrane repolarization and for abbreviating the cardiac action potential, especially during sympathetic activation and at high heart rates. Mutations in either gene can cause long QT syndrome, which can lead to fatal arrhythmias. To understand better the elementary behavior of this slowly activating channel complex, we quantitatively analyzed direct measurements of single-channel I_Ks. Single-channel recordings from transiently transfected mouse ltk⁻ cells confirm a channel that has long latency periods to opening (1.67 ± 0.073 s at +60 mV) but that flickers rapidly between multiple open and closed states in non-deactivating bursts at positive membrane potentials. Channel activity is cyclic with periods of high activity followed by quiescence, leading to an overall open probability of only ∼0.15 after 4 s under our recording conditions. The mean single-channel conductance was determined to be 3.2 pS, but unlike any other known wild-type human potassium channel, long-lived subconductance levels coupled to activation are a key feature of both the activation and deactivation time courses of the conducting channel complex. Up to five conducting levels ranging from 0.13 to 0.66 pA could be identified in single-channel recordings at 60 mV. Fast closings and overt subconductance behavior of the wild-type I_Ks channel required modification of existing Markov models to include these features of channel behavior.     

Modulating the voltage sensor of a cardiac potassium channel shows antiarrhythmic effects

Cardiac arrhythmias are the most common cause of sudden cardiac death worldwide. Lengthening the ventricular action potential duration (APD), either congenitally or via pathologic or pharmacologic means, predisposes to a life-threatening ventricular arrhythmia, Torsade de Pointes. I_Ks (KCNQ1+KCNE1), a slowly activating K⁺ current, plays a role in action potential repolarization. In this study, we screened a chemical library in silico by docking compounds to the voltage-sensing domain (VSD) of the I_Ks channel. Here, we show that C28 specifically shifted I_Ks VSD activation in ventricle to more negative voltages and reversed the drug-induced lengthening of APD. At the same dosage, C28 did not cause significant changes of the normal APD in either ventricle or atrium. This study provides evidence in support of a computational prediction of I_Ks VSD activation as a potential therapeutic approach for all forms of APD prolongation. This outcome could expand the therapeutic efficacy of a myriad of currently approved drugs that may trigger arrhythmias.

