Inhibition of the cardiac inward rectifier potassium currents by KB-R7943

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium–calcium exchanger (NCX) with potential experimental and therapeutic use. However, KB-R7943 is shown to be a potent blocker of several ion currents including inward and delayed rectifier K⁺ currents of cardiomyocytes. To further characterize KB-R7943 as a blocker of the cardiac inward rectifiers we compared KB-R7943 sensitivity of the background inward rectifier (I_K1) and the carbacholine-induced inward rectifier (I_KACh) currents in mammalian (Rattus norvegicus; rat) and fish (Carassius carassius; crucian carp) cardiac myocytes. The basal I_K1 of ventricular myocytes was blocked with apparent IC50-values of 4.6 × 10^− 6 M and 3.5 × 10^− 6 M for rat and fish, respectively. I_KACh was almost an order of magnitude more sensitive to KB-R7943 than I_K1 with IC₅₀-values of 6.2 × 10^− 7 M for rat and 2.5 × 10^− 7 M for fish. The fish cardiac NCX current was half-maximally blocked at the concentration of 1.9–3 × 10^− 6 M in both forward and reversed mode of operation. Thus, the sensitivity of three cardiac currents to KB-R7943 block increases in the order I_K1 ~ I_NCX < I_KACh. Therefore, the ability of KB-R7943 to block inward rectifier potassium currents, in particular I_KACh, should be taken into account when interpreting the data with this inhibitor from in vivo and in vitro experiments in both mammalian and fish models.     

Inward-rectifier K<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript> Current in Guinea-pig Ventricular Myocytes Exposed to Hyperosmotic Solutions

Superfusion of heart cells with hyperosmotic solution causes cell shrinkage and inhibition of membrane ionic currents, including delayed-rectifer K⁺ currents. To determine whether osmotic shrinkage also inhibits inwardly-rectifying K⁺ current (I_K1), guinea-pig ventricular myocytes in the perforated-patch or ruptured-patch configuration were superfused with a Tyrodes solution whose osmolarity (T) relative to isosmotic (1T) solution was increased to 1.3–2.2T by addition of sucrose. Hyperosmotic superfusate caused a rapid shrinkage that was accompanied by a negative shift in the reversal potential of Ba²⁺-sensitive I_K1, an increase in the amplitude of outward I_K1, and a steepening of the slope of the inward I_K1-voltage (V) relation. The magnitude of these effects increased with external osmolarity. To evaluate the underlying changes in chord conductance (G_K1) and rectification, G_K1-V data were fitted with Boltzmann functions to determine maximal G_K1 (G_K1max) and voltage at one-half G_K1max (V_0.5). Superfusion with hyperosmotic sucrose solutions led to significant increases in G_K1max (e.g., 28±2% with 1.8T), and significant negative shifts in V_0.5 (e.g., –6.7±0.6 mV with 1.8T). Data from myocytes investigated under hyperosmotic conditions that do not induce shrinkage indicate that G_K1max and V_0.5 were insensitive to hyperosmotic stress per se but sensitive to elevation of intracellular K⁺. We conclude that the effects of hyperosmotic sucrose solutions on I_K1 are related to shrinkage-induced concentrating of intracellular K⁺.     

The vicissitudes of the pacemaker current <Emphasis Type="Italic">I</Emphasis><Subscript>Kdd</Subscript> of cardiac purkinje fibers

