Tsuguhisa Ehara

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 (IK) 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 IKr inhibitor E‐4031 (5 μm) 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 IK (IKr and IKs, respectively) in guinea‐pig SA node cells. The activation range for IKr, defined as the E‐4031‐sensitive current (half‐maximal activation voltage (V1/2) of −26.2 mV) was much more negative than that for IKs, defined as the E‐4031‐resistant current (V1/2 of +17.2 mV). IKr exhibited a marked inward rectification at potentials positive to −50 mV, whereas IKs showed only a slight rectification. In the current‐clamp experiments, bath application of E‐4031 (0.5 and 5 μm) 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 IKr in guinea‐pig SA node cells. External application of the selective IKs inhibitor 293B (30 μm) 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 IKr and IKs are present and play crucial roles in the spontaneous electrical activity of guinea‐pig SA node pacemaker cells.

Swelling-induced Cl- current in guinea-pig atrial myocytes: inhibition by glibenclamide.

1 Whole‐cell currents were recorded from guinea‐pig atrial myocytes using the patch‐clamp technique under conditions designed to block K+ channels, Ca2+ channels and electrogenic transporters. 2 Exposure of atrial myocytes to the hyposmotic external solution (Na+ reduction to about 70% of control) resulted in hyposmotic cell swelling which was associated with activation of an outwardly rectifying Cl− current (ICl,swell). 3 Whereas the activation of ICl,swell was not significantly affected by replacement of ATP in the pipette solution with the non‐hydrolysable ATP analogue 5′‐adenylyl‐imidodiphosphate (AMP‐PNP), its activation was greatly reduced in cells dialysed with an ATP‐free pipette solution, thus indicating that the activation process of ICl,swell requires the presence of intra‐cellular ATP, but not its hydrolysis. 4 Bath application of glibenclamide produced a concentration‐dependent block of ICl,swell with a half‐maximal inhibitory concentration (IC50) of 60.0 μm and a Hill coefficient of 2.1. The maximal effect (100% inhibition) was obtained with 500 μm glibenclamide. The steady‐state inhibition showed little voltage dependence, while glibenclamide at concentrations of more than 100 μm inhibited the outward ICl,swell more rapidly than the inward ICl,swell. The glibenclamide inhibition was fully reversible after removal of the drug, even when a maximal effect (full inhibition) was achieved at a high drug concentration (500 μm). 5 These results show that (i) glibenclamide is one of the most potent inhibitors of guinea‐pig atrial ICl,swell and (ii) atrial ICl,swell and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− currents are almost equally sensitive to inhibition by glibenclamide.

Activation of the muscarinic K+ channel by P2-purinoceptors via pertussis toxin-sensitive G proteins in guinea-pig atrial cells.

1. Whole‐cell voltage clamp and cell‐attached patch‐clamp techniques were applied to single atrial myocytes enzymatically dissociated from adult guinea‐pig hearts. 2. In whole‐cell clamp conditions, external applications, of ATP activated the muscarinic K+ (KACh) current, identified by its inward rectification, its reversal potential near the calculated K+ equilibrium potential (EK) and its relaxation properties during step changes of whole‐cell membrane potential. Theophylline, an antagonist for Pi‐purinoceptors, did not affect the action of ATP on the KACh current, indicating that the response was evoked through P2‐purinoceptors. 3. The concentration‐response relationship for ATP was well described by a Hill equation with a half‐maximal concentration of 1.84 microM and a Hill coefficient of 0.94. ATP (100 microM) produced a maximal increase of the KACh current to 10.92 microA microF‐1, which corresponds to 44.9 and 80.9% of the maximal increases evoked by ACh (10 microM) and adenosine (100 microM), respectively. 4. The activation of KACh current gradually declined to a steady level despite the continuous presence of ATP (desensitization). Recovery from the desensitization was relatively rapid with a half‐time of approximately 1.5 min. 5. The activation of KACh current by ATP was completely abolished by pre‐incubating myocytes with pertussis toxin (PTX, 5 micrograms ml‐1), indicating that P2‐purinoceptors are coupled to PTX‐sensitive G proteins to activate the KACh channel. 6. In the cell‐attached patch recording, ATP (5 microM) applied to the pipette solution enhanced the activity of a channel with single‐channel conductance of 52.7 +/‐ 0.9 pS (mean +/‐ S.E.M., n = 10), reversal potential near EK and mean open time of 1.1 +/‐ 0.1 ms. These conductance and kinetic properties are identical to those of the KACh channel in the heart. In contrast, ATP applied to the bath solution did not significantly affect the basal activity of KACh channel openings. These observations suggest that the mechanism coupling the P2‐purinoceptor to the activation of the KACh channel involves membrane‐delimited component(s) rather than soluble second messenger(s). 7. These results strongly suggest a direct coupling of the P2‐purinoceptor to the KACh channel through PTX‐sensitive G proteins, analogous to the coupling mechanism of the muscarinic ACh receptor and Pi‐purinoceptor to this channel.