I_Ks (KCNQ1+KCNE1), a slowly activating delayer rectifier in the heart, is important in controlling cardiac action potential duration (APD) and adaptation of heart rate in various physiological conditions (1). The I_Ks potassium channel has slow activation kinetics, and the activation terminates cardiac action potentials (APs) (2). This channel is formed by the voltage-gated potassium (K_V) channel subunit KCNQ1 and the regulatory subunit KCNE1. The association of KCNE1 drastically alters the phenotype of the channel, including a shift of voltage dependence of activation to more positive voltages, a slower activation time course, a changed ion selectivity, and different responses to drugs and modulators (3–6). Similar to other K_V channels, KCNQ1 has six transmembrane segments, S1 to S6, in which S1 to S4 form the voltage-sensing domain (VSD), while S5 and S6 form the pore domain (PD); four KCNQ1 subunits comprise the KCNQ1 channel (7, 8). KCNQ1 and I_Ks channels are activated by voltage. The VSD in response to membrane depolarization changes conformation, triggered by the movements of the S4 segment that contains positively charged residues (9–15). This conformational change alters the interactions between the VSD and the pore, known as the VSD–pore coupling, to induce pore opening (12–15).The ventricular APD depends on the balance of outward and inward currents flowing at plateau potentials. The outward currents include the delayed rectifiers I_Kr and I_Ks, while the inward currents include a persistent sodium current (I_NaP) (16). Specific mutations in any of these channel proteins that cause a reduction in outward current or increase in inward current are associated with congenital long QT syndrome (LQTS). The QT interval is the time between the initial depolarization of the ventricle until the time to full repolarization. LQTS is a condition in which the APD is abnormally prolonged, predisposing the afflicted patients to a lethal cardiac arrhythmia called Torsades de Pointes (TdP) (17). In fact, mutations in multiple genes that alter the function of various ion channels have been associated with LQTS (18). There is also a much more prevalent problem called acquired LQTS (aLQTS) that is most often associated with off target effects of drugs. Many drugs are marketed with a QT prolongation warning, and the drug concentrations that can be used therapeutically are limited by this potentially lethal side effect. Some effective drugs have been removed from the market (19) because of QT prolongation, and others are abandoned before clinical trials even began. Therefore, aLQTS is costly for the pharmaceutical industry both in drug development (to avoid this side effect) and when it results in removal from the market of compounds that have effectively treated other diseases (20, 21). At present, the I_Kr (HERG) potassium channel (20) and the phosphoinositide 3-kinase (PI3K) (22, 23) have been identified as the most prominent off targets of these drugs for the association with aLQTS.We hypothesized that in LQTS, the normal heart function can be restored and QT prolongation prevented by compensating for the change in net current from any of the channels produced by the myocyte; all that is required is that a reasonable facsimile of normal net current flow be restored. We applied this approach to aLQTS based on a computational study to show that a shift of voltage-dependent activation of I_Ks to more negative voltages would increase I_Ks during ventricular APs; this increase of I_Ks would revert drug-induced APD prolongation to normal. More importantly, a change in I_Ks voltage-dependent activation might affect the normal APD to a much smaller degree because of its slow activation kinetics in healthy ventricular myocytes, thereby posing minimal risk of cardiac toxicity on its own. To apply this approach experimentally, we needed a compound that could specifically shift the voltage dependence of I_Ks activation. Previous studies showed that the benzodiazepine R-L3 (24) and polyunsaturated fatty acids (25) activate I_Ks channels, while more recently, rottlerin was shown to act similarly to R-L3 (26). However, the effects of these compounds on I_Ks are complex, likely through binding to more than one site in the channel protein instead of simply acting on voltage-dependent activation (24, 27, 28). In addition, these compounds showed poor specificity for I_Ks, also affecting other ion channels in the heart (27, 29, 30), which make these compounds unsuitable to test our hypothesis. Recently, we have identified CP1 as an activator for I_Ks, which mimics the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) to mediate the VSD–pore coupling (13, 31). CP1 enhances I_Ks primarily by increasing current amplitude with some shift of voltage dependence of activation, which is not suitable for our test either.In this study, we identified a compound, C28, using an approach that combines in silico and experimental screening, that interacts with the KCNQ1 VSD and shifts voltage dependence of VSD activation to more negative voltages. C28 increases both exogenously expressed I_Ks and the current in native cardiac myocytes. As predicted by computational modeling, C28 can prevent or reverse the drug-induced APD prolongation back to normal while having a minimal effect on the control APD at the same concentration in healthy cardiac myocytes. This study demonstrates that the KCNQ1 VSD can be used as a drug target for developing a therapy for LQTS, and C28 identified in this study may be used as a lead for this development. Furthermore, our results provide support for the use of docking computations based on ion channel structure and cellular physiology, in combination with functional studies based on molecular mechanisms, as an effective approach for rational drug design.     

Mutation of an A-kinase-anchoring protein causes long-QT syndrome

A-kinase anchoring proteins (AKAPs) recruit signaling molecules and present them to downstream targets to achieve efficient spatial and temporal control of their phosphorylation state. In the heart, sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD), mediated by β-adrenergic receptor (βAR) activation, requires assembly of AKAP9 (Yotiao) with the I_Ks potassium channel α subunit (KCNQ1). KCNQ1 mutations that disrupt this complex cause type 1 long-QT syndrome (LQT1), one of the potentially lethal heritable arrhythmia syndromes. Here, we report identification of (i) regions on Yotiao critical to its binding to KCNQ1 and (ii) a single putative LQTS-causing mutation (S1570L) in AKAP9 (Yotiao) localized to the KCNQ1 binding domain in 1/50 (2%) subjects with a clinically robust phenotype for LQTS but absent in 1,320 reference alleles. The inherited S1570L mutation reduces the interaction between KCNQ1 and Yotiao, reduces the cAMP-induced phosphorylation of the channel, eliminates the functional response of the I_Ks channel to cAMP, and prolongs the action potential in a computational model of the ventricular cardiocyte. These reconstituted cellular consequences of the inherited S1570L-Yotiao mutation are consistent with delayed repolarization of the ventricular action potential observed in the affected siblings. Thus, we have demonstrated a link between genetic perturbations in AKAP and human disease in general and AKAP9 and LQTS in particular.     