Summary The mechanisms underlying the pacemaker current in cardiac tissues is not agreed upon. The pacemaker potential in Purkinje fibers has been attributed to the decay of the potassium current I _Kdd. An alternative proposal is that the hyperpolarization-activated current I _f underlies the pacemaker potential in all cardiac pacemakers. The aim of this review is to retrace the experimental development related to the pacemaker mechanism in Purkinje fibers with reference to findings about the pacemaker mechanism in the SAN as warranted. Experimental data and their interpretation are critically reviewed. Major findings were attributed to K⁺ depletion in narrow extracellular spaces which would result in a time dependent decay of the inward rectifier current I _K1. In turn, this decay would be responsible for a “fake” reversal of the pacemaker current. In order to avoid such a postulated depletion, Ba²⁺ was used to block the decay of I _K1. In the presence of Ba²⁺ the time-dependent current no longer reversed and instead increased with time and more so at potentials as negative as −120 mV. In this regard, the distinct possibility needs to be considered that Ba²⁺ had blocked I _Kdd (and not only I _K1). That indeed this was the case was demonstrated by studying single Purkinje cells in the absence and in the presence of Ba²⁺. In the absence of Ba²⁺, I _Kdd was present in the pacemaker potential range and reversed at E _K. In the presence of Ba²⁺, I _Kdd was blocked and I _f appeared at potentials negative to the pacemaker range. The pacemaker potential behaves in a manner consistent with the underlying I _Kdd but not with I _f. The fact that I _f is activated on hyperpolarization at potential negative to the pacemaker range makes it suitable as a safety factor to prevent the inhibitory action of more negative potentials on pacemaker discharge. It is concluded that the large body of evidence reviewed proves the pacemaker role of I _Kdd (but not of I _f) in Purkinje fibers.     

Diversity of K+ channels in the basolateral membrane of restingnecturus oxyntic cells

Summary Patch-clamp techniques have been applied to characterize the channels in the basolateral membrane of resting (cimetidine-treated, nonacid secreting) oxyntic cells isolated from the gastric mucosa ofNecturus maculosa. In cell-attached patches with pipette solution containing 100mm KCl, four major classes of K⁺ channels can be distinguished on the basis of their kinetic behavior and conductance: (1) 40% of the patches contained either voltage-independent (a) or hyperpolarization-activated (b), inward-rectifying channels with short mean open times (16 msec fora, and 8 msec forb). Some channels showed subconductance levels. The maximal inward conductanceg _max was 31±5 pS (n=13) and the reversal potentialE _rev was atV _p=–34±6 mV (n=9). (2) 10% of the patches contained depolarization-activated and inward-rectifying channels withg _max=40 ±18 pS (n=3) andE _rev was atV _p=–31±5 mV (n=3). With hyperpolarization, the channels open in bursts with rapid flickerings within bursts. Addition of carbachol (1mm) to the bath solution in cell-attached patches increased the open probabilityP _o of these channels. (3) 10% of the patches contained voltage-independent inward-rectifying channels withg _max=21±3 pS (n=4) andE _rev was atV _p=–24±9 mV (n=4). These channels exhibited very high open probability (P _o=0.9) and long mean open time (1.6 sec) at the resting potential. (4) 20% of the patches contained voltage-independent channels with limiting inward conductance of 26±2 pS (n=3) andE _rev atV _p=–33±3 mV (n=3). The channels opened in bursts consisting of sequential activation of multiple channels with very brief mean open times (10 msec). In addition, channels with conductances less than 6 pS were observed in 20% of the patches. In all nine experiments with K⁺ in the pipette solution replaced by Na⁺, unitary currents were outward, and inward currents were observed only for large hyperpolarizing potentials. This indicates that the channels are more selective for K⁺ over Na⁺ and Cl^–. A variety of K⁺ channels contributes to the basolateral K⁺ conductance of resting oxyntic cells.     

GATA-4 induces changes in electrophysiological properties of rat mesenchymal stem cells

Background

Transplanted mesenchymal stem cells (MSC) can differentiate into cardiac cells that have the potential to contribute to heart repair following ischemic injury. Overexpression of GATA-4 can significantly increase differentiation of MSC into cardiomyocytes (CM). However, the specific impact of GATA-4 overexpression on the electrophysiological properties of MSC-derived CM has not been well documented.

Methods

Adult rat bone marrow MSC were retrovirally transduced with GATA-4 (MSC^GATA-4) and GFP (MSC^Null) and subsequently co-cultured with neonatal rat ventricular cardiomyocytes (CM). Electrophysiological properties and mRNA levels of ion channels were assessed in MSC using patch-clamp technology and real-time PCR.

