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[The role of sulphonylurea derivatives in treatment of diabetes type 2. The role of glimepiride].

Type 2 diabetes is characterised by some kind of duality. In its pathogenesis an important role have as well genetic as environmental factors. Both of them influence the insulin secretion at the one side and the insulin resistance at the other one. Very important group of antidiabetic drugs are sulphonylureas. They bind to the sulphonylurea receptor localized at the potassium channel in the cellular membrane. Contemporary sulphonylurea derivatives should be characterised by rather weak binding with the receptor, action during mealtime only (prandial regulation of glycemia), not to strong insulin secretion and for the quality of life reason once daily application. This criteria are fulfilled by e.g. slow-releasing preparations of glipizide and glikiazide and also by glimepiride.

ATP-Binding Cassette Transporters↗

In vivo androgen treatment shortens the QT interval and increases the densities of inward and delayed rectifier potassium currents in orchiectomized male rabbits.

OBJECTIVES: Women have longer rate-corrected QT intervals (QTc) and are at higher risk for developing life-threatening torsades de pointes ventricular arrhythmias than men, especially after taking medications that block cardiac human ether-a-go-go-related gene (HERG)-encoded K(+) channels. The purpose of the present study was to determine if the male sex steroid hormone, dihydrotestosterone (DHT), influences QT intervals in orchiectomized (Orch) male rabbits. METHODS: ECG and whole-cell patch-clamp analyses were employed to evaluate cardiac repolarization and K(+) currents in hearts isolated from orchiectomized (Orch) male New Zealand White rabbits receiving subcutaneous sustained release pellets for either dihydrotestosterone (DHT) or placebo. The efficacy of the treatment paradigm was monitored by measuring plasma DHT concentrations before and after the treatment period (10-14 days). RESULTS: The results show that rate- and drug-induced QT-lengthening is attenuated in hearts from DHT-treated rabbits relative to placebo-treated controls. No significant changes in QRS were observed in response to DHT, thereby indicating that DHT influences QT primarily through an effect on ventricular repolarization. In addition, hearts from DHT-treated rabbits displayed significantly less QT lengthening in response to quinidine challenge compared to placebo controls. Current densities for two important cardiac repolarizing K(+) currents, I(K1) and I(Kr), were found to be significantly increased in ventricular myocardium of DHT-treated rabbits. Further, the half-maximal voltage of activation (V(1/2)) for I(Kr) was significantly shifted to more negative potentials in myocytes from DHT vs. placebo hearts (21.2+/-1.2 vs. 30.2+/-1.4 mV, respectively, n=12, P<0.001). Corresponding changes in rabbit ether-a-go-go-related gene (RERG) mRNA were not found when examined by Northern blot hybridization. CONCLUSIONS: These results suggest that the presence of male sex steroid hormones in male rabbits helps to suppress rate- and drug-induced delays in cardiac repolarization. DHT action produces increased current densities for I(K1) and I(Kr) and a left-shift in the V(1/2) for I(Kr) that could account, at least in part, for the observed QTc differences between males and females. Since little change was seen in ventricular RERG gene expression, DHT action in the heart may influence I(Kr) via post-transcriptional and/or post-translational mechanisms.

Animals↗

Potassium channels of myenteric neurons in guinea-pig small intestine.

