Yuji Imaizumi

Comparison of potassium currents in rabbit atrial and ventricular cells.

1. In rabbit and human hearts there are significant differences in the action potential configuration in atrium and ventricle, and the action potential waveform exhibits marked frequency dependence in both tissues. To study the ionic mechanism(s) of these phenomena, the size and time course of the potassium (K+) currents responsible for repolarization have been recorded from single cells using a whole‐cell microelectrode voltage clamp method. 2. At physiological heart rates, the action potential in atrial cells has a short plateau phase; however, the rapid early repolarization is strongly frequency dependent. Ventricular myocytes have a long plateau (400‐700 ms at 23 degrees C), and the late repolarizing phase of the action potential is much faster in ventricle than in atrium. 3. In both cell types, four different outward currents can be recorded: (i) a large transient outward current, It; (ii) IK(Ca), a smaller Ca2+‐dependent K+ current; (iii) IK, a small, maintained time‐ and voltage‐dependent delayed rectifier K+ current; (iv) IK1, an inwardly rectifying K+ current. 4. It, which is responsible for early repolarization, is much larger in atrium than in ventricle. It has very rapid activation and inactivation kinetics but a very slow time course of recovery from inactivation (tau = 5.4 s at 23 degrees C). Our results show that the reactivation kinetics of It are responsible for the pronounced dependence of the shape of the atrial action potential on stimulus frequency. 5. IK(Ca) is variable from cell to cell and is larger in atrium than in ventricle. In both cell types, IK(Ca) is much smaller than It. 6. The delayed rectifier current, IK, is very small and turns on relatively slowly in both cell types. It is therefore not activated strongly during the relatively short plateau of the atrial action potential. Even in ventricle, it contributes only a small repolarizing current. 7. IK1, the inward rectifier K+ current, is much larger in ventricle than in atrium. The current‐voltage relationship for IK1 in ventricle exhibits a negative slope conductance between ‐50 and 0 mV. IK1 is the outward current which generates the resting membrane potential and it modulates the final repolarization phase of the action potential in both cell types. 8. These data strongly suggest that the action potential configuration and its frequency dependence in rabbit atrial and ventricular cells are mainly due to the differences in sizes and kinetics of It and IK1.

Ionic currents in single smooth muscle cells from the ureter of the guinea‐pig.

1. Ionic currents underlying the action potential were recorded from enzymatically isolated smooth muscle cells of guinea‐pig ureter. 2. The action potential recorded from a single cell was similar to that from a multicellular preparation. It showed repetitive spikes on a plateau potential which followed the first spike. Treatment with 10 mM‐tetraethylammonium (TEA) increased the amplitude and duration of the plateau phase and abolished the repetitive spikes. 3. Under voltage clamp mode, at least two (maybe three) kinds of outward currents were activated during depolarizing pulses. The main outward current was Ca2+‐dependent K+ current (IK(Ca], which was mostly blocked in Ca2+‐free solution, or by application of 1 mM‐cadmium (Cd2+) or 2 mM‐tetraethylammonium (TEA). IK(Ca) was greatly decreased by treatment with 5 mM‐caffeine or an addition of 10 mM‐EGTA in a pipette solution. 4. In the presence of 1 mM‐Cd2+ and 2 mM‐TEA, a small transient outward current remained. 4‐Aminopyridine (1 mM) suppressed the transient outward current by about 40%. Time‐ and voltage‐dependent delayed rectifier outward currents were small in ureter cells. An inwardly rectifying K+ current was not detected. 5. An application of 1 mM‐Cd2+, 5 mM‐cobalt (Co2+), 1 mM‐lanthanum (La3+) or 0.1 microM‐nifedipine completely blocked the action potential. Replacement of 80‐90% of extracellular Na+ with Li+ or Tris almost abolished the plateau potential and repetitive spikes but did not change significantly the first spike. 6. In the presence of 30 mM‐TEA, the inward current elicited by depolarization was monophasic and lasted for more than 1 s. Application of 1 mM‐Cd2+, 1 mM‐La3+, 0.1 microM‐nifedipine, or 5 mM‐Co2+ completely blocked inward current. The replacement (87%) of extracellular Na+ ions with Li+, Tris, sucrose or TEA speeded up the decay of inward current; the inward current decreased by 10‐60% at the end of a 500 ms pulse. 7. Even in low‐Na+ solution (120 mM‐TEA), the inactivation of ICa had a quite slow component (tau = 1 s), in addition to another faster component (tau = 100 ms) at 0 mV. When short depolarizing clamp pulses (50 ms) were repetitively applied at short intervals (50 ms) and with interpulse voltage of ‐10 or ‐20 mV to mimic the repetitive spikes on the plateau of the action potential, the decline of peak Ca2+ current during the train of pulses was smaller than the decay of Ca2+ current during a long pulse.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of the transient outward current in rabbit atrial cells.