Results

MSC^GATA-4 exhibited higher levels of the TTX-sensitive Na⁺ current (I_Na.TTX), L-type calcium current (I_Ca.L), transient outward K⁺ current (I_to), delayed rectifier K⁺ current (IK_DR) and inwardly rectifying K⁺ current (I_K1) channel activities reflective of electrophysiological characteristics of CM. Real-time PCR analyses showed that MSC^GATA-4 exhibited upregulated mRNA levels of Kv1.2, Kv2.1, SCN2a1, CCHL2a, KV1.4 and Kir1.1 channels versus MSC^Null. Interestingly, MSC^GATA-4 treated with IGF-1 neutralizing antibodies resulted in a significant decrease in Kir1.1, Kv2.1, KV1.4, CCHL2a and SCN2a1 channel mRNA expression. Similarly, MSC^GATA-4 treated with VEGF neutralizing antibodies also resulted in an attenuated expression of Kv2.1, Kv1.2, Kv1.4, Kir1.1, CCHL2a and SCN2a1 channel mRNAs.

Conclusions

GATA-4 overexpression increases I_to, IK_DR, I_K1, I_Na.TTX and I_Ca.L currents in MSC. Cytokine (VGEF and IGF-1) release from GATA-4 overexpressing MSC can partially account for the upregulated ion channel mRNA expression.

General significance

Our results highlight the ability of GATA4 to boost the cardiac electrophysiological potential of MSC.     

Effect of external K+, Ca2+, and Ba2+ on membrane potential and ionic conductance in rat astrocytes

Summary 1. The purpose of this study was (a) to identify if astrocytes show a similar non-Nernstian depolarization in low K⁺ or low Ca²⁺ solutions as previously found in human glial and glioma cells, and (b) to analyze the influence of the K⁺ conductance on the membrane potential of astrocytes.2. The membrane potential (E_m) and the ionic conductance were studied with whole-cell patch-clamp technique in neonatal rat astrocytes (5–9 days in culture) and in human glioma cells (U-251MG).3. In 3.0 mM K⁺, E_m was –75 ± 1.0 mV (mean ± SEM,n=39) in rat astrocytes and –79 ± 0.7 mV (n=5) in U-251MG cells. In both cell types E_m changed linearly to the logarithm of [K⁺]₀ between 3.0 and 160 mM K⁺. K⁺ free medium caused astrocytes to hyperpolarize to –93 ± 2.7 mV (n=21) and U-251MG cells to depolarize to –27 ± 2.1 mV (n=3).4. The I-E curve did not show inward rectification in astrocytes at this developmental stage. The slope conductance (g) exhibited only a small decrease (–19%) in K⁺ free solution and no significant change in 160 mM K⁺.5. Ba²⁺ (1.0 mM) depolarized astrocytes to –45 ± 2.9 mV (n=11), decreasing the slope conductance (g) by 42.4 ± 8.3% (n=11). Ca²⁺ free solution depolarized astrocytes to –53 ± 3.4 mV (n=12) and resulted in a positive shift of the I-E curve, increasing g by 15.3 ± 8.2% (n=8).6. Calculations indicated that a block of K⁺ channels explains the depolarizing effect of Ba²⁺. The effects of K⁺ free or Ca²⁺ free solutions on E_m can be explained by a transformation of K⁺ channels to non-specific leakage channels. That astrocytes show a different reaction to low K⁺ than glioma cells can be related to the lack of inwardly rectifying K⁺ channels in astrocytes at this developmental stage.     

pH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH₄Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD _m ) and membrane resistance (R _m) in bicarbonate-free Ringer. The removal of extracellular NH₄Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g _K+), reflected by a decrease of K⁺ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR _m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg _K+ by 3 mmol/liter BaCl₂ enhances the total membrane conductance, being shown byR _m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR _m and hyperpolarizes PD _m . The replacement of extracellular sodium by choline in the presence of BaCl₂ significantly hyperpolarizes PD _m and increasesR _m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10^–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na⁺-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl^–. The low-conductive channel is 4.8 times more permeable for Na⁺ than for K⁺. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).     

