Reduced volume-regulated outwardly rectifying anion channel activity in ventricular myocyte of type 1 diabetic mice.

The currents through the volume-regulated outwardly rectifying anion channel (VRAC) were measured in single ventricular myocytes obtained from streptozotocin (STZ)-induced diabetic mice, using whole-cell voltage-clamp method. In myocytes from STZ-diabetic mice, the density of VRAC current induced by hypotonic perfusion was markedly reduced, compared with that in the cells form normal control mice. Video-image analysis showed that the regulatory volume decrease (RVD), which was seen in normal cells after osmotic swelling, was almost lost in myocytes from STZ-diabetic mice. Some mice were pretreated with 3-O-methylglucose before STZ injection, to prevent the STZ's beta cell toxicity. In the myocytes obtained from such mice, the magnitude of VRAC current and the degree of RVD seen during hypotonic challenge were almost normal. Incubation of the myocytes from STZ-diabetic mice with insulin reversed the attenuation of VRAC current. These findings suggested that the STZ-induced chronic insulin-deficiency was an important causal factor for the attenuation of VRAC current. Intracellular loading of the STZ-diabetic myocytes with phosphatidylinositol 3,4,5-trisphosphate (PIP3), but not phosphatidylinositol 4,5-bisphosphate (PIP2), also reversed the attenuation of VRAC current. Furthermore, treatment of the normal cells with wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, suppressed the development of VRAC current. We postulate that an impairment PI3K-PIP3 pathway, which may be insulin-dependent, is responsible for the attenuation of VRAC currents in STZ-diabetic myocytes.

Regulation of volume-regulated outwardly rectifying anion channels by phosphatidylinositol 3,4,5-trisphosphate in mouse ventricular cells.

Volume-regulated outwardly rectifying anion channel (VRAC) plays an important role in cell-volume regulation in many types of cells. Little is known about the regulation of VRAC by phosphatidylinositides (PIs), which include phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2). We examined the effect of PIs on the VRAC current activated in hypotonic solution in mouse ventricular cells. VRAC current was inhibited strongly by intracellular application of LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or anti-PIP3 antibody (PIP3-Ab), and less strongly by anti-PIP2 antibody (PIP2-Ab). LY294002 inhibited regulatory volume decrease in hypotonically swollen cells, which was in parallel with the VRAC inhibition. Intracellular PIP3 or PIP2 influenced neither the basal background current in isotonic solution nor the VRAC current in hypotonic solution. However, PIP3, but not PIP2, restored the VRAC current suppressed by LY294002 or PIP2-Ab. These results suggest that the activation of VRAC current requires the presence of intracellular PIP3, that PI3K-mediated increase in PIP3 level is sufficient to fully activate VRAC current, and that PIP3 alone without osmotic stimulation cannot induce VRAC current. We propose that VRAC in mouse ventricular cells is regulated by PIP3 and/or its down stream signaling pathways.

Regulation of extracellular UTP-activated Cl- current by P2Y-PLC-PKC signaling and ATP hydrolysis in mouse ventricular myocytes.

The intracellular signaling pathways responsible for extracellualr uridine-5'-triphosphate (UTPo)-induced chloride (Cl-) currents (I(Cl.UTP)) were studied in mouse ventricular myocytes with the whole-cell clamp technique. UTPo (0.1 to 100 microM) activated a whole-cell current that showed a time-independent activation, a linear current-voltage relationship in symmetrical Cl- solutions, an anion selectivity of Cl- > iodide > aspartate, and an inhibition by a thiazolidinone-derived specific inhibitor (CFTR(inh)-172, 10 microM) of cystic fibrosis transmembrane conductance regulator (CFTR), but not by a disulfonic stilbene derivative (DIDS, 100 microM), these properties matching those of CFTR Cl- channels. The potency order of nucleotides for an activation of the Cl- current was UTP = ATP > uridine-5'-diphosphate (UDP) = ADP. Suramin (100 microM), a P2Y receptor antagonist, strongly inhibited the UTPo -activation of the Cl- current, whereas pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 100 microM), another P2Y receptor antagonist, induced little inhibition of I(Cl.UTP). The activation of I(Cl.UTP) was sensitive to protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, intracellular GDPbetaS (nonhydrolyzable GDP analogue) or anti-Gq/11 antibody. UTPo failed to activate the Cl- current when the cells were dialyzed with nonhydrolyzable ATP analogues (ATPS or AMP-PNP) without ATP, suggesting that ATP hydrolysis is a prerequisite for the current activation. I(Cl.UTP) was persistently activated with a mixture of ATPgammaS + ATP in the pipette, suggesting the involvement of phosphorylation reaction in the current activation process. Our results strongly suggest that I(Cl.UTP) is due to the activation of CFTR Cl- channels through Gq/11-coupled P2Y2 receptor-PLC-PKC signaling and ATP hydrolysis in mouse heart.

