Current Transients Associated with BK Channels in Human Glioma Cells

We have previously demonstrated the expression of BK channels in human glioma cells. There was a curious feature to the whole-cell currents of glioma cells seen during whole-cell patch-clamp: large, outward current transients accompanied repolarization of the cell membrane following an activating voltage step. This transient current, I _transient, activated and inactivated rapidly (1 ms). The I-V relationship of I _transient had features that were inconsistent with simple ionic current through open ion channels: (i) I _transient amplitude peaked with a –80 mV voltage change and was invariant over a 200 mV range, and (ii) I _transient remained large and outward at –140 mV. We provide evidence for a direct relationship of I _transient to glioma BK currents. They had an identical time course of activation, identical pharmacology, identical voltage-dependence, and small, random variations in the amplitude of the steady-state BK current and I _transient seen over time were often perfectly in phase. Substituting intracellular K⁺ with Cs⁺, Li⁺, or Na ⁺ ions reversibly reduced I _transient and BK currents. I _transient was not observed in recordings of other BK currents (hbr5 expressed in HEK cells and BK currents in rat neurons), suggesting I _transient is unique to BK currents in human glioma cells. We conclude that I _transient is generated by a mechanism related to the deactivation, and level of prior activation, of glioma BK channels. To account for these findings we propose that K⁺ ions are trapped within glioma BK channels during deactivation and are forced to exit to the extracellular side in a manner independent of membrane potential.     

Mechanisms for nucleosome mobilization

Nucleosomes are the ubiquitous and fundamental packaging for eukaryotic genomes, and are the substrate for many processes in the nucleus. Nucleosomes are not static entities but can readily be moved by thermal energy and ATP-dependent chromatin remodeling complexes in a process known as sliding or shifting. We summarize from a mechanical perspective the twist defect and bulge diffusion mechanisms proposed as the most likely pathway for nucleosome mobilization. We then consider the elastic properties of DNA and how this affects the potential for each mechanism, concentrating on kinetic aspects of twist diffusion and possible planar bulge sizes and summarize the experimental evidence reflecting on each. Either, or both, mechanisms could occur, and careful experimentation focusing on their uniquely distinguishing features will be required to determine their relative contributions to chromatin dynamics.     

Mechanosensitive channel of Thermoplasma, the cell wall-less archaea

By using a functional approach of reconstituting detergent-solubilized membrane proteins into liposomes and following their function in patch-clamp experiments, we identified a novel mechanosensitive (MS) channel in the thermophilic cell wall-less archaeon Thermoplasma volcanium. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enriched protein fractions revealed a band of approx 15 kDa comparable to MscL, the bacterial MS channel of large conductance. 20 N-terminal residues determined by protein microsequencing, matched the sequence to an unknown open reading frame in the genome of a related species Thermoplasma acidophilum. The protein encoded by the T. acidophilum gene was cloned and expressed in Escherichia coli and reconstituted into liposomes. When examined for function, the reconstituted protein exhibited properties typical of an MS ion channel: 1) activation by negative pressure applied to the patch-clamp pipet, 2) blockage by gadolinium, and 3) activation by the anionic amphipath trinitrophenol. In analogy to the nomenclature used for bacterial MS channels, the MS channel of T. acidophilum was termed MscTA. Secondary structural analysis indicated that similar to MscL, the T. acidophilum MS protein may have two transmembrane domains, suggesting that MS channels of thermophilic Archaea belong to a family of structurally related MscL-like ion channels with two membrane-spanning regions. When the mscTA gene was expressed in the mscL ⁻ knockout strain and the MscTA protein reconstituted into liposomes, the gating of MscTA was charaterized by very brief openings of variable conductance. In contrast, when the mscTA gene was expressed in the wild-type mscL ⁺ strain of E. coli, the gating properties of the channel resembled MscL. However, the channel had reduced conductance and differed from MscL in its kinetics and in the free energy of activation, suggesting that MscTA and MscL can form functional complexes and/or modulate each other activity. Similar to MscL, MscTA exhibited an increase in activity in liposomes made of phospholipids having shorter acyl chain, suggesting a role of hydrophobic mismatch in the function of prokaryotic MS channels.     