Impaired Adrenergic/Protein Kinase A Response of Slow Delayed Rectifier Potassium Channels as a Long QT Syndrome Motif: Importance and Unknowns

The slow delayed rectifier potassium current (I_Ks) significantly contributes to cardiac repolarization under specific conditions, particularly at stimulation by the protein kinase A (PKA) during increased sympathetic tone. Impaired PKA-mediated stimulation of I_Ks channels may considerably aggravate dysfunction of the channels induced by mutations in the KCNQ1 gene that encodes the structure of the α-subunit of I_Ks channels. These mutations are associated with several subtypes of inherited arrhythmias, mainly long QT syndrome type 1, less commonly short QT syndrome type 2, and atrial fibrillation. The impaired PKA reactivity of I_Ks channels may significantly increase the risk of arrhythmia in these patients. Unfortunately, only approximately 2.7% of the KCNQ1 variants identified as putatively clinically significant have been studied with respect to this problem. This review summarizes the current knowledge in the field to stress the importance of the PKA-mediated regulation of I_Ks channels, and to appeal for further analysis of this regulation in KCNQ1 mutations associated with inherited arrhythmogenic syndromes. On the basis of the facts summarized in our review, we suggest several new regions of the α-subunit of the I_Ks channels as potential contributors to PKA stimulation, namely the S4 and S5 segments, and the S2-S3 and S4-S5 linkers. Deeper knowledge of mechanisms of the impaired PKA response in mutated I_Ks channels may help to better understand this regulation, and may improve risk stratification and management of patients suffering from related pathologies.     

The anti-ischemic effect of trimetazidine in patients with postprandial myocardial ischemia is unrelated to meal composition

Background

Previous studies provide evidence for a significant reduction of coronary flow reserve after ingestion of meals of different compositions. A possible role of hyperinsulinemia and increased free fatty acid levels, which are deleterious during acute myocardial ischemia and reperfusion, has been hypothesized. We assessed in patients with stable coronary disease the effects of high-fat meals (HFMs) and high-carbohydrate meals (HCMs) on ischemic threshold and stress left ventricular function on placebo and after partial fatty acid inhibition by trimetazidine (TMZ).

Methods

Ten patients (9 men, age 68 ± 7 years) were allocated to placebo and TMZ (40 mg TID), both administered in the 24 hours preceding testing, according to a randomized double-blind study design. All patients underwent stress (treadmill exercise testing according to the Bruce protocol) echocardiography after fasting (8 hours) and after an HFM and HCM (2 hours) either on placebo or on TMZ. Time to 1-mm ST-segment depression (time to 1 mm) and stress wall motion score index (WMSI) were evaluated.

Results

An HFM did not affect exercise variables compared with fasting, whereas an HCM resulted in a reduction of the ischemic threshold (time to 1 mm from 402 ± 141 to 292 ± 123 seconds, P = .025). Compared with placebo, TMZ improved time to 1 mm after fasting, HFM, and HCM (432 ± 153 vs 402 ± 141, 439 ± 118 vs 380 ± 107, 377 ± 123 vs 292 ± 123, F_1,9 = 26.91, P = .0006). Compared with placebo, on TMZ, stress WMSI decreased from 1.55 ± 0.25 to 1.29 ± 0.14 after fasting, from 1.57 ± 0.10 to 1.39 ± 0.28 after HFM, and from 1.64 ± 0.21 to 1.39 ± 0.21 after HCM (F_1,9 = 37.04, P = .0002). Interestingly, stress WMSI on TMZ was never different from rest WMSI on placebo.

Conclusions

In patients with coronary disease, exercise testing after an HCM results in more severe myocardial ischemia compared with that after an HFM. The observed beneficial effects of the partial fatty acid inhibitor TMZ seem to be unrelated to meal composition and are possibly caused by the better glucose use induced by the drug.     

Individual IKs channels at the surface of mammalian cells contain two KCNE1 accessory subunits