Acidic extracellular pH-activated outwardly rectifying chloride current in mammalian cardiac myocytes.

Extracellular acidic pH was found to induce an outwardly rectifying Cl- current (I(Cl,acid)) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed the permeability sequence for anions to be SCN- > Br- > I- > Cl- > F- > aspartate, while it exhibited a time-dependent activation at large positive potentials. Similar currents were also observed in mouse atrial cells and in atrial and ventricular cells from guinea pig. Some Cl- channel blockers (DIDS, niflumic acid, and glibenclamide) inhibited ICl,acid, whereas tamoxifen had little effect on it. Unlike volume-regulated Cl- current (ICl,vol) and CFTR Cl- current (ICl,CFTR), ICl,acid was independent of the presence of intracellular ATP. Activation of ICl,acid appeared to be also independent of intracellular Ca2+ and G protein. ICl,acid and ICl,vol could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced ICl,CFTR was inhibited by acidification in a pH-dependent manner in guinea pig ventricular cells. Our results support the view that ICl,acid and ICl,vol stem from two distinct populations of anion channels and that the ICl,acid channels are present in cardiac cells. ICl,acid may play a role in the control of action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis.

Different intracellular polyamine concentrations underlie the difference in the inward rectifier K(+) currents in atria and ventricles of the guinea-pig heart.

The outward component of the strong inward rectifier potassium current, I(K1), is significantly larger in ventricles than in atria of the heart, resulting in faster repolarization at the final phase of the action potential in ventricles. However, the underlying mechanism of the difference in I(K1) remains poorly understood. I(K1) channels are composed of subunits from the Kir2 subfamily, and I(K1) amplitude is determined by the voltage-dependent blockade of the channel by the intracellular polyamines spermine and spermidine, and by Mg(2+). Using a perforated patch-clamp method, which minimizes changes in the intracellular polyamine and Mg(2+) concentrations, we detected repolarization-induced outward I(K1) transients, which are caused by competition between Mg(2+) and spermine to block the channel, in ventricular but not in atrial myocytes from guinea-pig heart. The contribution of the Kir2.3 subunit to the I(K1) channel was found to be minor in the guinea-pig heart, because the activation time course of the Kir2.3 currents was approximately 10-fold slower than those of I(K1), and the marked external pH sensitivity of the Kir2.3 currents was not found in I(K1). Both the Kir2.1 and Kir2.2 currents recorded from inside-out patches exhibited outward transients similar to those of ventricular I(K1) in the presence of 5-10 microM spermine and 0.6-1.1 mM Mg(2+), and their amplitudes were diminished by increasing the spermine or spermidine concentrations. The total and free polyamine concentrations in guinea-pig cardiac tissues were higher in atria than ventricles. These results strongly suggest that different intracellular polyamine concentrations are responsible for the difference in atrial and ventricular I(K1) of the guinea-pig heart.

Cell-volume regulation by swelling-activated chloride current in guinea-pig ventricular myocytes.

