Inwardly rectifying K(+) channels in esophageal smooth muscle

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.     

Regulation of Na(+) pump expression by vascular smooth muscle cells

The Na(+) pump and its regulation is important for maintaining membrane potential and transmembrane Na(+) gradient in all mammalian cells and thus is essential for cell survival and function. Vascular smooth muscle cells (VSMC) have a relatively low number of pump sites on their membrane compared with other cells. We wished to determine the mechanisms for regulating the number of pump sites in these cells. We used canine saphenous vein VSMC cultured in 10% serum and passaged one time. These cells were subcultured in 5% serum media with low K(+) (1 mM vs. control of 5 mM), and their pump expression was assessed. These VSMC upregulated their pump sites as early as 4 h after treatment (measured by [(3)H]ouabain binding). At this early time point, there was no detectable increase in protein expression of either alpha(1)- or beta(1)-subunits of the pump shown by Western blots. When the cells were treated with the phosphoinositide 3-kinase (PI-3-K) inhibitor LY-294002 (which is known to inhibit cytoplasmic transport processes) in low-K(+) media, the pump site upregulation was inhibited. These data suggest that the low-K(+)-induced upregulation of Na(+) pump number can occur by translocation of preformed pumps from intracellular stores.     

Characterization of stretch-activated cation current in coronary smooth muscle cells

Stretch-activated ion currents were recorded from vascular smooth muscle (VSM) after enzymatic isolation of single cells from porcine coronary arterioles. Patch pipettes were used to record whole cell current and control cell length. Under voltage clamp in physiological saline solution, an inward cation current (I(CAT)) was activated by 105--135% longitudinal stretch. I(CAT) coincided with an increase in intracellular Ca(2+) concentration. Under current clamp, membrane depolarization was induced by stretch. The magnitude of I(CAT) varied from -0.8 to -6.9 pA/pF at a holding potential of -60 mV. I(CAT) was graded with stretch, inactivated on release, and could be repeatedly induced. A potassium current (I(K)) activated in unstretched cells by depolarization was also enhanced by stretch. In Ca(2+)-free bath solution, stretch-induced enhancement of I(K) was blocked, but I(CAT) was still present. Hexamethyleneamiloride (50 microM), a reputed inhibitor of mechanosensitive channels, blocked I(CAT) and the stretch-induced increase in I(K) but not basal I(K). Grammostolla spatulata venom (1:100,000) blocked basal I(K), blocked stretch-induced increases in I(K), and blocked I(CAT). Iberiotoxin, a specific Ca(2+)-activated K(+) channel blocker, did not alter I(CAT) but blocked the stretch-induced increase in I(K) and increased the magnitude of stretch-induced depolarization. We concluded that longitudinal stretch directly activates a cation current and secondarily activates a Ca(2+)-activated K(+) current in isolated coronary myocytes. Although these two currents would partially counteract each other, the predominance of I(CAT) at physiological potentials is likely to explain the depolarization and contraction observed in intact coronary VSM during pressure elevation.     

Vascular smooth muscle expresses a truncated Na+, K(+)-ATPase alpha-1 subunit isoform.

By regulating transmembrane Na+ and K+ concentrations and membrane potential, the Na+,K(+)-ATPase plays an important role in regulating cardiac, skeletal, and smooth muscle function. A high degree of amino acid sequence and structural identity characterizes the three Mr 100,000 Na+,K(+)-ATPase alpha subunit isoforms expressed in cardiac and skeletal muscle. Strikingly, vascular smooth muscle utilizes alternative RNA processing of the alpha-1 gene to express a structurally distinct Mr approximately 65,000 isoform, alpha 1-T (truncated). Analysis of both its mRNA and protein structure reveals that alpha-1-T represents a major, evolutionarily conserved, truncated Na+,K(+)-ATPase isoform expressed in vascular smooth muscle. This demonstrates an unexpected complexity in the regulation of vascular smooth muscle Na+,K(+)-ATPase gene expression and suggests that a structurally novel, truncated alpha subunit may play a role in vascular smooth muscle active ion transport.     