Polarized distribution of Na, K-ATPase α-subunit isoforms in electrocyte membranes

We have previously demonstrated that Na⁺, K⁺-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the α subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of α isoforms (α1 and α2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na⁺, K⁺-ATPase activity 2.6-fold more sensitive to ouabain (I₅₀=1.0±0.1 μM) than the activity of innervated membranes (I₅₀=2.6±0.2 μM). As depicted in [³H]ouabain binding experiments, when the [³H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na⁺, K⁺-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of α1 and α2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na⁺, K⁺-ATPase α-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.     

Contribution of slowly inactivating potassium current to delayed firing of action potentials in NG108-15 neuronal cells: experimental and theoretical studies

The properties of slowly inactivating delayed-rectifier K⁺ current (I_Kdr) were investigated in NG108-15 neuronal cells differentiated with long-term exposure to dibutyryl cyclic AMP. Slowly inactivating I_Kdr could be elicited by prolonged depolarizations from −50 to +50 mV. These outward K⁺ currents were found to decay at potentials above −20 mV, and the decay became faster with greater depolarization. Cell exposure to aconitine resulted in the reduction of I_Kdr amplitude along with an accelerated decay of current inactivation. Under current-clamp recordings, a delay in the initiation of action potentials (APs) in response to prolonged current stimuli was observed in these cells. Application of aconitine shortened the AP initiation in combination with an increase in both width of spike discharge and firing frequency. The computer model, in which state-dependent inactivation of I_Kdr was incorporated, was also implemented to predict the firing behavior present in NG108-15 cells. As the inactivation rate constant of I_Kdr was elevated, the firing frequency was progressively increased along with a shortening of the latency for AP appearance. Our theoretical work and the experimental results led us to propose a pivotal role of slowly inactivating I_Kdr in delayed firing of APs in NG108-15 cells. The results also suggest that aconitine modulation of I_Kdr gating is an important molecular mechanism through which it can contribute to neuronal firing.     

Epithelial transport and barrier function in occludin-deficient mice

Background and Aims

This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model.

Methods

Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (R^e, epithelial resistance). Conductance scanning differentiated transcellular (G^c) and tight junctional conductance (G^tj). The pH-stat technique quantified gastric acid secretion.

Results

In occludin+/+ mice, R^e was 23±5 Ω cm² in jejunum, 66±5 Ω cm² in distal colon and 33±6 Ω cm² in gastric corpus and was not altered in heterozygotic occludin+/− or homozygotic occludin−/− mice. Additionally, [³H]mannitol fluxes were unaltered. In the control colon, G^c and G^tj were 7.6±1.0 and 0.3±0.1 mS/cm² and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin−/− mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control R^t was 5.8±0.8 kΩ cm² in urinary bladder and 33±6 Ω cm² in stomach and not altered in occludin−/− mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin−/− mice.

Conclusion

Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.     

Modifications of current properties by expression of a foreign potassium channel gene in Xenopus embryonic cells

The pH-sensitivity of transepithelial K⁺ transport was studied in vitro in isolated vestibular dark cell epithelium from the gerbil ampulla. The cytosolic pH (pH _iwas measured microfluorometrically with the pH-sensitive dye 2,7-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) and the equivalent short-circuit current (I _sc), which is a measure for transepithelial K⁺ secretion, was calculated from measurements of the transepithelial voltage (V _t)and the transepithelial resistance (R _t) in a micro-Ussing chamber. All experiments were conducted in virtually HCO ₃ ^– -free solutions. Under control conditions, pH _iwas 7.01±0.04 (n=18), V _twas 9.1±0.5 mV, R _t16.7±0.09 cm², and I _sc was 587±30 A/cm² (n=49). Addition of 20 mm propionate^– caused a biphasic effect involving an initial acidification of pH _i, increase in V _tand I _sc and decrease in R _tand a subsequent alkalinization of pH _i, decrease of V _tand increase of R _t. Removal of propionate^– caused a transient effect involving an alkalinization of pH _i, a decrease of V _tand I _sc and an increase in R _t. pH _iin the presence of propionate^– exceeded pH _iunder control conditions. Effects of propionate ^– on V _t, R _tand I _sc were significantly larger when propionate^– was applied to the basolateral side rather than to the apical side of the epithelium. The pH _i-sensitivityof I _sc between pH 6.8 and 7.5 was –1089 A/(cm² · pH-unit) suggesting that K⁺ secretion ceases at about pH _i7.6. Acidification of the extracellular pH (pH _o)caused an increase of V _tand I _sc and a decrease of R _tmost likely due to acidification of pH _i. Effects were significantly larger when the extracellular acidification was applied to the basolateral side rather than to the apical side of the epithelium. The pH _osensitivity of I _sc between pH 7.4 and 6.4 was –155 A/(cm² · pH unit). These results demonstrate that transepithelial K⁺ transport is sensitive to pH _iand pH _oand that vestibular dark cells contain propionate^– uptake mechanism. Further, the data suggest that cytosolic acidification activates and that cytosolic alkalinization inactivates the slowly activating K⁺ channel (I _sK)in the apical membrane. Whether the effect of pH _ion the I _sK channel is a direct or indirect effect remains to be determined.The authors wish to thank Drs. Daniel C. Marcus, Zhjiun Shen and Hiroshi Sunose for helpful discussions. This work was supported by grants NIH-R29-DC01098 and NIH-R01-DC00212.     