The cell-volume regulation by swelling-activated Cl- current (I(Cl,swell)) was studied in guinea pig ventricular myocytes, using a microscopic video-image analysis. We have previously shown that in ventricular cells depolarized in high-K+ ([K+]o>45 mM) solution, an activation of the cyclic AMP-dependent Cl- current (I(Cl,cAMP)) leads to cell swelling. We first investigated the mechanism underlying the I(Cl,cAMP)-independent recovery (shrinkage) of the swollen cells. They shrank when the membrane potential (Vm) was made negative to the equilibrium potential of Cl- (ECl) by lowering [K+]o or [Cl-]o in the high-K+ solution. This shrinkage was attenuated by the inhibitors (DIDS, glibenclamide, furosemide) of swelling-activated Cl- current (I(Cl,swell)). These findings suggested an involvement of I(Cl,swell) in the observed isosmotic cell shrinkage. On the other hand, an application of hyposmotic (70% of control) solution to the cells at normal [K+]o (ECl>Vm) induced a cell swelling, and the swollen cells underwent a slight but definite spontaneous cell shrinkage during hyposmotic challenge, indicating the operation of the mechanism of regulatory volume decrease (RVD). This RVD was pronounced at low [Cl-]o, at which ECl was much more positive than Vm. On the contrary, when the hyposmotic solution was applied to the cells at high [K+]o, at which ECl was negative to Vm, the cells swelled vigorously and monotonically without showing RVD, the swelling being much greater than that seen at normal [K+]o. Both the RVD at normal [K+]o and the extra cell swelling at high [K+]o were suppressed by DIDS. These results suggest that I(Cl,swell) activated by cell swelling can shrink or inflate the cardiac cells under hyposmotic as well as isosmotic conditions, depending on Vm and ECl.

Two modes of polyamine block regulating the cardiac inward rectifier K+ current IK1 as revealed by a study of the Kir2.1 channel expressed in a human cell line.

The strong inward rectifier K(+) current, I(K1), shows significant outward current amplitude in the voltage range near the reversal potential and thereby causes rapid repolarization at the final phase of cardiac action potentials. However, the mechanism that generates the outward I(K1) is not well understood. We recorded currents from the inside-out patches of HEK 293T cells that express the strong inward rectifier K(+) channel Kir2.1 and studied the blockage of the currents caused by cytoplasmic polyamines, namely, spermine and spermidine. The outward current-voltage (I-V) relationships of Kir2.1, obtained with 5-10 microm spermine or 10-100 microm spermidine, were similar to the steady-state outward I-V relationship of I(K1), showing a peak at a level that is approximately 20 mV more positive than the reversal potential, with a negative slope at more positive voltages. The relationships exhibited a plateau or a double-hump shape with 1 microm spermine/spermidine or 0.1 microm spermine, respectively. In the chord conductance-voltage relationships, there were extra conductances in the positive voltage range, which could not be described by the Boltzmann relations fitting the major part of the relationships. The extra conductances, which generated most of the outward currents in the presence of 5-10 microm spermine or 10-100 microm spermidine, were quantitatively explained by a model that considered two populations of Kir2.1 channels, which were blocked by polyamines in either a high-affinity mode (Mode 1 channel) or a low-affinity mode (Mode 2 channel). Analysis of the inward tail currents following test pulses indicated that the relief from the spermine block of Kir2.1 consisted of an exponential component and a virtually instantaneous component. The fractions of the two components nearly agreed with the fractions of the blockages in Mode 1 and Mode 2 calculated by the model. The estimated proportion of Mode 1 channels to total channels was 0.9 with 0.1-10 microm spermine, 0.75 with 1-100 microm spermidine, and between 0.75 and 0.9 when spermine and spermidine coexisted. An interaction of spermine/spermidine with the channel at an intracellular site appeared to modify the equilibrium of the two conformational channel states that allow different modes of blockage. Our results suggest that the outward I(K1) is primarily generated by channels with lower affinities for polyamines. Polyamines may regulate the amplitude of the outward I(K1), not only by blocking the channels but also by modifying the proportion of channels that show different sensitivities to the polyamine block.

Identification of human Kir2.2 (KCNJ12) gene encoding functional inward rectifier potassium channel in both mammalian cells and Xenopus oocytes.

Arginine residue at position 285 (R285) in the intracellular C-terminal domain of inward rectifier potassium channel Kir2.2 is conserved in many species, but missing in previously reported human Kir2.2 sequences. We here identified the human Kir2.2 gene in normal individuals, which contained R285 in the deduced amino-acid sequence (hKir2.2/R285). All 30 individuals we examined were homozygous for Kir2.2/R285 gene. The hKir2.2/R285 was electrophysiologically functional in both mammalian cells and Xenopus oocytes. However, the hKir2.2 missing R285 was functional only in Xenopus oocytes, but not in mammalian cells. Thus, R285 in Kir2.2 is important for its functional expression in mammalian cells.

