Natural and synthetic modulators of SK (K(ca)2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7

BACKGROUND AND PURPOSE

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells.

EXPERIMENTAL APPROACH

We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments.

KEY RESULTS

TRPM7 currents were inhibited by modulators of small conductance Ca²⁺-activated K⁺ channels (K_Ca2.1–2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC₅₀ 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg²⁺-dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

This study reveals a tight overlap in the pharmacological profiles of TRPM7 and K_Ca2.1–2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7.     

Protein kinase C-independent inhibition of arterial smooth muscle K(+) channels by a diacylglycerol analogue

BACKGROUND AND PURPOSE

Analogues of the endogenous diacylglycerols have been used extensively as pharmacological activators of protein kinase C (PKC). Several reports show that some of these compounds have additional effects that are independent of PKC activation, including direct block of K⁺ and Ca²⁺ channels. We investigated whether dioctanoyl-sn-glycerol (DiC8), a commonly used diacylglycerol analogue, blocks K⁺ currents of rat mesenteric arterial smooth muscle in a PKC-independent manner.

EXPERIMENTAL APPROACH

Conventional whole-cell and inside-out patch clamp was used to measure the inhibition of K⁺ currents of rat isolated mesenteric smooth muscle cells by DiC8 in the absence and presence of PKC inhibitor peptide.

KEY RESULTS

Mesenteric artery smooth muscle K_v currents inactivated very slowly with a time constant of about 2 s following pulses from −65 to +40 mV. Application of 1 µM DiC8 produced an approximate 40-fold increase in the apparent rate of inactivation. Pretreatment of the cells with PKC inhibitor peptide had a minimal effect on the action of DiC8, and substantial inactivation still occurred, indicating that this effect was mainly independent of PKC. We also found that DiC8 blocked BK and K_ATP currents, and again a significant proportion of these blocks occurred independently of PKC activation.

CONCLUSIONS AND IMPLICATIONS

These results show that DiC8 has a direct effect on arterial smooth muscle K⁺ channels, and this precludes its use as a PKC activator when investigating PKC-mediated effects on vascular K⁺ channels.     

Activation of endothelial and epithelial K(Ca) 2.3 calcium-activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles

BACKGROUND AND PURPOSE

Small (K_Ca2) and intermediate (K_Ca3.1) conductance calcium-activated potassium channels (K_Ca) may contribute to both epithelium- and endothelium-dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we investigated the expression of K_Ca2.3 and K_Ca3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries.

EXPERIMENTAL APPROACH

Channel expression and functional studies were conducted in human isolated small pulmonary arteries and bronchioles. K_Ca2 and K_Ca3.1 currents were examined in human small airways epithelial (HSAEpi) cells by whole-cell patch clamp techniques.

RESULTS

While K_Ca2.3 expression was similar, K_Ca3.1 protein was more highly expressed in pulmonary arteries than bronchioles. Immunoreactive K_Ca2.3 and K_Ca3.1 proteins were found in both endothelium and epithelium. K_Ca currents were present in HSAEpi cells and sensitive to the K_Ca2.3 blocker UCL1684 and the K_Ca3.1 blocker TRAM-34. In pulmonary arteries contracted by U46619 and in bronchioles contracted by histamine, the K_Ca2.3/ K_Ca3.1 activator, NS309, induced concentration-dependent relaxations. NS309 was equally potent in relaxing pulmonary arteries, but less potent in bronchioles, than salbutamol. NS309 relaxations were blocked by the K_Ca2 channel blocker apamin, while the K_Ca3.1 channel blocker, charybdotoxin failed to reduce relaxation to NS309 (0.01–1 µM).

CONCLUSIONS AND IMPLICATIONS

K_Ca2.3 and K_Ca3.1 channels are expressed in the endothelium of human pulmonary arteries and epithelium of bronchioles. K_Ca2.3 channels contributed to endo- and epithelium-dependent relaxations suggesting that these channels are potential targets for treatment of pulmonary hypertension and chronic obstructive pulmonary disease.     