Patch-clamp recording was used to study rectifying K+ currents in myenteric neurons in short-term culture. In conditions that suppressed Ca2+ -activated K+ current, three kinds of voltage-activated K+ currents were identified by their voltage range of activation, inactivation, kinetics and pharmacology. These were A-type current, delayed outwardly rectifying current (I(K),dr) and inwardly rectifying current (I(K),ir). I(K),ir consisted of an instantaneous component followed by a time-dependent current that rapidly increased at potentials negative to -80 mV. Time-constant of activation was voltage-dependent with an e-fold decrease for a 31-mV hyperpolarization amounting to a decrease from 800 to 145 ms between -80 and -100 mV. I(K),ir did not inactivate. I(K),ir was abolished in K+ -free solution. Increases in external K+ increased I(K),ir conductance in direct relation to the square root of external K+ concentration. Activation kinetics were accelerated and the activation range shifted to more positive K+ equilibrium potentials. I(K),ir was suppressed by external Cs+ and Ba2+ in a concentration-dependent manner. Ca2+ and Mg+ were less effective than Ba2+. I(K),ir was unaffected by tetraethylammonium ions. I(K),dr was activated at membrane potentials positive to - 30 mV with an e-fold decrease in time-constant of activation from 145 to 16 ms between -20 and 30 mV. It was half-activated at 5 mV and fully activated at 50 mV. Inactivation was indiscernible during 2.5 s test pulses. I(K),dr was suppressed in a concentration-, but not voltage-dependent manner by either tetraethylammonium or 4-aminopyridine and was insensitive to Cs+. The results suggest that I(K),ir may be important in maintaining the high resting membrane potentials found in afterhyperpolarization-type enteric neurons. They also suggest importance of I(K),ir channels in augmentation of the large hyperpolarizing after-potentials in afterhyperpolarization-type neurons and the hyperpolarization associated with inhibitory postsynaptic potentials. I(K),dr in afterhyperpolarization-type enteric neurons has overall kinetics and voltage behaviour like delayed rectifier currents in other excitable cells where the currents can also be distinguished from A-type and Ca2+ -activated K+ current.

Animals↗

Up-regulation of the Kv3.4 potassium channel subunit in early stages of Alzheimer's disease.

Gene expression throughout the different stages of Alzheimer's disease was analysed in samples from cerebral cortex. The gene encoding the voltage-gated potassium channel Kv3.4 was already overexpressed in early stages of the disease, and in advanced stages Kv3.4 was present at high levels in neurodegenerative structures. This subunit regulates delayed-rectifier currents, which are primary determinants of spike repolarization in neurones. In unique samples from a patient with Alzheimer's disease whose amount of amyloid plaques was decreased by beta amyloid immunization, Kv3.4 was overexpressed. The channel subunit was expressed in the neuropil, in the remaining conventional plaques in the frontal cortex and in collapsed plaques in the orbitary cortex. Therefore, amyloid deposition in plaques does not seem to be responsible for the increase in Kv3.4 levels. Nevertheless, Kv3.4 up-regulation is related to amyloid pathology, given that transgenic mice with the Swedish mutation of amyloid precursor protein showed increased expression of Kv3.4. Up-regulation of voltage-gated potassium channel subunits alters potassium currents in neurones and leads to altered synaptic activity that may underlie the neurodegeneration observed in Alzheimer's disease. Thus, Kv3.4 likely represents a novel therapeutic target for the disease.

Aged↗

Identification of ether à go-go and calcium-activated potassium channels in human melanoma cells.

Ion channels and intracellular Ca2+ are thought to be involved in cell proliferation and may play a role in tumor development. We therefore characterized Ca(2+)-regulated potassium channels in the human melanoma cell lines IGR1, IPC298, and IGR39 using electrophysiological and molecular biological methods. All cell lines expressed outwardly rectifying K+ channels. Rapidly activating delayed rectifier channels were detected in IGR39 cells. The activation kinetics of voltage-gated K+ channels in IRG1 and IPC298 cells displayed characteristics of ether à go-go (eag) channels as they were much slower and depended both on the holding potential and on extracellular Mg2+. In addition, they could be blocked by physiological concentrations of intracellular Ca2+. In accordance with these electrophysiological results, analysis of mRNA revealed the expression of a gene coding for h-eag1 channels in IGR1 and IPC298 cells, but not in IGR39 cells. At elevated Ca2+ concentrations various types of Ca(2+)-activated K+ channels with single-channel characteristics similar to IK and SK channels were detected in IGR1 cells. The whole-cell Ca(2+)-activated K+ currents were not voltage dependent, insensitive for 100 nm apamin and 200 microm d-tubocurarine, but were blocked by charybdotoxin (100 nm) and clotrimazole (50 nm). Analysis of mRNA revealed the expression of hSK1, hSK2, and hIK channels in IGR1 cells.

