Seiko Kawano

Calmodulin Mediates Calcium-Dependent Inactivation of the Cerebellar Type 1 Inositol 1,4,5-Trisphosphate Receptor

The dependency of purified mouse cerebellar type 1 inositol 1,4,5-trisphosphate receptor (IP3R1)/Ca2+ channel function on cytoplasmic Ca2+ was examined. In contrast to the channels in crude systems, the purified IP3R1 reconstituted into planar lipid bilayers did not show the bell-shaped dependence on Ca2+. It was activated with increasing Ca2+ sublinearly without inhibition even up to 200 microM. The addition of calmodulin to the cytoplasmic side inhibited the channel at high Ca2+ concentrations. Calmodulin antagonists reversed the Ca2+-dependent inactivation of the native channels in cerebellar microsomes. These results indicate that the bell-shaped dependence on cytoplasmic Ca2+ is not an intrinsic property of the IP3R1, and the Ca2+-dependent inactivation is directly mediated by calmodulin.

Role of cardiac chloride currents in changes in action potential characteristics and arrhythmias

Various types of Cl- currents have been recorded in cardiac myocytes from different regions of the heart and in different species. With few exceptions, most of these currents are not active under basal conditions, but are activated under the influence of various agonists and by physical stress. These channels are distributed nonuniformly, depending on the cell type, tissue and region of the heart. Therefore, Cl- current activation may influence membrane potential and impulse formation differently in different cells, and may play a role in arrhythmogenesis. Among these Cl- currents, the protein kinase A-activated Cl- current (I Cl.PKA), the stretch- or swelling-activated Cl- current (I Cl.SWELL) and the Ca(2+)-activated Cl current (I Cl.Ca) comprise the major anion currents that modify cardiac electrical activity. These currents exhibit outward-going rectification, or are predominantly activated at depolarized voltages and, thus, contribute significantly to shortening of the action potential duration but little to diastolic depolarization. The action potential shortening by Cl- current activation may not only perpetuate reentry by shortening the refractory period in a reentry pathway, but may also prevent the development of early afterdepolarization and triggered activity caused by the prolongation of action potentials. I Cl.Ca contributes to delayed afterdepolarization at diastolic potentials in Ca(2+)-overloaded cells. Another factor limiting the influence of Cl- currents on diastolic potentials is the presence of a predominantly opposing background K+ current, except at the nodal regions that lack these K+ channels, or under conditions of decreased K+ conductance. Therefore, the contribution of Cl- currents to the genesis of arrhythmias may depend on their association with the conductance of other ions, especially that of K+.

Ca(2+) oscillations regulated by Na(+)-Ca(2+) exchanger and plasma membrane Ca(2+) pump induce fluctuations of membrane currents and potentials in human mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. We have demonstrated spontaneous [Ca(2+)](i) oscillations in hMSCs without agonist stimulation, which result primarily from release of Ca(2+) from intracellular stores via InsP(3) receptors. In this study, we further investigated functions and contributions of Ca(2+) transporters on plasma membrane to generate [Ca(2+)](i) oscillations. In confocal Ca(2+) imaging experiments, spontaneous [Ca(2+)](i) oscillations were observed in 193 of 280 hMSCs. The oscillations did not sustain in the Ca(2+) free solution and were completely blocked by the application of 0.1mM La(3+). When plasma membrane Ca(2+) pumps (PMCAs) were blocked with blockers, carboxyeosin or caloxin, [Ca(2+)](i) oscillations were inhibited. Application of Ni(2+) or KBR7943 to block Na(+)-Ca(2+) exchanger (NCX) also inhibited [Ca(2+)](i) oscillations. Using RT-PCR, mRNAs were detected for PMCA type IV and NCX, but not PMCA type II. In the patch clamp experiments, Ca(2+) activated outward K(+) currents (I(KCa)) with a conductance of 170+/-21.6pS could be recorded. The amplitudes of I(KCa) and membrane potential (V(m)) periodically fluctuated liked to [Ca(2+)](i) oscillations. These results suggest that in undifferentiated hMSCs both Ca(2+) entry through plasma membrane and Ca(2+) extrusion via PMCAs and NCXs play important roles for [Ca(2+)](i) oscillations, which modulate the activities of I(KCa) to produce the fluctuation of V(m).