KCNE1 (E1) β-subunits assemble with KCNQ1 (Q1) voltage-gated K⁺ channel α-subunits to form I_Kslow (I_Ks) channels in the heart and ear. The number of E1 subunits in I_Ks channels has been an issue of ongoing debate. Here, we use single-molecule spectroscopy to demonstrate that surface I_Ks channels with human subunits contain two E1 and four Q1 subunits. This stoichiometry does not vary. Thus, I_Ks channels in cells with elevated levels of E1 carry no more than two E1 subunits. Cells with low levels of E1 produce I_Ks channels with two E1 subunits and Q1 channels with no E1 subunits—channels with one E1 do not appear to form or are restricted from surface expression. The plethora of models of cardiac function, transgenic animals, and drug screens based on variable E1 stoichiometry do not reflect physiology.Voltage-gated potassium (K_V) channels include four α-subunits that form a single, central ion conduction pathway with four peripheral voltage sensors (1–3). Incorporation of accessory β-subunits modifies the function of K_V channels to suit the diverse requirements of different tissues. KCNE genes encode minK-related peptides (MiRPs) (4–6), β-subunits with a single transmembrane span that assemble with a wide array of K_V α-subunits (7, 8) to control surface expression, voltage dependence, and kinetics of gating transitions, unitary conductance, ion selectivity, and pharmacology of the resultant channel complexes (4, 9–15). I_Kslow (I_Ks) channels in the heart and inner ear are formed by the α-subunit encoded by KCNQ1 (called Q1, K_VLQT1, K_V7.1, or KCNQ1) and the β-subunit encoded by KCNE1 (called E1, mink, or KCNE1) (16, 17). Inherited mutations in Q1 and E1 are associated with cardiac arrhythmia and deafness.The number of E1 subunits in I_Ks channels has been a longstanding matter of disagreement. We first argued for two E1 subunits per channel based on the suppression of current by an E1 mutant (18). Subsequently, we reached the same conclusion by determining the total number of channels using radiolabeled charybdotoxin (CTX), a scorpion toxin that blocks channels when one molecule binds in the external conduction pore vestibule, and an antibody-based luminescence assay to tally E1 subunits (19). Morin and Kobertz (20) used iterative chemical linkage between CTX in the pore and E1, and they also assigned two accessory subunits to >95% of I_Ks channels without gathering evidence for variation in subunit valence. Furthermore, when we formed I_Ks channels from separate E1 and Q1 subunits and compared them with channels enforced via genetic encoding to contain two or four E1 subunits (19), we observed the natural I_Ks channels to have the same gating attributes, small-molecule pharmacology, and CTX on and off rates (a reflection of pore vestibule structure) as channels encoded with two E1 subunits but not those with four. These findings support the conclusion that two E1 subunits are necessary, sufficient, and the normal number in I_Ks channels.In contrast, others have argued that I_Ks channels have variable stoichiometry with one to four E1 subunits, or even more (21–24). Recently, Nakajo et al. (25) applied single-particle spectroscopy to the question; this powerful “gold-standard” tool has been a valuable strategy to assess the subunit composition of ion channels (26–28) and should be expected to improve on prior investigations conducted on populations of I_Ks channels and subject, therefore, to the simplifying assumptions that attend macroscopic studies (29). Nakajo et al. (25) reported a variable number of E1 subunits, from one to four, in I_Ks channels studied in Xenopus laevis oocytes. The impact of this result has been striking because it has engendered new models of cardiac physiology, altered models of I_Ks channel biosynthesis and function, stimulated the use of transgenic animals artificially enforced to express I_Ks channels with four E1 subunits (by expression of a fused E1–Q1 subunit), and prompted cardiac drug design based on the assumption that I_Ks channels can form with one E1 subunit (23, 30–32).We were concerned that the conclusions of Nakajo et al. (25) were in error because they appraised only a limited fraction of particles that were immobile in the oocyte membrane; counted E1 and Q1 asynchronously rather than simultaneously (increasing the risk that particles moved into or out of the field of view); and studied Q1 and E1 appended not only with the fluorescent proteins (FP) required to count subunits by photobleaching but also with a common trafficking motif that suppressed channel mobility by interacting with an overexpressed anchoring protein, thereby risking nonnatural aggregation of subunits.Here, to resolve mobility problems and obviate the need for modification of subunits with targeting motifs, we describe and perform single-fluorescent-particle photobleaching at the surface of live mammalian cells, demonstrating three spectroscopic counting approaches: standard, asynchronous subunit counting; simultaneous, two-color subunit counting; and toxin-directed, simultaneous, two-color photobleaching. To analyze the data, we use two statistical approaches—one to assess the degree of colocalization of objects in dual-color images (33) and the other to infer stoichiometry from single-molecule photobleaching (34). These methods also allow determination of the surface density of assemblies of defined subunit composition and are therefore useful to assess the formation and life cycle of membrane protein complexes.We report that single I_Ks channels at the surface of mammalian cells contain two E1 subunits—no more and no less. This finding refutes the single-particle studies of Nakajo et al. (25) in oocytes and macroscopic studies (21–24, 30–32), arguing that forcing cells to express excess E1 produces I_Ks channels containing more than two E1 subunits and that low levels of E1 yields I_Ks channels with less than two E1 subunits. Not once did we observe an I_Ks channel with three or four E1 subunits. Moreover, simultaneous, two-color subunit counting revealed that low amounts of E1 relative to Q1 [ratios like those reported in human cardiac ventricle (35, 36)] produced two types of channels on the cell surface: I_Ks channels (with two E1 subunits) and Q1 channels (with no E1 subunits). Finally, E1 was shown to increase in I_Ks channel surface expression threefold, as we predicted based on assessment of I_Ks channel unitary conductance (11), whereas few E1 subunits were on the surface outside of I_Ks channels, even when E1 was expressed alone. This finding indicates that E1 does not travel to the surface readily on its own, that two E1 subunits facilitate I_Ks channel trafficking to the surface (or enhance surface residence time compared with Q1 channels), and that I_Ks channels with only one E1 subunit do not form, do not reach the surface, or are rapidly recycled.     