Regional differences in cholinergic regulation of potassium current in feline esophageal circular smooth muscle

Potassium channels are important contributors to membrane excitability in smooth muscles. There are regional differences in resting membrane potential and K(+)-channel density along the length of the feline circular smooth muscle esophagus. The aim of this study was to assess responses of K(+)-channel currents to cholinergic (ACh) stimulation along the length of the feline circular smooth muscle esophageal body. Perforated patch-clamp technique assessed K(+)-channel responses to ACh stimulation in isolated smooth muscle cells from the circular muscle layer of the esophageal body at 2 (distal)- and 4-cm (proximal) sites above the lower esophageal sphincter. Western immunoblots assessed ion channel and receptor expression. ACh stimulation produced a transient increase in outward current followed by inhibition of spontaneous transient outward currents. These ACh-induced currents were abolished by blockers of large-conductance Ca(2+)-dependent K(+) channels (BK(Ca)). Distal cells demonstrated a greater peak current density in outward current than cells from the proximal region and a longer-lasting outward current increase. These responses were abolished by atropine and the specific M(3) receptor antagonist 4-DAMP but not the M(1) receptor antagonist pirenzipine or the M(2) receptor antagonist methoctramine. BK(Ca) expression along the smooth muscle esophagus was similar, but M(3) receptor expression was greater in the distal region. Therefore, ACh can differentially activate a potassium channel (BK(Ca)) current along the smooth muscle esophagus. This activation probably occurs through release of intracellular calcium via an M(3) pathway and has the potential to modulate the timing and amplitude of peristaltic contraction along the esophagus.     

Characterization of outward K(+) currents in isolated smooth muscle cells from sheep urethra

The perforated-patch techniquewas used to measure membrane currents in smooth muscle cells from sheepurethra. Depolarizing pulses evoked large transient outward currentsand several components of sustained current. The transient current anda component of sustained current were blocked by iberiotoxin, penitremA, and nifedipine but were unaffected by apamin or 4-aminopyridine,suggesting that they were mediated by large-conductanceCa²⁺-activated K⁺ (BK) channels. When the BKcurrent was blocked by exposure to penitrem A (100 nM) andCa²⁺-free bath solution, there remained a voltage-sensitiveK⁺ current that was moderately sensitive to blockade withtetraethylammonium (TEA; half-maximal effective dose = 3.0 ± 0.8 mM) but not 4-aminopyridine. Penitrem A (100 nM) increasedthe spike amplitude and plateau potential in slow waves evoked insingle cells, whereas addition of TEA (10 mM) further increased theplateau potential and duration. In conclusion, bothCa²⁺-activated and voltage-dependent K⁺currents were found in urethral myocytes. Both of these currents arecapable of contributing to the slow wave in these cells, suggesting that they are likely to influence urethral tone under certain conditions.

     

Expression of a voltage-dependent potassium current precedes fusion of human muscle satelite cells (myoblasts)

Using the whole-cell recording patch clamp tehnique in clonal cultures of human muscle satellite cells (SC), we studied a voltage-gated potassium current analogous to the delayed rectifier current (I_kdr) described in adult human skeletal muscle. This current was absent in proliferating SC cultured in a growth medium containing 15% serum, except when the SC approached the end of their replicative life (between 77 and 124 days in culture); at that time, approximately 50% of the SC possessed I_kdr In contrast, I_kdr was expressed within less than 4 days in approximately 70% of the SC cultured in a serum-free medium (SFM) and within 24 h in differetiating medium. We belive that I_kdr may be a characteristic feature of fusion-component SC and that it may be involved in the fusion process for the following resons: (1) after the transfer in differentiating medium, cultures of SC in which the expression of I_kdr was previously promoted by exposure to SFM were found to fuse immediately, without the initial 24 h lag time observed in control sister cultures; (2) in the latter “naive” SC, I_kdr was expressed during the first day in differentiating medium, before SC began to fuse; (3) most of the SC that did not fuse even after weeks of exposure to differentiating medium did not express I_kdr; (4) TEA, at a concentration of 3 mM, reduces the amplitude of I_kdr by 55% and the fusion index by 55–67%. © 1995 Wiley-Liss, Inc.     