Calcium↗

A discriminant block among K+ channel types by phenytoin in neuroblastoma cells.

The action of the anticonvulsant drug phenytoin on K+ channels was investigated in neuroblastoma cells (N2A) by using the single-channel patch-clamp technique. N2A cells expressed three types of delayed rectifier K+ channels, which were found to have a conductance of 10-20 pS in a 'physiological' K+ gradient. When added to the external solution at concentrations ranging between 1 and 200 microM, phenytoin decreased single channel activity, whereas the unitary current amplitude was unaffected in all three types of channels. The open probability of the biggest channel decreased, according to an exponential distribution of open and closed times, from 40% in control conditions to 10% in the presence of 50 microM phenytoin (Vm=40 mv). The reduction in the open-channel probability was concentration-dependent with a IC50 = 27.2+/-0.9 microM. A transient type of K channel was identified that was affected by cumulative inactivation and had a conductance of a mean value equal to 26 pS. Finally, a voltage-and Ca2+-dependent K+ channel with a unitary conductance of 95 pS was recorded. Both the channel's amplitude and kinetics were unaffected by phenytoin. These results confirm the phenytoin effect on K+ currents and suggest that the drug may be considered a selective blocker of delayed rectifier K+ channels.

Animals↗

A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel.

Mutations in HERG cause an inherited cardiac arrhythmia, long QT syndrome (LQT). To define the function of HERG, we expressed the protein in Xenopus oocytes. The biophysical properties of expressed HERG are nearly identical to the rapidly activating delayed rectifier K+ current (IKr) in cardiac myocytes. HERG current is K+ selective, declines with depolarizations above 0 mV, is activated by extracellular K+, and is blocked by lanthanum. Interestingly, HERG current is not blocked by drugs that specifically block IKr in cardiac myocytes. These data indicate that HERG proteins form IKr channels, but that an additional subunit may be required for drug sensitivity. Since block of IKr is a known mechanism for drug-induced cardiac arrhythmias, the finding that HERG encodes IKr channels provides a mechanistic link between certain forms of inherited and acquired LQT.

Animals↗

A mouse brain homolog of the Drosophila Shab K+ channel with conserved delayed-rectifier properties.

We have cloned and expressed a mouse brain K+ channel that is the homolog of the Drosophila Shab K+ channel. Mouse and Drosophila Shab K+ channels (mShab and fShab, respectively) represent an instance of K+ channels and structurally related species that are both functionally and structurally conserved; most kinetic, voltage-sensitive, and pharmacological properties are similar for the 2 channels. The greatest functional difference between the currents is recovery from inactivation, which is several times slower for mShab than for fShab currents. In addition to conserved structure, the mShab polypeptide has an unusually long nonconserved region at the carboxyl end of the protein. Truncation of 293 residues from the carboxyl end produced no noticeable change in voltage-sensitive, kinetic, or pharmacological properties. Thus, the measured functional properties of mShab are determined by the remaining 564 residues, most of which are conserved. The mShab and fShab channels are naturally occurring structural variants having substitutions in conserved portions that appear relatively neutral with respect to all measured properties except for, possibly, the rate of recovery from inactivation. The mShab current closely resembles a native delayed-rectifier-type potassium current, IK, in hippocampal neurons.

Amino Acid Sequence↗

Simulation of Brugada syndrome using cellular and three-dimensional whole-heart modeling approaches.