KCNQ1 Antibodies for Immunotherapy of Long QT Syndrome Type 2

BackgroundPatients with long QT syndrome (LQTS) are predisposed to life-threatening arrhythmias. A delay in cardiac repolarization is characteristic of the disease. Pharmacotherapy, implantable cardioverter-defibrillators, and left cardiac sympathetic denervation are part of the current treatment options, but no targeted therapy for LQTS exists to date. Previous studies indicate that induced autoimmunity against the voltage-gated KCNQ1 K⁺ channels accelerates cardiac repolarization.ObjectivesHowever, a causative relationship between KCNQ1 antibodies and the observed electrophysiological effects has never been demonstrated, and thus presents the aim of this study.MethodsThe authors purified KCNQ1 antibodies and performed whole-cell patch clamp experiments as well as single-channel recordings on Chinese hamster ovary cells overexpressing I_Ks channels. The effect of purified KCNQ1 antibodies on human cardiomyocytes derived from induced pluripotent stem cells was then studied.ResultsThe study demonstrated that KCNQ1 antibodies underlie the previously observed increase in repolarizing I_Ks current. The antibodies shift the voltage dependence of activation and slow the deactivation of I_Ks. At the single-channel level, KCNQ1 antibodies increase the open time and probability of the channel. In models of LQTS type 2 (LQTS2) using human induced pluripotent stem cell-derived cardiomyocytes, KCNQ1 antibodies reverse the prolonged cardiac repolarization and abolish arrhythmic activities.ConclusionsHere, the authors provide the first direct evidence that KCNQ1 antibodies act as agonists on I_Ks channels. Moreover, KCNQ1 antibodies were able to restore alterations in cardiac repolarization and most importantly to suppress arrhythmias in LQTS2. KCNQ1 antibody therapy may thus present a novel promising therapeutic approach for LQTS2.     