Effects of quinidine on plateau currents of guinea-pig ventricular myocytes

Effects of quinidine on membrane currents forming the plateau of action potentials were studied using an isolated single ventricular cell from guinea-pig hearts. Quinidine (5 mg/l) produced a fall and shortening of the early part of the plateau, and delayed its later part and final repolarization, without changes in resting membrane potential. Application of quinidine caused a reversible depression of the peak Ca2+ current by about 30% of the control. Delayed outward K+ current, iK, also decreased to less than 20% of the control. Thus, an outward tail current upon repolarization to -40 mV from depolarizing voltage steps of the plateau ranges became inward. Current values at the end of 200 ms pulses in response to voltage steps to -60-0 mV were always positive and were not changed by the drug. The inward current elicited at potentials negative to resting potential level, also, decreased by 13% to 23% of the control in the presence of the drug, but the effect was not reversible upon wash-out of the drug. These results suggest that quinidine causes a non-specific depression of inward rectifier K+ current, iK1, with minor degree but has little effect on the window sodium current. Therefore, changes in the action potential repolarization produced by quinidine can be explained by its effects on both calcium current and delayed outward K+ current.

Activation mechanism of Ca(2+)-sensitive transient outward current in rabbit ventricular myocytes.

1. The mechanism of activation of the Ca(2+)‐sensitive and 4‐aminopyridine (4‐AP)‐insensitive transient outward current, I(to)(Ca), was examined in single rabbit ventricular myocytes using the whole‐cell patch‐clamp technique. 2. When the steady‐state intracellular Ca2+ (Ca2+i) concentration ([Ca2+]i) was < 1 nM, I(to)(Ca) could not be activated by applying pulses at 0.1 Hz. When [Ca2+]i was increased to > or = 10 nM, I(to)(Ca) was activated by 0.1 Hz depolarizing pulses in all control experiments. 3. I(to)(Ca) was completely blocked by an anion transport blocker, DIDS, or by replacement of NaCl with sodium aspartate. Upon changing extracellular [Cl‐], the reversal potential was shifted as predicted for a chloride‐selective conductance. When intracellular K+ was replaced with Cs+, I(to)(Ca) was also observed. From these results it was concluded that I(to)(Ca) was carried by Cl‐. 4. Anion selectivity of I(to)(Ca) was investigated by the replacement of C.‐ with various anions. The sequence of permeability was SCN‐ > I‐ > Br‐ > Cl‐. 5. The amplitude of I(to)(Ca) was enhanced in a [Ca2+]i‐dependent manner between 10 nM and 1 microM Ca2+i, while steady‐state inactivation curves and the voltage‐dependent activation curves were unchanged. The half‐inactivation and half‐activation potentials were ‐35 mV and +37 mV, respectively, at all [Ca2+]i. 6. I(to)(Ca) was inhibited by blocking Ca2+ influx or Ca2+ release from sarcoplasmic reticulum, suggesting that a ‘Ca(2+)‐induced Ca(2+)‐release’ mechanism is essential for the activation of I(to)(Ca). 7. A steady‐state Ca(2+)‐activated Cl‐ current with a linear I‐V relationship was observed at 1 microM Ca2+, while the current activated by depolarization was strictly dependent on Ca2+ entry or Ca2+ release from the sarcoplasmic reticulum. These results suggest that the I(to)(Ca) channel is purely ligand (Ca2+) gated and its time course reflects the concentration of Ca2+i.

Identification and Characterization of the Slowly Exchanging pH-dependent Conformational Rearrangement in KcsA

Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K+ channel KcsA under near-physiological conditions, using solution-state NMR. In a series of 1H15N-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K+ channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a “pH-sensor” for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed “dual gating” properties of the K+ channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.

Transient outward currents and action potential alterations in rabbit ventricular myocytes