Methods for the study of ionic currents and Ca2+-signals in isolated colonic crypts

Isolated epithelial cells from intestinal mucosae are a suitable object for the study of the regulation of ion transport in the gut. This regulation possesses a great importance for human and veterinary medicine, as diarrheal diseases, which often are caused by an inadequate activation of intestinal anion secretion, are one of the major lethal diseases of children or young animals. The aim of this paper is to describe a method for the isolation of intact colonic crypts, e.g. for the subsequent investigation of the regulation of anion secretion by the intracellular second messenger, Ca²⁺ using electrophysiological and imaging techniques. Published: April 8, 2002     

The interdigitated beta-helix domain of the P22 tailspike protein acts as a molecular clamp in trimer stabilization

The P22 tailspike adhesin is an elongated thermostable trimer resistant to protease digestion and to denaturation in sodium dodecyl sulfate. Monomeric, dimeric, and protrimeric folding and assembly intermediates lack this stability and are thermolabile. In the native trimer, three right-handed parallel beta-helices (residues 143-540), pack side-by-side around the three-fold axis. After residue 540, these single chain beta-helices terminate and residues 541-567 of the three polypeptide chains wrap around each other to form a three-stranded interdigitated beta-helix. Three mutants located in this region -- G546D, R563Q, and A575T -- blocked formation of native tailspike trimers, and accumulated soluble forms of the mutant polypeptide chains within cells. The substitutions R563Q and A575T appeared to prevent stable association of partially folded monomers. G546D, in the interdigitated region of the chain, blocked tailspike folding at the transition from the partially-folded protrimer to the native trimer. The protrimer-like species accumulating in the G546D mutant melted out at 42 degrees C and was trypsin and SDS sensitive. The G546D defect was not corrected by introduction of global suppressor mutations, which correct kinetic defects in beta-helix folding. The simplest interpretation of these results is that the very high thermostability (T(m) = 88 degrees C), protease and detergent resistance of the native tailspike acquired in the protrimer-to-trimer transition, depends on the formation of the three-stranded interdigitated region. This interdigitated beta-helix appears to function as a molecular clamp insuring thermostable subunit association in the native trimer.     

Fine gating properties of channels responsible for persistent sodium current generation in entorhinal cortex neurons

The gating properties of channels responsible for the generation of persistent Na(+) current (I(NaP)) in entorhinal cortex layer II principal neurons were investigated by performing cell-attached, patch-clamp experiments in acutely isolated cells. Voltage-gated Na(+)-channel activity was routinely elicited by applying 500-ms depolarizing test pulses positive to -60 mV from a holding potential of -100 mV. The channel activity underlying I(NaP) consisted of prolonged and frequently delayed bursts during which repetitive openings were separated by short closings. The mean duration of openings within bursts was strongly voltage dependent, and increased by e times per every approximately 12 mV of depolarization. On the other hand, intraburst closed times showed no major voltage dependence. The mean duration of burst events was also relatively voltage insensitive. The analysis of burst-duration frequency distribution returned two major, relatively voltage-independent time constants of approximately 28 and approximately 190 ms. The probability of burst openings to occur also appeared largely voltage independent. Because of the above "persistent" Na(+)-channel properties, the voltage dependence of the conductance underlying whole-cell I(NaP) turned out to be largely the consequence of the pronounced voltage dependence of intraburst open times. On the other hand, some kinetic properties of the macroscopic I(NaP), and in particular the fast and intermediate I(NaP)-decay components observed during step depolarizations, were found to largely reflect mean burst duration of the underlying channel openings. A further I(NaP) decay process, namely slow inactivation, was paralleled instead by a progressive increase of interburst closed times during the application of long-lasting (i.e., 20 s) depolarizing pulses. In addition, long-lasting depolarizations also promoted a channel gating modality characterized by shorter burst durations than normally seen using 500-ms test pulses, with a predominant burst-duration time constant of approximately 5-6 ms. The above data, therefore, provide a detailed picture of the single-channel bases of I(NaP) voltage-dependent and kinetic properties in entorhinal cortex layer II neurons.     