KCNE3 acts by promoting voltage sensor activation in KCNQ1

KCNE β-subunits assemble with and modulate the properties of voltage-gated K⁺ channels. In the colon, stomach, and kidney, KCNE3 coassembles with the α-subunit KCNQ1 to form K⁺ channels important for K⁺ and Cl⁻ secretion that appear to be voltage-independent. How KCNE3 subunits turn voltage-gated KCNQ1 channels into apparent voltage-independent KCNQ1/KCNE3 channels is not completely understood. Different mechanisms have been proposed to explain the effect of KCNE3 on KCNQ1 channels. Here, we use voltage clamp fluorometry to determine how KCNE3 affects the voltage sensor S4 and the gate of KCNQ1. We find that S4 moves in KCNQ1/KCNE3 channels, and that inward S4 movement closes the channel gate. However, KCNE3 shifts the voltage dependence of S4 movement to extreme hyperpolarized potentials, such that in the physiological voltage range, the channel is constitutively conducting. By separating S4 movement and gate opening, either by a mutation or PIP₂ depletion, we show that KCNE3 directly affects the S4 movement in KCNQ1. Two negatively charged residues of KCNE3 (D54 and D55) are found essential for the effect of KCNE3 on KCNQ1 channels, mainly exerting their effects by an electrostatic interaction with R228 in S4. Our results suggest that KCNE3 primarily affects the voltage-sensing domain and only indirectly affects the gate.Voltage-gated K⁺ (Kv) channels are essential membrane proteins with a variety of crucial physiological roles. Most Kv channels are expressed in excitable cells where, e.g., they regulate and modulate the resting potential and the threshold and duration of the action potential (1). The KCNQ1 channel (also called Kv7.1 or KvLQT1) differs from most other Kv channels in that it has key physiological roles in both excitable cells, such as cardiomyocytes (2, 3) and pancreatic β-cells (4, 5), and in nonexcitable cells, such as in epithelia (3, 6). The KCNQ1 channels display diverse biophysical properties in different cell types, a diversity thought to be mainly due to the KCNQ1 channel’s association with five tissue-specific, single-transmembrane segment KCNE β-subunits (KCNE1–5) (7–13). KCNQ1 α-subunit expressed by itself forms a voltage-dependent K⁺ channel that opens at negative voltages (Fig. 1 A and D). However, coexpression of KCNQ1 with KCNE1 slows the kinetics of activation and shifts the voltage dependence of activation to positive voltages (Fig. 1 B and D) (7, 8), thereby generating the slowly activating, voltage-dependent I_Ks current that controls the repolarization phase of cardiac action potentials. In contrast, coexpression of KCNQ1 with KCNE3 results in a constitutively conducting channel in the physiological voltage range of –80 to +40 mV (Fig. 1 C and D), which is important for transport of water and salt in epithelial tissues, including those of the colon, small intestine, and airways (9, 14, 15). In addition, mutations of KCNE3 have been associated with cardiac arrhythmia (16, 17) and diseases in the inner ear, such as Meniere’s disease and tinnitus (18, 19). Because KCNQ1/KCNE3 channels are necessary for water and salt secretion in the colon, they are a potential drug target in the treatment of secretory diarrhea (20, 21).Open in a separate windowFig. 1.KCNE β-subunits alter the voltage dependence and kinetics of KCNQ1 channels. Representative current traces from (A) KCNQ1, (B) KCNQ1/KCNE1, and (C) KCNQ1/KCNE3 channels for the indicated voltage protocol. (D) Normalized G(V) of recordings from (□) KCNQ1, (▲) KCNQ1/KCNE1, and (●) KCNQ1/KCNE3 channels (mean ± SEM ; n = 7). Black lines are Boltzmann fit. (E) Topology of KCNQ1 and KCNE3. Residues mutated in this study are indicated.Here, we investigate the mechanism by which KCNE3 modifies KCNQ1 channel gating. KCNQ1 comprises six transmembrane segments (S1–S6; Fig. 1E). Four KCNQ1 α subunits form a tetrameric channel. The central pore domain of that channel is formed by the S5–S6 segments from all four subunits. The central pore domain is flanked by four peripheral voltage-sensing domains, each composed of the S1–S4 segments of a subunit (22). The fourth transmembrane segment S4, which has several positively charged amino acid residues, has been shown to move in response to voltage and thereby function as voltage sensor (23–27). In Kv channels, it is thought that the S4 movement upon membrane depolarization causes a movement of the S4–S5 linker, which forms several interactions with the S6 gate. The movement of the S4–S5 linker thereby pulls open the S6 gate (22).Three-dimensional structure information on KCNQ1/KCNE channel complexes is not available. However, disulfide cross-linking studies suggest that KCNE1 localizes laterally of the central pore domain in the otherwise lipid-filled crevices between voltage sensor domains of KCNQ1 channels (28–30), such that KCNE1 could act on voltage sensors, the pore, or the coupling between voltage sensors and pore. Because of the high sequence similarity within the KCNE family, KCNE3 is likely positioned in the KCNQ1 channel structure much like KCNE1. However, how KCNE3 modulates KCNQ1 channels in a manner so distinct from that of KCNE1 to produce voltage-insensitive KCNQ1/KCNE3 channels is unclear. KCNE3 could, e.g., lock the S4 segment in the activated position and thereby lock the gate open, or KNCE3 could decouple S4 movement from the gate so that the gate is always open, independent of the S4 position.Alanine substitutions of positively charged residues in the S4 segment of KCNQ1 result in KCNQ1/KCNE3 channels that are constitutively conducting to varying degrees (31). The authors of this study reasoned that S4 mutants would not affect gate opening if KCNE3 uncoupled the S4 movement from the gate of the pore; therefore, they concluded that KCNE3 does not uncouple the S4 movement from the gate, but might lock S4 segments in their active positions. In addition, cysteine accessibility experiments showed that the time course of modification of residues in S4 (mutated to cysteines) in KCNQ1/KCNE3 channels was independent of voltage (28, 32). These data suggested that KCNE3 stabilizes S4 segments in a configuration that allows accessibility to externally applied MTS reagents at all membrane voltages (28, 32). Together, these experiments suggest that the S4 segments of KCNQ1/KCNE3 channels are in their active positions independent of voltage, and that S4 and the gate are not decoupled in KCNQ1/KCNE3 channels. However, it is not clear whether KCNE3 locks the gate open directly (and only indirectly locks S4 in its active conformation), KCNE3 locks S4 directly (and only indirectly locks the gate open), or KCNE3 locks both S4 and the gate in their active and open conformations, respectively. In addition, the molecular mechanism for the action of KCNE3 on KCNQ1 is unclear.Here, we simultaneously track changes in voltage sensor movement and in gate opening of KCNQ1/KCNE3 channels using voltage clamp fluorometry (VCF) to determine the molecular mechanism by which KCNE3 alters KCNQ1 channel gating. We find that KCNE3 primarily affects the S4 segment, so that S4 movement occurs negative to the physiological voltage range. The KCNQ1/KCNE3 channels thus become voltage-independent in the physiological voltage range. However, strong negative voltages (more negative than −120 mV) start to move S4 segments back to their resting state and close KCNQ1/KCNE3 channels. We also find that two negatively charged residues, D54 and D55, at the external end of the KCNE3 transmembrane segment are necessary for the KCNE3 effect on KCNQ1 channel activation, and that D54 and D55 in KCNE3 interact with the outer most arginine, R228, in S4 of KCNQ1.     