Exercise-induced translocation of Na(+)-K(+) pump subunits to the plasma membrane in human skeletal muscle

Six human subjects performed one-legged knee extensor exercise (90 +/- 4 W) until fatigue (exercise time 4.6 +/- 0.8 min). Needle biopsies were obtained from vastus lateralis muscle before and immediately after exercise. Production of giant sarcolemmal vesicles from the biopsy material was used as a membrane purification procedure, and Na(+)-K(+) pump alpha- and beta-subunits were quantified by Western blotting. Exercise significantly increased (P < 0.05) the vesicular membrane content of the alpha(2)-, total alpha-, and beta(1)-subunits by 70 +/- 29, 35 +/- 10, and 26 +/- 5%, respectively. The membrane content of alpha(1) was not changed by exercise, and the densities of subunits in muscle homogenates were unchanged. The ratio of vesicular to crude muscle homogenate content of the alpha(2)-, total alpha-, and beta(1)-subunits was elevated during exercise by 67 +/- 33 (P < 0.05), 23 +/- 6 (P < 0.05), and 40 +/- 14% (P = 0.06), respectively. It is concluded that translocation of subunits is an important mechanism involved in the short time upregulation of the Na(+)-K(+) pumps in association with human muscle activity.     

Ouabain augments Ca(2+) transients in arterial smooth muscle without raising cytosolic Na(+)

Ouabain and other cardiotonic steroids (CTS) inhibit Na(+) pumps and are widely believed to exert their cardiovascular effects by raising the cytosolic Na(+) concentration ([Na(+)](cyt)) and Ca(2+). This view has not been rigorously reexamined despite evidence that low-dose CTS may act without elevating [Na(+)](cyt); also, it does not explain the presence of multiple, functionally distinct isoforms of the Na(+) pump in many cells. We investigated the effects of Na(+) pump inhibition on [Na(+)](cyt) (with Na(+) binding benzofuran isophthalate) and Ca(2+) transients (with fura 2) in primary cultured arterial myocytes. Low concentrations of ouabain (3-100 nM) or human ouabain-like compound or reduced extracellular K(+) augmented hormone-evoked mobilization of stored Ca(2+) but did not increase bulk [Na(+)](cyt). Augmentation depended directly on external Na(+), but not external Ca(2+), and was inhibited by 10 mM Mg(2+) or 10 microM La(3+). Evoked Ca(2+) transients in pressurized small resistance arteries were also augmented by nanomolar ouabain and inhibited by Mg(2+). These results suggest that Na(+) enters a tiny cytosolic space between the plasmalemma (PL) and the adjacent sarcoplasmic reticulum (SR) via an Mg(2+)- and La(3+)-blockable mechanism that is activated by SR store depletion. The Na(+) and Ca(2+) concentrations within this space may be controlled by clusters of high ouabain affinity (alpha3) Na(+) pumps and Na/Ca exchangers located in PL microdomains overlying the SR. Inhibition of the alpha3 pumps by low-dose ouabain should raise the local concentrations of Na(+) and Ca(2+) and augment hormone-evoked release of Ca(2+) from SR stores. Thus the clustering of small numbers of specific PL ion transporters adjacent to the SR can regulate global Ca(2+) signaling. This mechanism may affect vascular tone and blood flow and may also influence Ca(2+) signaling in many other types of cells.     

不同牵张刺激对豚鼠胃窦环行肌细胞电压依赖性钙电流的影响

利用全细胞膜片钳技术,在胃窦环行肌细胞上观察了不同方式的牵张刺激对电压依赖性钙电流的影响,探讨牵张刺激对胃窦平滑肌细胞电压依赖性钙电流的作用。用低渗性溶液灌流细胞引起的牵张刺激首先增加电压依赖性钙电流,接着激活一种内向性钳制电流。钙电流的增加发生在灌流后１ｍｉｎ内,而内向性钳制电流在细胞明显膨胀之后缓慢激活。低渗和正压引起的细胞膨胀明显增加电压依赖性钙离子电流,而利用两个电极直接牵细胞则不出现钙电     