Treatment with the Kv7 potassium channel activator flupirtine is beneficial in two independent mouse models of pulmonary hypertension

Background and purpose:

Voltage-gated potassium (K_v) channels contribute to resting membrane potential in pulmonary artery smooth muscle cells and are down regulated in patients with pulmonary arterial hypertension (PAH) and a contribution from K_v7 channels has been recently proposed. We investigated the effect of the K_v7 channel activator, flupirtine, on PAH in two independent mouse models: PAH induced by hypoxia and spontaneous PAH in mice over-expressing the 5-HT transporter (SERT⁺ mice).

Experimental approach:

Right ventricular pressure was assessed in vivo in mice chronically treated with flupirtine (30 mg·kg⁻¹·day⁻¹). In separate in vitro experiments, pulmonary arteries from untreated mice were mounted in a wire myograph. Relaxations to acute administration of flupirtine and contractions to K_v channel blocking drugs, including the K_v7 channel blocker linopirdine, were measured.

Key results:

In wild-type (WT) mice, hypoxia increased right ventricular pressure, pulmonary vascular remodelling and right ventricular hypertrophy. These effects were attenuated by flupirtine, which also attenuated these indices of PAH in SERT⁺ mice. In the in vitro experiments, flupirtine induced a potent relaxant response in arteries from untreated WT and SERT⁺ mice. The relaxation was fully reversed by linopirdine, which potently contracted mouse pulmonary arteries while other K_v channel blockers did not.

Conclusions and implications:

Flupirtine significantly attenuated development of chronic hypoxia-induced PAH in mice and reversed established PAH in SERT⁺ mice, apparently via K_v7 channel activation. These results provide the first direct evidence that drugs activating K_v7 channels may be of benefit in the treatment of PAH with different aetiologies.     

New tricks for old dogs: KCNQ expression and role in smooth muscle

Ion channels encoded by the KCNQ gene family (K_v7.1–7.5) are major determinants of neuronal membrane potential and the cardiac action potential. This key physiological role is highlighted by the existence of a number of hereditary disorders caused by mutations to KCNQ genes. Recently, KCNQ gene expression has been identified in vascular and non-vascular smooth muscles. In addition, experiments with an array of pharmacological modulators of KCNQ channels have supported a crucial role for these channels in regulating smooth muscle contractility. This article will provide an overview of present understanding in this nascent area of KCNQ research and will offer guidance as to future directions.     

Concomitant facilitation of GABAA receptors and KV7 channels by the non-opioid analgesic flupirtine

BACKGROUND AND PURPOSE

Flupirtine is a non-opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study.

EXPERIMENTAL APPROACH

Effects of flupirtine on native and recombinant voltage- and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant K_IR3 and K_V7 channels and α3β4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na⁺, Ca²⁺, inward rectifier K⁺, K_V7 K⁺, and TRPV1 channels, as well as GABA_A, glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons.

KEY RESULTS

Therapeutic flupirtine concentrations (≤10 µM) did not affect voltage-gated Na⁺ or Ca²⁺ channels, inward rectifier K⁺ channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of K_V7 K⁺ channels to more negative potentials and the gating of GABA_A receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on K_V7 channels and GABA_A receptors was comparable, whereas in hippocampal neurons the effects on K_V7 channels were more pronounced.

CONCLUSIONS AND IMPLICATIONS

These results indicate that flupirtine exerts its analgesic action by acting on both GABA_A receptors and K_V7 channels.     

Patents related to therapeutic activation of KATP and K2P potassium channels for neuroprotection: ischemic/hypoxic/anoxic injury and general anesthetics