A dual action of taurine on the delayed rectifier K+ current in embryonic chick cardiomyocytes

Summary Effects of taurine on the delayed rectifier K⁺ channel in isolated 10-day-old embryonic chick ventricular cardiomyocytes were examined at different intracellular Ca²⁺ concentrations ([Ca]_i), using whole-cell voltage and current clamp techniques. Experiments were performed at room temperature (22°C). Test pulses were applied between -20 to +90m V from a holding potential of -40mV. When [Ca]_i was pCa 7, addition of 10 and 20 mM taurine to the bath solution reduced the delayed rectifier K⁺ current (I_K) at +90mV by 17.4 ± 2.8% (n = 5, P < 0.01) and 25.5 ± 2.6% (n = 5, P < 0.001), respectively. In contrast, when [Ca]_i was pCa 10, I_K at +90 mV was enhanced by 19.1 ± 3.1% (n = 7, P < 0.01) at 10mM taurine, and by 29.3 ± 2.4% (n = 7, P < 0.001) at 20mM taurine. The voltage of half-maximum activation (V_1/2) was shifted in a hyperpolarizing direction; at pCa 7, the value was +0.2 ± 2.2mV (n = 5) in control and -10.6 ± 1.8mV (n = 5) in 20mM taurine. At pCa 10, the V_1/2 value was +18.5 ± 4.6mV (n = 5) in control and +6.6 ± 5.2mV (n = 5) in taurine (20mM). Taurine decreased the action potential duration (APD) at pCa 10, but at pCa 7 did not affect it. In addition, taurine enhanced the transient outward current in a concentration-dependent manner. These results indicate that taurine modulates the delayed rectifier K⁺ channel, an effect dependent on [Ca]_i and capable of regulating APD.     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

Hemolymph ion regulation and kinetic characteristics of the gill (Na⁺, K⁺)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na⁺, K⁺)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg⁻¹ H₂O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg⁻¹ H₂O). Hemolymph [Na⁺] (323.0 ± 2.5 mmol L⁻¹) and [Mg²⁺] (34.6 ± 1.0 mmol L⁻¹) are hypo-regulated while [Ca²⁺] (22.5 ± 0.7 mmol L⁻¹) is hyper-regulated; [K⁺] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L⁻¹) but hypo-regulated (6.2 ± 0.7 mmol L⁻¹) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na⁺, K⁺)-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 ± 3.5 U mg⁻¹; K_0.5 = 7.07 ± 0.01 μmol L⁻¹) and a low-affinity ATP binding site (Vm = 108.1 ± 2.5 U mg⁻¹; K_0.5 = 0.11 ± 0.3 mmol L⁻¹), both obeying cooperative kinetics, were disclosed. Modulation of (Na⁺, K⁺)-ATPase activity by Mg²⁺, K⁺ and NH₄⁺ also exhibits site-site interactions, but modulation by Na⁺ shows Michaelis-Menten kinetics. (Na⁺, K⁺)-ATPase activity is synergistically stimulated up to 45% by NH₄⁺ plus K⁺. Enzyme catalytic efficiency for variable [K⁺] and fixed [NH₄⁺] is 10-fold greater than for variable [NH₄⁺] and fixed [K⁺]. Ouabain inhibited ≈80% of total ATPase activity (K_I = 464.7 ± 23.2 μmol L⁻¹), suggesting that ATPases other than (Na⁺, K⁺)-ATPase are present. While (Na⁺, K⁺)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min⁻¹ mg⁻¹) and 45‰-acclimated crabs (around 154 nmol Pi min⁻¹ mg⁻¹), ATP affinity decreases 110-fold and Na⁺ and K⁺ affinities increase 2-3-fold in 45‰-acclimated crabs.     