Inhibition of voltage-dependent Na+ current in cell-fusion hybrids containing activated c-Ha-ras

Summary The electrophysiological properties of EJ (human bladder carcinoma), GM2291 (human fetal lung fibroblast), and of three hybrid cell lines obtained from their cell fusion were investigated using the patch-clamp technique. GM2291 cells, which are nontumorigenic, express voltage-dependent Na⁺ channels. The pharmacology and gating properties of the Na⁺ channels in GM2291 cells are distinct from neuronal and cardiac Na⁺ channels. EJ cells, which are tumorigenic and contain activated c-Ha-ras, express inward rectifier K⁺ channels. The three cell-fusion hybrid lines, named 145 (nontumorigenic), 145L (non-tumorigenic but morphologically altered), and 147TR2 (fully tumorigenic segregant), have been previously shown to express levels of activated c-Ha-ras similar to those of the EJ parental line. Voltage-dependent Na⁺ channels were observed in none of the hybrid cell lines, while inward rectifier K⁺ channels were observed in each of the hybrid cell lines. The possibility that c-Ha-ras inhibits expression of a voltage-dependent Na⁺ channel is discussed.     

Characterization of a novel voltage-dependent outwardly rectifying anion current in Caenorhabditis elegans oocytes

An inwardly rectifying swelling- and meiotic cell cycle-regulated anion current carried by the ClC channel splice variant CLH-3b dominates the whole cell conductance of the Caenorhabditis elegans oocyte. Oocytes also express a novel outwardly rectifying anion current termed I_Cl,OR. We recently identified a worm strain carrying a null allele of the clh-3 gene and utilized oocytes from these animals to characterize I_Cl,OR biophysical properties. The I_Cl,OR channel is strongly voltage dependent. Outward rectification is due to voltage-dependent current activation at depolarized voltages and rapid inactivation at voltages more hyperpolarized than approximately +20 mV. Apparent channel open probability is zero at voltages less than +20 mV. The channel has a 4:1 selectivity for Cl^– over Na⁺ and an anion selectivity sequence of SCN^– > I^– > Br^– > Cl^– > F^–. I_Cl,OR is relatively insensitive to most conventional anion channel inhibitors including DIDS, 4,4'-dinitrostilbene-2,2'-disulfonic acid, 9-anthracenecarboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid. However, the current is rapidly inhibited by niflumic acid, metal cations including Gd³⁺, Cd²⁺, and Zn²⁺, and bath acidification. The combined biophysical properties of I_Cl,OR are distinct from those of other anion currents that have been described. During oocyte meiotic maturation, I_Cl,OR activity is rapidly downregulated, suggesting that the channel may play a role in oocyte Cl^– homeostasis, development, cell cycle control, and/or ovulation. chloride channel; ovulation; cell cycle; meiotic maturation     

Cytoskeletal modulation of sodium current in human jejunal circular smooth muscle cells

ANa⁺ current is present in human jejunal circular smoothmuscle cells. The aim of the present study was to determine the role ofthe cytoskeleton in the regulation of the Na⁺ current.Whole cell currents were recorded by using standard patch-clamptechniques with Cs⁺ in the pipette to block K⁺currents. Cytochalasin D and gelsolin were used to disrupt the actincytoskeleton and phalloidin to stabilize it. Colchicine was used todisassemble the microtubule cytoskeleton (and intermediate filaments)and paclitaxel to stabilize it. Acrylamide was used to disrupt theintermediate filament cytoskeleton. Perfusion of the recording chamberat 10 ml/min increased peak Na⁺ current recorded fromjejunal smooth muscle cells by 27 ± 3%. Cytochalasin D andgelsolin abolished the perfusion-induced increase in Na⁺current, whereas incubation with phalloidin, colchicine, paclitaxel, oracrylamide had no effect. In conclusion, the Na⁺ currentexpressed in human jejunal circular smooth muscle cells appears to beregulated by the cytoskeleton. An intact actin cytoskeleton is requiredfor perfusion-induced activation of the Na⁺ current.