Brugada syndrome (BS) is a genetic disease identified by an abnormal electrocardiogram (ECG) (mainly abnormal ECGs associated with right bundle branch block and ST-elevation in right precordial leads). BS can lead to increased risk of sudden cardiac death. Experimental studies on human ventricular myocardium with BS have been limited due to difficulties in obtaining data. Thus, the use of computer simulation is an important alternative. Most previous BS simulations were based on animal heart cell models. However, due to species differences, the use of human heart cell models, especially a model with three-dimensional whole-heart anatomical structure, is needed. In this study, we developed a model of the human ventricular action potential (AP) based on refining the ten Tusscher et al (2004 Am. J. Physiol. Heart Circ. Physiol. 286 H1573-89) model to incorporate newly available experimental data of some major ionic currents of human ventricular myocytes. These modified channels include the L-type calcium current (I(CaL)), fast sodium current (I(Na)), transient outward potassium current (I(to)), rapidly and slowly delayed rectifier potassium currents (I(Kr) and I(Ks)) and inward rectifier potassium current (I(Ki)). Transmural heterogeneity of APs for epicardial, endocardial and mid-myocardial (M) cells was simulated by varying the maximum conductance of I(Ks) and I(to). The modified AP models were then used to simulate the effects of BS on cellular AP and body surface potentials using a three-dimensional dynamic heart-torso model. Our main findings are as follows. (1) BS has little effect on the AP of endocardial or mid-myocardial cells, but has a large impact on the AP of epicardial cells. (2) A likely region of BS with abnormal cell AP is near the right ventricular outflow track, and the resulting ST-segment elevation is located in the median precordium area. These simulation results are consistent with experimental findings reported in the literature. The model can reproduce a variety of electrophysiological behaviors and provides a good basis for understanding the genesis of abnormal ECG under the condition of BS disease.

Action Potentials↗

Properties of the cromakalim-induced potassium conductance in smooth muscle cells isolated from the rabbit portal vein.

1. Single smooth muscle cells were isolated freshly from the rabbit portal vein and membrane currents were recorded by the whole-cell or excised patch configurations of the patch-clamp technique at room temperature. 2. Cromakalim (Ckm, 10 microM) induced a potassium current (ICkm) that showed no pronounced voltage-dependence and had low current noise. 3. This current, ICkm, was inhibited by (in order of potency): phencyclidine greater than quinidine greater than 4-aminopyridine greater than tetraethylammonium ions (TEA). These drugs inhibited the delayed rectifier current, IdK, which is activated by depolarization of the cell, with the same order of potency. 4. Large conductance calcium-activated potassium channels (LKCa) in isolated membrane patches were blocked by (in order of potency) quinidine greater than TEA approximately phencyclidine. 4-Aminopyridine was ineffective. A similar order of potency was found for block of spontaneous transient outward currents thought to represent bursts of openings of LKCa channels. 5. The low current noise of ICkm at positive potentials, and its susceptibility to inhibitors indicated that it was not carried by LKCa channels, and that it may be carried by channels which underlie IdK. It was observed that when ICkm was activated, IdK was reduced. However, in two experiments, ICkm was much more susceptible to glibenclamide than IdK; possible reasons for this are discussed.

Animals↗

Selective blockade of the delayed rectifier potassium current by tacrine in Drosophila.

Tetrahydroaminoacridine (tacrine) is an anticholinesterase agent used in the treatment of Alzheimer's disease. Its effectiveness against dementia is attributed to its inhibition of acetylcholine breakdown in the synaptic cleft. Tacrine has also been shown to block ionic currents, including many types of potassium (K+) currents, calcium currents, and sodium currents. However, the physiologic significance of this blockade, especially with respect to its effectiveness against Alzheimer's disease, is not clear because of relatively high (several hundred micromolar to millimolar) concentrations of tacrine employed in many studies of channel blockade, and because it blocks several types of currents. A complete mutational and pharmacologic resolution of ionic currents in the larval muscles of Drosophila allowed us to examine the selectivity of tacrine's effects at very low concentrations. At concentrations as low as 10 microM, tacrine selectively blocked the delayed rectifier K+ current without affecting the three other K+ currents or the calcium channel current in these cells. It also increased the duration of the action potentials significantly. An interesting aspect of tacrine's selectivity is that the current blocked by it is the quinidine-sensitive delayed rectifier K+ current rather than the 4-aminopyridine (4-AP)-sensitive transient K+ current. This is in contrast to the generally emphasized structural relationship between tacrine and 4-AP. Since tacrine is structurally related to quinidine as well, these observations suggest a structural basis for the selectivity of tacrine, 4-AP, and quinidine for specific K+ channels. Furthermore, the data are consistent with the possibility of increased neurotransmitter release, due to prolonged presynaptic action potentials, acting synergistically with the anticholinesterase activity of tacrine to increase its therapeutic effectiveness.