Effects of Exenatide Combined with Lifestyle Modification in Patients with Type 2 Diabetes

Objective

To determine the effect of a lifestyle modification program plus exenatide versus lifestyle modification program plus placebo on weight loss in overweight or obese participants with type 2 diabetes treated with metformin and/or sulfonylurea.

Methods

In this 24-week, multicenter, randomized, double-blind, placebo-controlled study, 194 patients participated in a lifestyle modification program, consisting of goals of 600 kcal/day deficit and physical activity of at least 2.5 hours/week. Participants were randomized to 5 μg exenatide twice daily injection + lifestyle modification program (n = 96) or placebo + lifestyle modification program (n = 98), and after 4 weeks increased their exenatide dose to 10 μg twice daily or volume equivalent of placebo.

Results

Baseline characteristics: (mean ± standard deviation) age, 54.8 ± 9.5 years; weight, 95.5 ± 16.0 kg; hemoglobin A_1c, 7.6 ± 0.8%. At 24 weeks (least squares mean ± standard error), treatments showed similar decreases in caloric intake (−378 ± 58 vs −295 ± 58 kcal/day, exenatide + lifestyle modification program vs placebo + lifestyle modification program, P = .27) and increases in exercise-derived energy expenditure. Exenatide + lifestyle modification program showed greater change in weight (−6.16 ± 0.54 kg vs −3.97 ± 0.52 kg, P = .003), hemoglobin A_1c (−1.21 ± 0.09% vs −0.73 ± 0.09%, P <.0001), systolic (−9.44 ± 1.40 vs −1.97 ± 1.40 mm Hg, P <.001) and diastolic blood pressure (−2.22 ± 1.00 vs 0.47 ± 0.99 mm Hg, P = .04). Nausea was reported more for exenatide + lifestyle modification program than placebo + lifestyle modification program (44.8% vs 19.4%, respectively, P <.001), with no difference in withdrawal rates due to adverse events (4.2% vs 5.1%, respectively, P = 1.0) or rates of hypoglycemia.

Conclusions

When combined with lifestyle modification, exenatide treatment led to significant weight loss, improved glycemic control, and decreased blood pressure compared with lifestyle modification alone in overweight or obese participants with type 2 diabetes on metformin and/or sulfonylurea treatment.     

Insulin sensitivity in young women with vasovagal syncope

Background

Insulin, in addition to its known metabolic effects, has sympatho-excitatory and vasodilatory actions on muscular blood vessels. The goal of this study was to evaluate insulin sensitivity in young women with vasovagal syncope and positive tilt test results (HUT+) and to compare it with that in patients with negative tilt test results (HUT-) and in control subjects without a history of syncope.

Methods

Different indices of insulin sensitivity were obtained by an oral glucose tolerance test (OGTT) in 13 young women with syncope and HUT+ (age 26.8 ± 9.1 years, body mass index 20.4 ± 2.1), 8 patients with HUT- (age 26 ± 5.6 years, body mass index 21.9 ± 2.4), and 13 control subjects without syncope and HUT- (age 28.9 ± 8.8 years, body mass index 23.1 ± 1.7). The following parameters were assessed: fasting glucose and insulin levels (G₀, I₀); G₀/I₀ ratio; G₀ × I₀; areas under the curve for glucose and insulin; homeostatic model assessment (HOMA); quantitative insulin sensitivity check index (QUICKI); and composite whole-body insulin sensitivity index (ISI).