Sensitivity of oocyte-expressed epithelial Na channel to glibenclamide

The effect of glibenclamide on heterologously expressed amiloride-sensitive sodium channels (ENaCs) was investigated in Xenopus oocytes. The ENaC is a heteromer and consists of α-, β- and γ-subunits and the α- and β-subunits have previously been shown to confer sensitivity to glibenclamide. We coexpressed either colonic rat α- (rα) or guinea-pig α-subunit (gpα) with Xenopus βγ-subunits. The gpαxβγ was significantly stimulated by glibenclamide (100 μM) (184±15%), whereas the rα-combination was slightly down-regulated by the sulfonylurea (79±4%). The stimulating effect did not interfere with Na⁺-self-inhibition resulting from intracellular accumulation of Na⁺-ions. We exchanged cytosolic termini between both orthologs but the gpα-chimera with the termini from rat retained sensitivity to glibenclamide. The effect of glibenclamide on Xenopus ENaC (xENaC) was inhibited by ADP-β-S but not by ATP-γ-S, when applied intracellularly. Intracellular loading with Na⁺-ions after inhibition of Na⁺/K⁺-ATPases with ouabain prevented an up-regulation of ENaC activity by glibenclamide. Pretreatment of oocytes expressing xENaC with edelfosine (ET-18-OCH₃) slightly reduced stimulation of I_ami (118±12%; control: 132±9%) while phosphatidylinositol-4,5-biphosphate (PIP₂) significantly reduced the effect of glibenclamide to 101±3%.     

Inhibition of the calcium-activated chloride current in cardiac ventricular myocytes by N-(p-amylcinnamoyl)anthranilic acid (ACA)

N-(p-amylcinnamoyl)anthranilic acid (ACA), a phospholipase A₂ (PLA₂) inhibitor, is structurally-related to non-steroidal anti-inflammatory drugs (NSAIDs) of the fenamate group and may also modulate various ion channels. We used the whole-cell, patch-clamp technique at room temperature to investigate the effects of ACA on the Ca²⁺-activated chloride current (I_Cl(Ca)) and other chloride currents in isolated pig cardiac ventricular myocytes. ACA reversibly inhibited I_Cl(Ca) in a concentration-dependent manner (IC₅₀ = 4.2 μM, n_Hill = 1.1), without affecting the L-type Ca²⁺ current. Unlike ACA, the non-selective PLA₂ inhibitor bromophenacyl bromide (BPB; 50 μM) had no effect on I_Cl(Ca). In addition, the analgesic NSAID structurally-related to ACA, diclofenac (50 μM) also had no effect on I_Cl(Ca), whereas the current in the same cells could be suppressed by chloride channel blockers flufenamic acid (FFA; 100 μM) or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS;100 μM). Besides I_Cl(Ca), ACA (50 μM) also suppressed the cAMP-activated chloride current, but to a lesser extent. It is proposed that the inhibitory effects of ACA on I_Cl(Ca) are PLA₂-independent and that the drug may serve as a useful tool in understanding the nature and function of cardiac anion channels.     