Background: Mechanisms of neuroprotection encompass energy deficits in brain arising from insufficient oxygen and glucose levels following respiratory failure; ischemia or stroke, which produce metabolic stresses that lead to unconsciousness and seizures; and the effects of general anesthetics. Foremost among those K⁺ channels viewed as important for neuroprotection are ATP-sensitive (K_ATP) channels, which belong to the family of inwardly rectifying K⁺ channels (K_ir) and contain a sulfonylurea subunit (SUR1 or SUR2) combined with either K_ir6.1 (KCNJ8) or K_ir6.2 (KCNJ11) channel pore-forming α-subunits, and various members of the tandem two-pore or background (K_2P) K⁺ channel family, including K_2P1.1 (KCNK1 or TWIK1), K_2P2.1 (KCNK2 or TREK/TREK1), K_2P3.1 (KCNK3 or TASK), K_2P4.1 (KCNK4 or TRAAK), and K_2P10.1 (KCNK10 or TREK2). Objectives: This review covers patents and patent applications related to inventions of therapeutics, compound screening methods and diagnostics, including K_ATP channel openers and blockers, as well as K_ATP and K_2P nucleic/amino acid sequences and proteins, vectors, transformed cells and transgenic animals. Although the focus of this patent review is on brain and neuroprotection, patents covering inventions of K_ATP channel openers for cardioprotection, diabetes mellitus and obesity, where relevant, are addressed. Results/conclusions: Overall, an important emerging therapeutic mechanism underlying neuroprotection is activation/opening of K_ATP and K_2P channels. To this end substantial progress has been made in identifying and patenting agents that target K_ATP channels. However, current K_2P channels patents encompass compound screening and diagnostics methodo-logies, reflecting an earlier ‘discovery’ stage (target identification/validation) than K_ATP in the drug development pipeline; this reveals a wide-open field for the discovery and development of K_2P-targeting compounds.     

Rosiglitazone selectively inhibits K(ATP) channels by acting on the K(IR) 6 subunit

BACKGROUND AND PURPOSE

Rosiglitazone is an anti-diabetic drug acting as an insulin sensitizer. We recently found that rosiglitazone also inhibits the vascular isoform of ATP-sensitive K⁺ channels and compromises vasodilatory effects of β-adrenoceptor activation and pinacidil. As its potency for the channel inhibition is in the micromolar range, rosiglitazone may be used as an effective K_ATP channel inhibitor for research and therapeutic purposes. Therefore, we performed experiments to determine whether other isoforms of K_ATP channels are also sensitive to rosiglitazone and what their sensitivities are.

EXPERIMENTAL APPROACH

K_IR6.1/SUR2B, K_IR6.2/SUR1, K_IR6.2/SUR2A, K_IR6.2/SUR2B and K_IR6.2ΔC36 channels were expressed in HEK293 cells and were studied using patch-clamp techniques.

KEY RESULTS

Rosiglitazone inhibited all isoforms of K_ATP channels in excised patches and in the whole-cell configuration. Its IC₅₀ was 10 µmol·L⁻¹ for the K_IR6.1/SUR2B channel and ∼45 µmol·L⁻¹ for K_IR6.2/SURx channels. Rosiglitazone also inhibited K_IR6.2ΔC36 channels in the absence of the sulphonylurea receptor (SUR) subunit, with potency (IC₅₀= 45 µmol·L⁻¹) almost identical to that for K_IR6.2/SURx channels. Single-channel kinetic analysis showed that the channel inhibition was mediated by augmentation of the long-lasting closures without affecting the channel open state and unitary conductance. In contrast, rosiglitazone had no effect on K_IR1.1, K_IR2.1 and K_IR4.1 channels, suggesting that the channel inhibitory effect is selective for K_IR6.x channels.

CONCLUSIONS AND IMPLICATIONS

These results suggest a novel K_ATP channel inhibitor that acts on the pore-forming K_IR6.x subunit, affecting the channel gating.

LINKED ARTICLE

This article is commented on by Dart, pp. 23–25 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01990.x     

Differential state-dependent modification of rat Na(v)1.6 sodium channels expressed in human embryonic kidney (HEK293) cells by the pyrethroid insecticides tefluthrin and deltamethrin