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, IK1, 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 IK1 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 5–10μm spermine or 10–100μm spermidine, were similar to the steady‐state outward I–V relationship of IK1, showing a peak at a level that is ∼20mV 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 1μm spermine/spermidine or 0.1μm 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 5–10μm spermine or 10–100μm 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 0.1–10μm spermine, 0.75 with 1–100μm 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 IK1 is primarily generated by channels with lower affinities for polyamines. Polyamines may regulate the amplitude of the outward IK1, not only by blocking the channels but also by modifying the proportion of channels that show different sensitivities to the polyamine block.

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+ (KIR) channel generates IK1, one of the major repolarizing currents of the cardiac action potential. The amplitude and the time dependence of the outward current that flows through KIR channels is determined by its blockage by cytoplasmic cations such as polyamines and Mg2+. Using the conventional whole‐cell recording technique, we recently showed that the outward IK1 can show a time dependence during repolarization due to competition of cytoplasmic particles for blocking KIR channels. We used the amphotericin B perforated patch‐clamp technique to measure the physiological amplitude and time dependence of IK1 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 IK1 was about 3.1 A F−1 at around −60 mV. The outward IK1 showed an instantaneous increase followed by a time‐dependent decay (outward IK1 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 IK1 transient. Our study provides the first evidence that an outward IK1 transient flows during cardiac action potentials.

Single‐channel study of the cyclic AMP‐regulated chloride current in guinea‐pig ventricular myocytes.

1. Properties of the cyclic AMP‐regulated Cl‐ channel were studied in guinea‐pig ventricular myocytes with the patch clamp technique. Cell‐attached patch recordings were performed, while the cell was dialysed with a cyclic AMP (0.2‐0.5 mM)‐containing internal solution through a second patch pipette. The latter pipette was also used to monitor the whole‐cell Cl‐ conductance. 2. The whole cell showed a large Cl‐ conductance for 10‐15 min after the beginning of cell dialysis. The activity of single Cl‐ channels began to appear in some of the cell‐attached patches during this time. 3. The channels showed a high open probability (0.69 +/‐ 0.14, mean +/‐ S.D., n = 12) at the time of their appearance, and the open probability did not appreciably increase thereafter, even when the whole‐cell Cl‐ conductance increased further with time. 4. An increase in the number of active channels was observed in some patches with progression of the cell dialysis. In such cases, the newly activated channels also showed a high open probability. 5. The above results are consistent with the hypothesis that the cyclic AMP system makes the ‘latent’ Cl‐ channels available without influencing their own kinetic behaviour. The available channels may intrinsically exhibit a high open probability. 6. Chloride channel currents could also be recorded in the outside‐out patches excised from the cyclic AMP‐loaded cells. The I‐V relation of these currents showed outward rectification under the condition of symmetrical Cl‐ gradients, suggesting that the channel itself or a related structure has the property of rectifying current flow. 7. The channel seemed to have at least one open state and two closed states; the open‐time histograms showed one exponential component with the values of time constant scattering around 1 s, while the closed‐time histograms showed two exponential components with the values of time constant scattering around 0.2 and 1 s. These time constants showed no clear voltage dependence.

A repolarization-induced transient increase in the outward current of the inward rectifier K+ channel in guinea-pig cardiac myocytes