To clarify ionic mechanisms underlying successive changes in action potential repolarization upon sudden increase in driving rate or initiation of rapid drive after a rest, membrane potentials and currents were recorded from isolated rabbit ventricular myocytes using the suction-pipette whole-cell clamp method. When 20 action potentials were elicited with a stimulus frequency of 2.0 Hz after a rest period of 20 s, the plateau and action potential duration showed complex changes in successive beats, whereas they were nearly constant with stimulation at 0.1 Hz. There were only weak correlations between changes in action potential parameters and preceding diastolic intervals. The changes were prominent in the first 10 beats but subsided gradually thereafter, attaining nearly steady configurations of action potentials. When depolarizing pulses were applied at a fast rate, under the voltage clamp, the amplitudes of the initial inward current in the presence of tetrodotoxin changed greatly depending on the pulse numbers and diastolic intervals, whereas the delayed outward K+ current changed little. Variations of the initial inward current in successive pulses were caused by different degrees of activation and recovery from inactivation in the Ca2+ current, the Ca(2+)-sensitive and -insensitive transient outward current. While inhibition of either one or two current components decreased the action potential alterations, blocking the three components completely abolished them. These results indicate that alterations of the Ca(2+)-sensitive and -insensitive transient outward current together with the Ca2+ current contribute to the action potential alterations after initiation of rapid drive or an increase in driving rates.

Planar bilayer recording of ryanodine receptors of sarcoplasmic reticulum

Publisher Summary This chapter describes methods to incorporate sarcoplasmic reticulum (SR) Ca 2+ channels, also called “ryanodine receptors,” into planar bilayers. Recordings of ryanodine receptors are best made using CsCI as current carrier, instead of the Ca-HEPES and Tris-HEPES solutions. The use of CsCI instead of Ca-HEPES and Tris-HEPES eliminate the need for perfusion and the need for large and unphysiological gradients of Ca 2+ , which severely inactivate the channel. SR Cl - channels can be separated from ryanodine receptors based on reversal potential. The polarity of channels incorporated into the bilayer is constant, in the majority of cases. The myoplasmic end of the receptor faces into the cis solution, and the intravesicular end faces into the trans solution. Polarity can be easily confirmed by the cis-activation of channels by adenosine triphosphate (ATP) and micromolar Ca 2+ , which are myoplasmic activators of ryanodine receptors.

Effects of Na+ channel blocker, pilsicainide, on HERG current expressed in HEK-293 cells.

Purpose: Pilsicainide, classified as a relatively pure Na+ channel blocker, occasionally causes QT prolongation, suggesting inhibitory actions on K+ currents. We studied effects of pilsicainide on the K+ channel current of the human ether‐a‐go‐go‐related gene (HERG) in heterologous expression system. Methods: The Patch‐clamp technique in whole‐cell configuration was used to record the channel current of HERG stably expressed in HEK293 cells. Results: Pilsicainide suppressed peak currents of HERG channel during depolarizing pulses and tail currents upon repolarization. Pilsicainide blocked HERG current with IC50 = 20.4 &mgr;M and Hill coefficient = 0.98. Voltage‐dependent activation was shifted in a negative direction by ˜10 mV by 10 to 20 &mgr;M pilsicainide. Block increased with depolarization to voltages between –20 and 0 mV and reached the maximum level at positive voltages to 0 mV without further increase. Following drug equilibration for 10 minutes (holding potential at –100 mV), the peak outward current upon the first depolarization showed time‐dependent block; tail current block was maximal. Frequency‐dependent block evaluated from tail current was absent with pulse frequencies of 1.33, 0.5, and 0.2 Hz. After a steady state block was achieved, time course of current activation and deactivation was slowed by pilsicainide, and steady‐state inactivation and time course of fast inactivation were mildly affected. Conclusions: Pilsicainide blocks HERG current with a preferential affinity, at least, to the open state of the channels with a fast access to binding sites.

Activation of Ca2+-sensitive Cl– current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

Abstract We investigated how Ca2+-sensitive transient outward current, Ito(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na+ (Na+i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na+-free solutions,the application of nicardipine (5 µM) to block L-type Ca2+ current (ICa) completely inhibited Ito(Ca). In cells dialysed with a [Na+]i≥5 mM, however, Ito(Ca) could be observed after blockade of ICa, indicating the activity of an ICa-independent component. The amplitude of ICa-independent Ito(Ca) increased with voltage in a [Na+]i-dependent manner. The block of Ca2+ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked ICa-independent Ito(Ca). In Ca2+-free bath solution Ito(Ca) was completely abolished. The application of 2 mM Ni2+ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na+/Ca2+ exchange, or perfusion with pipette solution containing XIP (10 µM), a selective blocker of the exchanger, blocked ICa-independent Ito(Ca). From these results we conclude that, in the presence of Na+i, Ito(Ca) can be activated via Ca2+-induced Ca2+ release triggered by Na+/Ca2+ exchange operating in the reverse mode after blockade of ICa.