4-Aminopyridine↗

Reversal of rectification and alteration of selectivity and pharmacology in a mammalian Kv1.1 potassium channel by deletion of domains S1 to S4.

1. A possible relation between the family of inwardly rectifying K+ channels and the Shaker superfamily of K+ channels was investigated using a deletion mutant (DelS1-S4) of a delayed rectifier Kv1.1 (RCK1) K+ channel. 2. The mutant DelS1-S4 was made by eliminating the sequence coding for transmembrane domains S1 to S4 of the Kv1.1 K+ channel, and re-ligating the sequence coding for the cytoplasmic amino terminus to transmembrane domain S5. Microelectrode voltage-clamp and patch-clamp experiments were performed on Xenopus laevis oocytes after injection of in vitro transcribed mRNA coding for mutant and wild-type channels. 3. The lack of transmembrane domains S1 to S4 converts a depolarization-activated wild-type Kv1.1 K+ channel with outward rectification into a hyperpolarization-activated channel with inward rectification. Although the pore region of the deletion mutant is identical to the wild-type channel, the mutant channel is a non-selective cation channel and is characterized by an altered pharmacology profile.

Animals↗

Human cardiac potassium channel DNA polymorphism modulates access to drug-binding site and causes drug resistance.

Expression of voltage-gated K channel, shaker-related subfamily, member 5 (KCNA5) underlies the human atrial ultra-rapid delayed rectifier K current (I(Kur)). The KCNA5 polymorphism resulting in P532L in the C terminus generates I(Kur) that is indistinguishable from wild type at baseline but strikingly resistant to drug block. In the present study, truncating the C terminus of KCNA5 generated a channel with wild-type drug sensitivity, which indicated that P532 is not a drug-binding site. Secondary structure prediction algorithms identified a probable alpha-helix in P532L that is absent in wild-type channels. We therefore assessed drug sensitivity of I(Kur) generated in vitro in CHO and HEK cells by channels predicted to exhibit or lack this C-terminal alpha-helix. All constructs displayed near-identical I(Kur) in the absence of drug challenge. However, those predicted to lack the C-terminal alpha-helix generated quinidine-sensitive currents (43-51% block by 10 microM quinidine), while the currents generated by those constructs predicted to generate a C-terminal alpha-helix were inhibited less than 12%. Circular dichroism spectroscopy revealed an alpha-helical signature with peptides derived from drug-resistant channels and no organized structure in those associated with wild-type drug sensitivity. In conclusion, we found that this secondary structure in the KCNA5 C terminus, absent in wild-type channels but generated by a naturally occurring DNA polymorphism, does not alter baseline currents but renders the channel drug resistant. Our data support a model in which this structure impairs access of the drug to a pore-binding site.

Amino Acid Substitution↗

A Kv1.5 to Kv1.3 switch in endogenous hippocampal microglia and a role in proliferation.

The proliferation of microglia is a normal process in CNS development and in the defense against pathological insults, although, paradoxically, it contributes to several brain diseases. We have examined the types of voltage-activated K(+) currents (Kv) and their roles in microglial proliferation. Microglia were tissue-printed directly from the hippocampal region using brain slices from 5- to 14-d-old rats. Immediately after tissue prints were prepared, unipolar and bipolar microglia expressed a large Kv current, and the cells were not proliferating. Surprisingly, this current was biophysically and pharmacologically distinct from Kv1.3, which has been found in dissociated, cultured microglia, but it was very similar to Kv1.5. After several days in culture the microglia became highly proliferative, and although the Kv prevalence and current density decreased, many cells exhibited a prominent Kv that was indistinguishable from Kv1.3. The Kv1.5-like current was present in nonproliferating cells, whereas proliferating cells expressed the Kv1.3-like current. Immunocytochemical staining showed a dramatic shift in expression and localization of Kv1.3 and Kv1.5 proteins in microglia: Kv1.5 moving away from the surface and Kv1.3 moving to the surface as the cells were cultured. K(+) channel blockers inhibited proliferation, and the pharmacology of this inhibition correlated with the type of Kv current expressed. Our study, which introduces a method for the physiological examination of microglia from identified brain regions, demonstrates the differential expression of two functional Kv subunits and shows that a functional delayed rectifier current is necessary for microglia proliferation.