1 Outward currents of the inwardly rectifying K+ current (IKir) in guinea‐pig ventricular myocytes were studied in the presence of 1 mM intracellular free Mg2+ using the whole‐cell patch‐clamp technique. 2 During repolarizing voltage steps following a large depolarizing pulse (> 0 mV), outward IKir increased transiently at voltages positive to the K+ equilibrium potential (EK, ‐84 mV for 5.4 mM extracellular [K+]). The rising phase was almost instantaneous, while the decay was exponential. The decay rate was faster at voltages closer to EK (time constants, 33.9 ± 9.8 and 4.8 ± 1.4 ms at ‐30 and ‐50 mV, respectively). 3 The transient outward IKir was absent when the preceding depolarization was applied from ‐40 mV. Larger transient currents developed as the voltage before the depolarization was shifted to more hyperpolarized levels. 4 Shift of the depolarizing voltage from > 0 mV to more negative ranges diminished the amplitudes of transient outward IKir and instantaneous inward IKir during the subsequent repolarizing steps positive and negative to EK, respectively. Since blockage of IKir by internal Mg2+ occurs upon large depolarization, and the block is instantaneously relieved at voltages negative to EK, the rising phase of the transient outward IKir was attributed to the relief of Mg2+ block at voltages positive to EK. Transient outward IKir was absent when intracellular [Mg2+] was reduced to 10 μM or lower. 5 Prolongation of the repolarizing voltage step increased the amplitude of time‐dependent inward IKir during the subsequent hyperpolarization, indicating the progress of a gating process (presumably the channel block by intracellular polyamine) during the decaying phase of outward IKir. 6 Progressive prolongation of the depolarizing pulse (> 0 mV) from 100 to 460 ms decreased the transient outward IKir amplitude during the subsequent repolarizing step due to slow progress of the gating (polyamine block) at > 0 mV. 7 Current‐voltage relations measured using repolarizing ramp pulses (‐3.4 mV ms−1) showed an outward hump at around ‐50 mV, the magnitude of which increased as the voltage before the conditioning depolarization (10 mV) was shifted to more negative levels. With slower ramp speeds (‐1.5 and ‐0.6 mV ms−1), the hump was depressed at voltages near EK. 8 Our study suggests that the relief of Mg2+ block may increase outward IKir during repolarization of cardiac action potentials, and that the resting potential, the level/duration of action potential plateau and the speed of repolarization influence the outward IKir amplitude. 9 A kinetic model incorporating a competition between polyamine block and Mg2+ block was able to account for the time dependence of outward IKir.

Conformational studies and pore-forming properties of an α-aminoisobutyric acid analogue of gramicidin B

Structure-function relation of a designed α-aminoisobutyric acid (Aib) analogue of gramicidin B (GBA), a model for helical protein structures, is examined by CD and patch-clamp experiments. This 16-residue peptide adopts stable helical structures in organic and aqueous solvent sytems, and phospholipid vesicles. The content of the helical structure in egg yolk phosphatidylcholine vesicles increases with the increase of lipid-to-peptide molar ratio, suggesting adsorption and incorporation processes. A possible helix–helix interaction is observable at low peptide-to-lipid molar ratios. The role of Trp side-chains for the rather high affinity of this peptide for membrane surfaces is stressed. Though shorter than the average thickness of phospholipid bilayers, GBA forms voltage-dependent multi-state ion-conducting pores in diphytanoyl phosphatidylcholine bilayers formed at the tip of micropipettes, at relatively low concentrations. A range of GBA conductances is comparable to membrane protein channels. GBA pores show a variety of conducting behaviours as well as rectifying properties.

Selective enhancement of the slow component of delayed rectifier K+ current in guinea-pig atrial cells by external ATP.

1 The effects of external ATP on the rapidly and slowly activating components (IKr and IKg, respectively) of the delayed rectifier K+ current (IK) in guinea‐pig atrial myocytes were determined using the whole‐cell configuration of the patch‐clamp technique. 2 An envelope of tails test was conducted by applying depolarizing pulses to +40 mV from a holding potential of −40 mV for various durations between 50 ms and 2 s under control conditions and during exposure to 50 μM ATP. The ATP‐induced IK, obtained by digital subtraction, exhibited a constant ratio (0.37) of the tail current to time‐dependent current, regardless of the pulse duration. This current ratio was compatible with the predicted ratio of the driving force at +40 and −40 mV for a non‐rectifying K+ conductance, suggesting that the ATP‐induced IK is due primarily to IKs. 3 The amplitude of IKr isolated from the IK enhanced by ATP, determined as an E‐4031 (5 μM)‐sensitive current, was similar to the control magnitude of IKr, thus showing that external ATP did not cause an increase in IKr. 4 The voltage‐dependent activation of the ATP‐induced IK during 500 ms depolarizing test pulses could be described by a Boltzmann equation with a half‐activation voltage (V1/2) of 11.5 mV and slope factor (k) of 12.0 mV, which were close to those of IKg(V1/4 of 12.1 mV and k of 12.3 mV), determined as an E‐4031‐resistant IK, under the same isochronal (500 ms) activation conditions. 5 These results provide evidence to suggest that extracellular ATP selectively potentiates the slow component of IK (IKg), with no measurable effects on IKr, in guinea‐pig atrial myocytes.

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.