Results

G₀ and I₀ values were significantly lower in patients with HUT+ than in control subjects (G₀ 4.9 vs 81.9, P < .05, I₀ 4.7 vs 9.1, P < .005). All the fasting values-based indices (ie, HOMA 0.9 vs 1.9, P < .005) and the ISI (12.8 vs 7.1, P = .01) differed significantly in both groups. None of the parameters showed significant differences between patients with HUT- and control subjects. Sixty-one percent of patients with HUT+ had a vasovagal reaction during OGTT.

Conclusions

Young women with vasovagal syncope and HUT+ have a greater insulin sensitivity. They have a propensity to reproduce symptoms during the OGTT. This hypersensitivity could be one of the predisposing factors for vasovagal episodes.     

Effects of benzyltetrahydropalmatine on two components of the delayed rectifier K+ current in Guinea pig ventricular myocytes

The effects of benzyltetrahydropalmatine (BTHP), a new class III antiarrhythmic agent, on the action potential in guinea pig papillary muscle and the rapidly activating component (I _Kr) and the slowly activating component (I _Ks) of the delayed rectifier potassium current (I _K) in isolated guinea pig ventricular myocytes were investigated. The action potentials of papillary muscles were studied using a standard microelectrode technique, while the K⁺ currents were recorded using the whole-cell patch clamp technique. The results showed that BTHP prolonged the action potential duration (APD) without altering other variables of the action potential in guinea pig papillary muscles. The 2 components of I _K were blocked by BTHP (1 100 mol·L^–1) in time-, concentration-, voltage-, and specifically frequency-dependent fashion. The IC₅₀ value for blockade ofI _Kr was 13.5 mol·L^–1, while the IC₅₀ value for blockade of I _Ks was 9.3 mol·L^–1. BTHP 30.0 mol·L^–1 reduced I _Kr and I _Kr,tail by 31 ± 4.3% and 36 ± 4.7% (n = 6, p < 0.01) and decreased I _Ks and I _Ks,tail by 40 ± 6.3% and 39 ± 4.6% (n = 7, p < 0.01) respectively. BTHP accelerated their deactivation course by reducing the time constants of deactivation of I _Kr and I _Ks. The activation kinetics of I _Kr or I _Ks were not affected by BTHP. It is concluded that BTHP prolonged the action potential duration with respect to its non-selective action on I _Kr and I _Ks in single guinea pig ventricular cell in a frequency-dependent fashion.     

Clinical diagnostic utility of adenosine deaminase, interferon-γ, interferon-γ-induced protein of 10 kDa, and dipeptidyl peptidase 4 levels in tuberculous pleural effusions

Objective

Current tools for the diagnosis of tuberculous pleural effusions are suboptimal. The study was undertaken to evaluate the accuracy of pleural fluid adenosine deaminase (ADA), interferon (IFN)-γ, interferon-γ-induced protein of 10 kDa (IP-10), and dipeptidyl peptidase (DPP) 4 levels in differentiating tuberculous pleural effusion (TPE) and non-TPE.

Methods

A total of 122 samples of pleural effusion were studied. Pleural fluid ADA activity was measured with the colorimetric method, and IP-10, IFN-γ, and DPP4 levels were measured with enzyme-linked immunosorbent assay.

Results

ADA activity and IP-10, IFN-γ, and DPP4 levels were significantly higher in TPE than in non-TPE (88.9 ± 62.7 U/L vs 18.1 ± 16.2 U/L, P < .05; 147.5 ± 117.3 ng/L vs 24.9 ± 19.7 ng/L, P < .05; 627.2 ± 345.3 ng/L vs 152.6 ± 71.4 ng/L, P < .05; and 560.6 ± 451.2 vs 56.8 ± 57.7, P < .05, respectively). The diagnostic sensitivity and specificity of ADA activity (cutoff value of 40 U/L) were 93.6% and 90.9%, respectively, and higher than those of IFN-γ (91.0% and 88.6% at the cutoff value of 225 ng/L, respectively), DPP4 (88.5% and 81.8% at the cutoff value of 75 ng/L, respectively), and IP-10 (83.3% and 86.4% at the cutoff value of 44 ng/L, respectively).

Conclusion

The roles of ADA and IFN-γ in the differential diagnosis of tuberculous pleurisy are pivotal. ADA or IFN-γ in combination with DPP4 or IP-10 can aid in differentiation between TPE and non-TPE with improved specificity and diagnostic efficiency.