Animals↗

Single channel studies of the phosphorylation of K+ channels in the squid giant axon. II. Nonstationary conditions.

The effects of phosphorylation on the properties of the 20-pS channel of the squid giant axon were studied using the cut-open axon technique. Phosphorylation of the channel was achieved by photoreleasing caged ATP (inside the patch pipette) in the presence of the catalytic subunit of the protein kinase A. An inverted K+ gradient (500 K+ external parallel 5 K+ internal) was used to study the activation process. Phosphorylation decreased the frequency of openings of the channel at most potentials by shifting the probability vs. voltage curve toward more positive potentials. The mean open times showed no voltage dependence and were not affected by phosphorylation. The distribution of first latencies, on the other hand, displayed a sharp voltage dependence. Phosphorylation increased the latency to the first opening at all potentials, shifting the median first latency vs. voltage curve toward more positive potentials. The slow inactivation process was studied in the presence of a physiological K+ gradient (10 K+ external parallel 310 K+ internal). Pulses to 40 mV from different holding potentials were analyzed. Phosphorylation increases the overall ensemble probability by decreasing the number of blank traces. A single channel inactivation curve was constructed by computing the relative appearance of blank traces at different holding potentials before and after photoreleasing caged ATP. As determined in dialyzed axons, the effect of phosphorylation consisted in a shift of the inactivation curve toward more positive potentials. The 20-pS channel has the same characteristics as the delayed rectifier current in activation kinetics, steady-state inactivation, and phosphorylation effects.

Animals↗

Mitogenic factors regulate ion channels in Schwann cells cultured from newborn rat sciatic nerve.

1. Patch clamp studies were carried out in Schwann cells cultured from newborn rat sciatic nerve to determine the effects of mitogens on voltage-gated currents without the confounding influences of axonal contact and myelin present in vivo. The relevance of the various Schwann cell currents to proliferation was assessed using assays of [3H]thymidine incorporation. 2. Treatment of cultured Schwann cells with known mitogens, namely axon fragments (AF), myelin fragments (MF), or glial growth factor in combination with forskolin (GGF+F), increased the magnitudes of delayed rectifying potassium (K+) and sodium (Na+) currents. 3. In both control and mitogen-treated cells, the magnitude of net outward current paralleled clearly the magnitude of the cells' proliferative response. 4. The K+ channel-blocking quaternary ammonium ions, tetrabutylammonium (TBuA), tetrapentylammonium (TPeA) and tetrahexylammonium (THeA), but not the Na+ channel blocker tetrodotoxin (TTX), reduced proliferation in a dose-dependent fashion offering further evidence for a role for K+ channels in Schwann cell proliferation. 5. Voltage-gated chloride (Cl-) currents were observed in both control and mitogen-treated cells. Addition of the Cl- channel blockers, 4-acetamido-4'-isocyanatostilbene-2,2'-disulphonate (SITS) or 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS), to the culture media enhanced proliferation. 6. The possible intermediary role of the Schwann cell resting potential was explored in ion substitution experiments by increasing the K+ concentration of the media and by adding ouabain. Both manipulations inhibited Schwann cell mitosis. 7. Comparison of the expression of functional ion channels in vitro with that previously described for Schwann cells in vivo suggests a difference in the Schwann cell response to the membrane fragment mitogens and their intact counterparts in regard to the regulation of ion channels. MF up-regulates the number of functional channels, whereas the elaboration of myelin (or a factor related to its presence) in vivo appears to down-regulate channel expression, at the cell soma of myelinating Schwann cells. In addition, axonal contact may be required for normal expression of functional inwardly rectifying K+ channels.

Animals↗