We expressed rat Na_v1.6 sodium channels in combination with the rat β1 and β2 auxiliary subunits in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on expressed sodium currents using the whole-cell patch clamp technique. Both pyrethroids produced concentration-dependent, resting modification of Na_v1.6 channels, prolonging the kinetics of channel inactivation and deactivation to produce persistent “late” currents during depolarization and tail currents following repolarization. Both pyrethroids also produced concentration dependent hyperpolarizing shifts in the voltage dependence of channel activation and steady-state inactivation. Maximal shifts in activation, determined from the voltage dependence of the pyrethroid-induced late and tail currents, were ~ 25 mV for tefluthrin and ~ 20 mV for deltamethrin. The highest attainable concentrations of these compounds also caused shifts of ~ 5–10 mV in the voltage dependence of steady-state inactivation. In addition to their effects on the voltage dependence of inactivation, both compounds caused concentration-dependent increases in the fraction of sodium current that was resistant to inactivation following strong depolarizing prepulses. We assessed the use-dependent effects of tefluthrin and deltamethrin on Na_v1.6 channels by determining the effect of trains of 1 to 100 5-ms depolarizing prepulses at frequencies of 20 or 66.7 Hz on the extent of channel modification. Repetitive depolarization at either frequency increased modification by deltamethrin by ~ 2.3-fold but had no effect on modification by tefluthrin. Tefluthrin and deltamethrin were equally potent as modifiers of Na_v1.6 channels in HEK293 cells using the conditions producing maximal modification as the basis for comparison. These findings show that the actions of tefluthrin and deltamethrin of Na_v1.6 channels in HEK293 cells differ from the effects of these compounds on Na_v1.6 channels in Xenopus oocytes and more closely reflect the actions of pyrethroids on channels in their native neuronal environment.     

KV7 channels are involved in hypoxia-induced vasodilatation of porcine coronary arteries

BACKGROUND AND PURPOSE

Hypoxia causes vasodilatation of coronary arteries, but the underlying mechanisms are poorly understood. We hypothesized that hypoxia reduces intracellular Ca²⁺ concentration ([Ca²⁺]_i) by opening of K channels and release of H₂S.

EXPERIMENTAL APPROACH

Porcine coronary arteries without endothelium were mounted for measurement of isometric tension and [Ca²⁺]_i, and the expression of voltage-gated K channels K_V7 channels (encoded by KCNQ genes) and large-conductance calcium-activated K channels (K_Ca1.1) was examined. Voltage clamp assessed the role of K_V7 channels in hypoxia.

KEY RESULTS

Gradual reduction of oxygen concentration from 95 to 1% dilated the precontracted coronary arteries and this was associated with reduced [Ca²⁺]_i in PGF_2α (10 μM)-contracted arteries whereas no fall in [Ca²⁺]_i was observed in 30 mM K-contracted arteries. Blockers of ATP-sensitive voltage-gated potassium channels and K_Ca1.1 inhibited hypoxia-induced dilatation in PGF_2α-contracted arteries; this inhibition was more marked in the presence of the K_v7 channel blockers, XE991 and linopirdine, while a K_V7.1 blocker, failed to change hypoxic vasodilatation. XE991 also inhibited H₂S- and adenosine-induced vasodilatation. PCR revealed the expression of K_V7.1, K_V7.4, K_V7.5 and K_Ca1.1 channels, and K_Ca1.1, K_V7.4 and K_V7.5 were also identified by immunoblotting. Voltage clamp studies showed the XE991-sensitive current was more marked in hypoxic conditions.

CONCLUSION

The K_V7.4 and K_V7.5 channels, which we identified in the coronary arteries, appear to have a major role in hypoxia-induced vasodilatation. The voltage clamp results further support the involvement of K_V7 channels in this vasodilatation. Activation of these K_V7 channels may be induced by H₂S and adenosine.     

The K+ channels KCa3.1 and Kv1.3 as novel targets for asthma therapy

Asthma affects 10% of the UK population and is an important cause of morbidity and mortality at all ages. Current treatments are either ineffective or carry unacceptable side effects for a number of patients; in consequence, development of new approaches to therapy are important. Ion channels are emerging as attractive therapeutic targets in a variety of non-excitable cells. Ion channels conducting K⁺ modulate the activity of several structural and inflammatory cells which play important roles in the pathophysiology of asthma. Two channels of particular interest are the voltage-gated K⁺ channel K_v1.3 and the intermediate conductance Ca²⁺-activated K⁺ channel K_Ca3.1 (also known as IK_Ca1 or SK4). K_v1.3 is expressed in IFNγ-producing T cells while K_Ca3.1 is expressed in T cells, mast cells, macrophages, airway smooth muscle cells, fibroblasts and epithelial cells. Both channels play important roles in cell activation, migration, and proliferation through the regulation of membrane potential and calcium signalling. We hypothesize that K_Ca3.1- and/or K_v1.3-dependent cell processes are one of the common denominators in asthma pathophysiology. If true, these channels might serve as novel targets for the treatment of asthma. Emerging evidence lends support to this hypothesis. Further validation through the study of the role that these channels play in normal and asthmatic airway cell (patho)physiology and in vivo models will provide further justification for the assessment of small molecule blockers of K_v1.3 and K_Ca3.1 in the treatment of asthma.     