A Single-Cell Model of Phase-Driven Control of Ventricular Fibrillation Frequency

The mechanisms controlling the rotation frequency of functional reentry in ventricular fibrillation (VF) are poorly understood. It has been previously shown that Ba²⁺ at concentrations up to 50 μmol/L slows the rotation frequency in the intact guinea pig (GP) heart, suggesting a role of the inward rectifier current (I_K1) in the mechanism governing the VF response to Ba²⁺. Given that other biological (e.g., sinoatrial node) and artificial systems display phase-locking behavior, we hypothesized that the mechanism for controlling the rotation frequency of a rotor by I_K1 blockade is phase-driven, i.e., the phase shift between transmembrane current and voltage remains constant at varying levels of I_K1 blockade. We measured whole-cell admittance in isolated GP myocytes and in transfected human embryonic kidney (HEK) cells stably expressing Kir 2.1 and 2.3 channels. The admittance phase, i.e., the phase difference between current and voltage, was plotted versus the frequency in control conditions and at 10 or 50 μmol/L Ba²⁺ (in GP heart cells) or 1 mM Ba²⁺ (in HEK cells). The horizontal distance between plots was called the “frequency shift in a single cell” and analyzed. The frequency shift in a single cell was −14.14 ± 5.71 Hz (n = 14) at 10 μM Ba²⁺ and −18.51 ± 4.00 Hz (n = 10) at 50 μM Ba²⁺, p < 0.05. The values perfectly matched the Ba²⁺-induced reduction of VF frequency observed previously in GP heart. A similar relationship was found in the computer simulations. The phase of Ba²⁺-sensitive admittance in GP cells was −2.65 ± 0.32 rad at 10 Hz and −2.79 ± 0.26 rad at 30 Hz. In HEK cells, the phase of Ba²⁺-sensitive admittance was 3.09 ± 0.03 rad at 10 Hz and 3.00 ± 0.17 rad at 30 Hz. We have developed a biological single-cell model of rotation-frequency control. The results show that although rotation frequency changes as a result of I_K1 blockade, the phase difference between transmembrane current and transmembrane voltage remains constant, enabling us to quantitatively predict the change of VF frequency resulting from I_K1 blockade, based on single-cell measurement.     

A comparison of galvanic skin conductance and skin wettedness as indicators of thermal discomfort during moderate and high metabolic rates

The relationship between local thermal comfort, local skin wettedness (w_local) and local galvanic skin conductance (GSC) in four body segments during two different exercise intensities was compared in 10 males. In a balanced order, participants walked at 35% VO_2max for 45 min (WALK) (29.0±1.9°C, 29.8±3.6% RH, no wind) in one test and in a separate test ran at 70% VO_2max for 45 min (RUN) (26.2±2.1°C, 31.1±7.0% RH, no wind). During both tests, participants wore a loose fitting 100% polyester long sleeve top and trouser ensemble with a low resistance to heat and vapour transfer (total thermal resistance of 0.154 m² K W⁻¹ and total water vapour resistance of 35.9 m² Pa W⁻¹). w_local, change from baseline in GSC (ΔGSC) and local thermal comfort were recorded every 5 min. The results suggest that both w_local and ΔGSC are strong predictors of thermal comfort during the WALK when sweat production is low and thermal discomfort minimal (r²>0.78 and r²>0.71, respectively). Interestingly, during the RUN w_local plateaued at ~0.6 to 0.8 due to the high sweat production, whilst ΔGSC gradually increased throughout the experiment. ΔGSC had a similar relationship with thermal comfort to w_local during the RUN (r²>0.95 and r²>0.94, respectively). Despite the strength of these relationships, the ability of w_local to predict local thermal comfort accurately dramatically reduces in the exponential part of the curve. In a situation of uncompensated heat stress such as high metabolic rate in hot climate, where sweat production is high, ΔGSC shows to be a better predictor of local thermal comfort than w_local. The w_local data shows regional differences in the threshold which triggers local discomfort during the WALK than RUN; lower values are found for upper arms (0.22±0.03 and 0.28 ±0.22) and upper legs (0.22±0.11 and 0.22±0.10), higher values for upper back (0.30±0.12 and 0.36 ±0.10) and chest (0.27±0.10 and 0.39 ±0.32), respectively. However, no regional differences in the threshold of discomfort are found in the ?GSC data. Instead, the data suggests that the degree of discomfort experienced appears to be related to the amount of sweat within and around the skin (as indirectly measured by ΔGSC) at each body site.