Rapidly and slowly activating components of delayed rectifier K(+) current in guinea-pig sino-atrial node pacemaker cells.

The components and properties of the delayed rectifier K(+) current (I(K)) in isolated guinea-pig sino-atrial (SA) node pacemaker cells were investigated using the whole-cell configuration of the patch-clamp technique. An envelope of tails test was conducted by applying depolarizing pulses from a holding potential of -50 mV to +30 mV for various durations ranging from 40 to 2000 ms. The ratio of the tail current amplitude elicited upon return to the holding potential to the magnitude of the time-dependent outward current activated during depolarizing steps was dependent on the pulse duration, while after exposure to the selective I(Kr) inhibitor E-4031 (5 microM) this current ratio became practically constant irrespective of the pulse duration. These observations are consistent with the presence of the E-4031-sensitive, rapidly activating and E-4031-resistant, slowly activating components of I(K) (I(Kr) and I(Ks), respectively) in guinea-pig SA node cells. The activation range for I(Kr), defined as the E-4031-sensitive current (half-maximal activation voltage (V(1/2)) of -26.2 mV) was much more negative than that for I(Ks), defined as the E-4031-resistant current (V(1/2) of +17.2 mV). I(Kr) exhibited a marked inward rectification at potentials positive to -50 mV, whereas I(Ks) showed only a slight rectification. In the current-clamp experiments, bath application of E-4031 (0.5 and 5 microM) initially slowed the repolarization at potentials negative to approximately -30 mV and produced a significant depolarization of the maximum diastolic potential, followed by the arrest of electrical activity, thus indicating that the late phase of the repolarization leading to the maximum diastolic potential at around -60 mV in spontaneous action potentials is primarily produced by I(Kr) in guinea-pig SA node cells. External application of the selective I(Ks) inhibitor 293B (30 microM) also delayed the repolarization process at potentials negative to about -20 mV and induced moderate depolarization of the maximum diastolic potential leading to the arrest of the spontaneous activity. These results provide evidence to suggest that both I(Kr) and I(Ks) are present and play crucial roles in the spontaneous electrical activity of guinea-pig SA node pacemaker cells.

Inward rectifier K(+) current under physiological cytoplasmic conditions in guinea-pig cardiac ventricular cells.

The outward current that flows through the strong inward rectifier K(+) (K(IR)) channel generates I(K1), one of the major repolarizing currents of the cardiac action potential. The amplitude and the time dependence of the outward current that flows through K(IR) channels is determined by its blockage by cytoplasmic cations such as polyamines and Mg(2+). Using the conventional whole-cell recording technique, we recently showed that the outward I(K1) can show a time dependence during repolarization due to competition of cytoplasmic particles for blocking K(IR) channels. We used the amphotericin B perforated patch-clamp technique to measure the physiological amplitude and time dependence of I(K1) during the membrane repolarization of guinea-pig cardiac ventricular myocytes. In 5.4 mM K(+) Tyrode solution, the density of the current consisting mostly of the sustained component of the outward I(K1) was about 3.1 A F(-1) at around -60 mV. The outward I(K1) showed an instantaneous increase followed by a time-dependent decay (outward I(K1) transient) on repolarization to -60 to -20 mV subsequent to a 200 ms depolarizing pulse at +37 mV (a double-pulse protocol). The amplitudes of the transients were large when a hyperpolarizing pre-pulse was applied before the double-pulse protocol, whereas they were small when a depolarizing pre-pulse was applied. The peak amplitudes of the transients elicited using a hyperpolarizing pre-pulse were 0.36, 0.63 and 1.01 A F(-1), and the decay time constants were 44, 14 and 6 ms, at -24, -35 and -45 mV, respectively. In the current-clamp experiments, a phase-plane analysis revealed that application of pre-pulses changed the current density at the repolarization phase to the extents expected from the changes of the I(K1) transient. Our study provides the first evidence that an outward I(K1) transient flows during cardiac action potentials.