PKC-dependent activation of human K(2P) 18.1 K(+) channels

BACKGROUND AND PURPOSE

Two-pore-domain K⁺ channels (K_2P) mediate K⁺ background currents that modulate the membrane potential of excitable cells. K_2P18.1 (TWIK-related spinal cord K⁺ channel) provides hyperpolarizing background currents in neurons. Recently, a dominant-negative loss-of-function mutation in K_2P18.1 has been implicated in migraine, and activation of K_2P18.1 channels was proposed as a therapeutic strategy. Here we elucidated the molecular mechanisms underlying PKC-dependent activation of K_2P18.1 currents.

EXPERIMENTAL APPROACH

Human K_2P18.1 channels were heterologously expressed in Xenopus laevis oocytes, and currents were recorded with the two-electrode voltage clamp technique.

KEY RESULTS

Stimulation of PKC using phorbol 12-myristate-13-acetate (PMA) activated the hK_2P18.1 current by 3.1-fold in a concentration-dependent fashion. The inactive analogue 4α-PMA had no effect on channel activity. The specific PKC inhibitors bisindolylmaleimide I, Ro-32-0432 and chelerythrine reduced PMA-induced channel activation indicating that PKC is involved in this effect of PMA. Selective activation of conventional PKC isoforms with thymeleatoxin (100 nM) did not reproduce K_2P18.1 channel activation. Current activation by PMA was not affected by pretreatment with CsA (calcineurin inhibitor) or KT 5720 (PKA inhibitor), ruling out a significant contribution of calcineurin or cross-talk with PKA to the PKC-dependent hK_2P18.1 activation. Finally, mutation of putative PKC phosphorylation sites did not prevent PMA-induced K_2P18.1 channel activation.

CONCLUSIONS AND IMPLICATIONS

We demonstrated that activation of hK_2P18.1 (TRESK) by PMA is mediated by PKC stimulation. Hence, PKC-mediated activation of K_2P18.1 background currents may serve as a novel molecular target for migraine treatment.     

Regulation of two-pore-domain (K2P) potassium leak channels by the tyrosine kinase inhibitor genistein

BACKGROUND AND PURPOSE: Two-pore-domain potassium (K2P) channels mediate potassium background (or 'leak') currents, controlling excitability by stabilizing membrane potential below firing threshold and expediting repolarization. Inhibition of K2P currents permits membrane potential depolarization and excitation. As expected for key regulators of excitability, leak channels are under tight control from a plethora of stimuli. Recently, signalling via protein tyrosine kinases (TKs) has been implicated in ion channel modulation. The objective of this study was to investigate TK regulation of K2P channels. EXPERIMENTAL APPROACH: The two-electrode voltage clamp technique was used to record K2P currents in Xenopus oocytes. In addition, K2P channels were studied in Chinese hamster ovary (CHO) cells using the whole-cell patch clamp technique. KEY RESULTS: Here, we report inhibition of human K2P3.1 (TASK-1) currents by the TK antagonist, genistein, in Xenopus oocytes (IC50=10.7 microM) and in CHO cells (IC50=12.3 microM). The underlying molecular mechanism was studied in detail. hK2P3.1 was not affected by genistin, an inactive analogue of genistein. Perorthovanadate, an inhibitor of tyrosine phosphatase activity, reduced the inhibitory effect of genistein. Current reduction was voltage independent and did not require channel protonation at position H98 or phosphorylation at the single TK phosphorylation site, Y323. Among functional hK2P family members, genistein also reduced K2P6.1 (TWIK-2), K2P9.1 (TASK-3) and K2P13.1 (THIK-1) currents, respectively. CONCLUSIONS AND IMPLICATIONS: Modulation of K2P channels by the TK inhibitor, genistein, represents a novel molecular mechanism to alter background K+ currents.     