Structural determinants for the action of grayanotoxin in D1 S4-S5 and D4 S4-S5 intracellular linkers of sodium channel alpha-subunits

We located a novel binding site for grayanotoxin on the cytoplasmic linkers of voltage-dependent cardiac (rH1) or skeletal-muscle (mu 1) Na(+) channel isoforms (segments S4-S5 in domains D1 and D4), using the alanine scanning substitution method. GTX-modification of Na(+) channels, transiently expressed in HEK 293 cells, was evaluated under whole-cell voltage clamp, from the ratio of maximum chord conductance for modified and unmodified Na(+) channels. In mu 1, mutations K237A, L243A, S246A, K248A, K249A, L250A, S251A, or T1463A, caused a moderate, but statistically significant decrease in this ratio. On making corresponding mutations in rH1, only L244A dramatically reduced the ratio. Because in mu 1, the serine at position 251 is the only heterologous residue with respect to rH1 (Ala-252), we made a double mutant L243A&S251A to match the sequence of mu 1 and rH1 in S4-S5 linkers of both domains. This double mutation resulted in a significant decrease in the ratio, to the same extent as L244A substitution in rH1 did, indicating that the site at Leu-244 in rH1 or at Leu-243 in mu 1 is a novel one, exhibiting a synergistic effect of grayanotoxin.     

Effect of cola intake on insulin resistance in moderate fat-fed weaning male rats

In recent years, the prevalence of type 2 diabetes mellitus has dramatically increased in Korea as the diet has rapidly become westernized. We determined the effect of a long-term cola intake for insulin resistance in weaning male Sprague Dawley rats consuming a moderate fat diet. Thirty male pubs born from 6 female rats were randomized into cola or water drinking groups. The rats of the cola group were freely provided with 33 energy percent fat diets and cola for 28 weeks, while the rats of the control group had the same diet with water instead of cola. The daily caloric intake did not differ between groups, while the rats in the cola group consumed more carbohydrates. However, the mean body weight of the cola group was lower than that of the control group from the second week of the study. Whole body glucose disposal rates measured by euglycemic hyperinsulinemic clamp were higher in the cola group. Compared to the control group, glycogen contents and fraction velocity of glycogen synthase of the quadriceps muscle in the cola group were higher by 39.4% and 40.3%, respectively. Uncoupling protein (UCP)-2 and GLUT 4 contents of soleus and quadriceps muscles were higher in the cola group than the control group. In conclusion, insulin action improved with increased peripheral glucose utilization in weaning male rats drinking cola, which was partly due to lower body weight. This latter was possibly as a result of increased thermogenesis in muscles.     

蛋白激酶A和蛋白激酶C对豚鼠心肌细胞延迟整流钾电流的作用

目的 :研究蛋白激酶A和蛋白激酶C对豚鼠心室肌细胞延迟整流钾电流 (Ik)的影响。方法 :采用电极内液浓度差扩散法进行细胞内给药 ,利用全细胞膜片箝技术测定单细胞Ik。结果 :cAMP15 0 μmol/L使Ik及Ik ,tail(pA/pF)从 13.7± 2 .1和 6 .1± 0 .3增至 18.5± 3.3和 6 .4± 2 .1(P <0 .0 1,n =6 ) ;8 CPT cAMP15 0 μmol/L使电流 (pA/pF)从 11.4± 1.8及 5 .3± 0 .6增至 17.9± 4 .0和 6 .2± 1.3,PKA的选择性抑制剂 6 2 2 1.0 μmol/L的可逆转二者的作用。cAMP使Ik的激活曲线左移 ,半激活电压 (V1/ 2 )从 2 3.3mV移至 18.7mV ,激活曲线斜率 (k)在用药前后变化较小。 10 μmol/LPMA可以分别使Ik和Ik ,tial(pA/pF)从 12 .9± 1.8和 5 .0± 1.7升至 2 3.7± 2 .8和 7.5±1.1。PMA使I V曲线幅值增加 ,并随去极化电压的升高其作用加强 ,同时PMA使通道的激活曲线k从 15 .3mV升到 2 5 .6mV ,但对V1/ 2 基本无影响。结论 :蛋白激酶A和蛋白激酶C均可增加豚鼠心肌细胞Ik,但二者作用特点有所不同     

Genistein inhibits the inward rectifying potassium current in guinea pig ventricular myocytes

Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. Previous studies have shown that genistein has additional effects, among which the direct blocking effects on various ionic channels have recently been disclosed. Using whole-cell voltage clamp and current clamp techniques, we demonstrate that micromolar concentrations of genistein dose-dependently and reversibly inhibit the inward rectifying K(+) current, and depolarize the resting membrane potential, resulting in abnormal automaticity in guinea pig ventricular myocytes. Interestingly, another potent tyrosine kinase inhibitor, tyrphostin 51, did not produce the same inhibitory effect, while the inactive analogue of genistein, daidzein, had a similar blocking effect. We suggest that genistein directly blocks the inward rectifying K(+) current in ventricular myocytes, and one should be cautious of its pro-arrhythmic effect in clinical use.