Carvedilol targets human K2P 3.1 (TASK1) K+ leak channels

BACKGROUND AND PURPOSE

Human K_2P3.1 (TASK1) channels represent potential targets for pharmacological management of atrial fibrillation. K_2P channels control excitability by stabilizing membrane potential and by expediting repolarization. In the heart, inhibition of K_2P currents by class III antiarrhythmic drugs results in action potential prolongation and suppression of electrical automaticity. Carvedilol exerts antiarrhythmic activity and suppresses atrial fibrillation following cardiac surgery or cardioversion. The objective of this study was to investigate acute effects of carvedilol on human K_2P3.1 (hK_2P3.1) channels.

EXPERIMENTAL APPROACH

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record hK_2P3.1 currents from Xenopus oocytes, Chinese hamster ovary (CHO) cells and human pulmonary artery smooth muscle cells (hPASMC).

KEY RESULTS

Carvedilol concentration-dependently inhibited hK_2P3.1 currents in Xenopus oocytes (IC₅₀= 3.8 µM) and in mammalian CHO cells (IC₅₀= 0.83 µM). In addition, carvedilol sensitivity of native I_K2P3.1 was demonstrated in hPASMC. Channels were blocked in open and closed states in frequency-dependent fashion, resulting in resting membrane potential depolarization by 7.7 mV. Carvedilol shifted the current–voltage (I–V) relationship by −6.9 mV towards hyperpolarized potentials. Open rectification, characteristic of K_2P currents, was not affected.

CONCLUSIONS AND IMPLICATIONS

The antiarrhythmic drug carvedilol targets hK_2P3.1 background channels. We propose that cardiac hK_2P3.1 current blockade may suppress electrical automaticity, prolong atrial refractoriness and contribute to the class III antiarrhythmic action in patients treated with the drug.     

Current understanding of K ATP channels in neonatal diseases: focus on insulin secretion disorders

ATP-sensitive potassium (K(ATP)) channels are cell metabolic sensors that couple cell metabolic status to electric activity, thus regulating many cellular functions. In pancreatic beta cells, K(ATP) channels modulate insulin secretion in response to fluctuations in plasma glucose level, and play an important role in glucose homeostasis. Recent studies show that gain-of-function and loss-of-function mutations in K(ATP) channel subunits cause neonatal diabetes mellitus and congenital hyperinsulinism respectively. These findings lead to significant changes in the diagnosis and treatment for neonatal insulin secretion disorders. This review describes the physiological and pathophysiological functions of K(ATP) channels in glucose homeostasis, their specific roles in neonatal diabetes mellitus and congenital hyperinsulinism, as well as future perspectives of K(ATP) channels in neonatal diseases.     

Alterations of insulin secretion following long-term manipulation of ATP-sensitive potassium channels by diazoxide and nateglinide

Previous studies have shown that prolonged exposure to drugs, which act via blocking KATP channels, can desensitize the insulinotropic effects of drugs and nutrients acting via KATP channels. In this study, effects of prolonged exposure to diazoxide, a KATP channel opener, on beta cell function were examined using clonal BRIN-BD11 cells. The findings were compared to the long-term effects of KATP channel blockers nateglinide and tolbutamide. Following 18 h exposure to 200 microM diazoxide, the amounts of insulin secreted in response to glucose, amino acids and insulinotropic drugs were increased. Secretory responsiveness to a variety of agents acting via KATP channels was retained following prolonged diazoxide exposure. In contrast, 18 h exposure to 100 microM nateglinide significantly attenuated the insulin secretory responses to tolbutamide, nateglinide and BTS 67 582. Glucose- and L-alanine-stimulated insulin release were unaffected by prolonged nateglinide exposure, however responsiveness to L-leucine and L-arginine was diminished. Prolonged exposure to nateglinide had no effect on forskolin- and PMA-stimulated insulin release, and the overall pattern of desensitization was similar to that induced by 100 microM tolbutamide. We conclude that in contrast to chronic long-term KATP channel blockade, long-term diazoxide treatment is not harmful to KATP channel mediated insulin secretion and may have beneficial protective effects on beta cell function.