1. Whole‐cell and patch clamp techniques have been used to study the steady‐state voltage dependence and the kinetics of a transient outward current, It, in single cells from rabbit atrium. 2. The steady‐state voltage dependence of both activation and inactivation of It are well described by Boltzmann functions. Inactivation is fully removed at potentials negative to ‐70 mV and it is complete near 0 mV. The threshold for activation of It is near ‐30 mV and it is fully activated at +30 mV. The region of overlap between the activation and inactivation curves indicates that a steady non‐inactivating current will be recorded over a membrane potential range from approximately ‐30 to 0 mV. 3. In general, the time course of inactivation at potentials in the range 0 to +50 mV is best described as a sum of two exponential functions. The kinetic parameters controlling these processes exhibit only very weak voltage dependence. 4. Comparison of the time course of the development of inactivation in response to long depolarizing voltage clamp steps with the development of inactivation in response to trains of brief depolarizing pulses indicates that inactivation develops very quickly and decays relatively slowly at potentials near the resting potential (e.g. ‐70 mV). Thus, in response to (i) a train of voltage‐clamp pulses or (ii) a series of action potentials, the magnitude of It decreases due to a progressive increase in the amount of inactivation. 5. A simple model of channel gating is presented: it can account for the major aspects of the voltage dependence and kinetics of It (cf. Aldrich, 1981). 6. Cell‐attached patch clamp recordings have been used to identify the single‐channel or unitary events underlying the current, It. In general, only one active channel is present per patch. The single‐channel conductance in normal Tyrode solution is approximately 14 pS and the current‐voltage relationship is approximately linear between +50 and +150 mV with respect to rest. This information, in combination with the fully activated current‐voltage characteristics from the whole‐cell data, can be used to estimate the number and density of It channels per cell: these are 1600 and one per 3‐4 micron 2, respectively. 7. Ensemble averages obtained from patch recordings are very similar in time course to the macroscopic or whole‐cell current itself: the ensemble current rises to a peak within approximately 5 ms and decays with a biexponential time course in response to depolarizations to approximately +50 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Sodium currents in smooth muscle cells freshly isolated from stomach fundus of the rat and ureter of the guinea-pig.

1. Inward currents elicited by depolarization from holding potentials of ‐80 to ‐10 mV in single smooth muscle cells isolated from stomach fundus of the rat and ureter of the guinea‐pig had two components. The initial fast component (Ifi) was activated and mostly inactivated within 1‐2 and 10 ms, respectively, at 21 degrees C. The following sustained component (Isi) lasted over 50 and 500 ms in fundus and ureter cells, respectively. Ifi was blocked by tetrodotoxin but not affected by 0.5 microM‐mu‐conotoxin in both types of cells. Isi was abolished by the substitution of extracellular Ca2+ with Mn2+. 2. The sensitivity of Ifis to TTX was markedly different in fundus and ureter cells. The half‐inhibition was obtained at 870 and 11 nM, respectively. The amplitude of Ifi was highly dependent on extracellular Na+ concentration in a solution containing 2.2 mM‐Mn2+ and 0 mM‐Ca2+ in both cells. It is concluded that Ifis in these cells are TTX‐sensitive and mu‐conotoxin‐insensitive Na+ currents. 3. Some of the kinetics of INa measured at 10 degrees C were markedly different in fundus and ureter cells. The current‐voltage relationships for Ifi in fundus and ureter cells had peaks at about ‐10 and 0 mV, respectively. The voltage dependence of the steady‐state inactivation of Ifi was also significantly different in these cell types. The half‐inactivation voltages were about ‐74 and ‐45 mV, respectively. The recovery time course from inactivation in fundus cells was about 10 times slower than that in ureter at ‐80 mV, where it was 25 ms. 4. The contribution of Ifi to the rising phase of an action potential was examined using TTX under current clamp mode at 21 degrees C. A fast notch‐like potential elicited by a subthreshold stimulus for action potential generation was blocked by TTX in both types of cells. Action potentials elicited by a stimulus around threshold were occasionally suppressed by TTX, whereas an action potential was never observed when extracellular Ca2+ was replaced with Mn2+. 5. In conclusion, the existence of at least two types of Na+ channel currents, which were distinguished by their TTX sensitivity and kinetics, was strongly suggested in smooth muscle cells from the rat fundus and the guinea‐pig ureter. INa in these cells may have a physiological role to accelerate the generation of an action potential by triggering a rapid activation of ICa, while not being essential for activation of action potentials.

TRIC-A Channels in Vascular Smooth Muscle Contribute to Blood Pressure Maintenance.

TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation channels postulated to mediate counter-ion movements facilitating physiological Ca(2+) release from internal stores. Tric-a-knockout mice developed hypertension during the daytime due to enhanced myogenic tone in resistance arteries. There are two Ca(2+) release mechanisms in vascular smooth muscle cells (VSMCs); incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization, while agonist-induced activation of inositol trisphosphate receptors (IP(3)Rs) evokes global Ca(2+) transients causing contraction. Tric-a gene ablation inhibited RyR-mediated hyperpolarization signaling to stimulate voltage-dependent Ca(2+) influx, and adversely enhanced IP(3)R-mediated Ca(2+) transients by overloading Ca(2+) stores in VSMCs. Moreover, association analysis identified single-nucleotide polymorphisms (SNPs) around the human TRIC-A gene that increase hypertension risk and restrict the efficiency of antihypertensive drugs. Therefore, TRIC-A channels contribute to maintaining blood pressure, while TRIC-A SNPs could provide biomarkers for constitutional diagnosis and personalized medical treatment of essential hypertension.

Serotonin Augments Gut Pacemaker Activity via 5-HT3 Receptors

Serotonin (5-hydroxytryptamine: 5-HT) affects numerous functions in the gut, such as secretion, muscle contraction, and enteric nervous activity, and therefore to clarify details of 5-HTs actions leads to good therapeutic strategies for gut functional disorders. The role of interstitial cells of Cajal (ICC), as pacemaker cells, has been recognised relatively recently. We thus investigated 5-HT actions on ICC pacemaker activity. Muscle preparations with myenteric plexus were isolated from the murine ileum. Spatio-temporal measurements of intracellular Ca2+ and electric activities in ICC were performed by employing fluorescent Ca2+ imaging and microelectrode array (MEA) systems, respectively. Dihydropyridine (DHP) Ca2+ antagonists and tetrodotoxin (TTX) were applied to suppress smooth muscle and nerve activities, respectively. 5-HT significantly enhanced spontaneous Ca2+ oscillations that are considered to underlie electric pacemaker activity in ICC. LY-278584, a 5-HT3 receptor antagonist suppressed spontaneous Ca2+ activity in ICC, while 2-methylserotonin (2-Me-5-HT), a 5-HT3 receptor agonist, restored it. GR113808, a selective antagonist for 5-HT4, and O-methyl-5-HT (O-Me-5-HT), a non-selective 5-HT receptor agonist lacking affinity for 5-HT3 receptors, had little effect on ICC Ca2+ activity. In MEA measurements of ICC electric activity, 5-HT and 2-Me-5-HT caused excitatory effects. RT-PCR and immunostaining confirmed expression of 5-HT3 receptors in ICC. The results indicate that 5-HT augments ICC pacemaker activity via 5-HT3 receptors. ICC appear to be a promising target for treatment of functional motility disorders of the gut, for example, irritable bowel syndrome.

Caveolin-1 facilitates the direct coupling between large-conductance Ca2+-activated K+ (BKCa) and Cav1.2 Ca2+ channels and their clustering to regulate membrane excitability in vascular myocytes.

Background: The contribution of caveolae to physiological interaction between two major ion channels, large conductance Ca2+-activated K+ (BKCa) and Ca2+ (Cav1.2) channels, is unknown in vascular myocytes. Results: The loss of caveola by caveolin-1 deficiency reduced BKCa-Cav1.2 coupling, Cav1.2 clustering, and membrane excitability regulation. Conclusion: Caveolin-1 provides platform for BKCa-Cav1.2 molecular complex. Significance: Caveolin-1-BKCa-Cav1.2 in caveola forms a novel Ca2+ signal domain for arterial tonus regulation. L-type voltage-dependent Ca2+ channels (LVDCC) and large conductance Ca2+-activated K+ channels (BKCa) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca2+ release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BKCa activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BKCa and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BKCa by Ca2+ influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BKCa co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BKCa can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BKCa and Cav1.2 but also the functional coupling between BKCa and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K+ revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BKCa-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca2+ dynamics including the negative feedback, to control the arterial excitability and contractility.

Molecular assembly and dynamics of fluorescent protein-tagged single KCa1.1 channel in expression system and vascular smooth muscle cells.

The large-conductance Ca(2+)-activated K(+) (K(Ca)1.1, BK) channel has pivotal roles in the regulation of vascular tone. To clarify the molecular dynamics of BK channels and their functionally coupled protein on the membrane surface, we examined single-molecule imaging of fluorescent-labeled BK subunits in the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. The dynamic mobility of yellow fluorescent protein (YFP)-tagged BKα subunit (BKα-YFP) expressed in human embryo kidney 293 (HEK) cells was detected in TIRF regions at the level of individual channels and their clusters on the plasma membrane with a diffusion coefficient of 6.7 × 10(3) nm(2)/s. When BKα-YFP was coexpressed with cyan fluorescent protein (CFP)-tagged BKβ1 subunit (BKβ1-CFP) in HEK cells, the mobility was reduced by ∼50%. Fluorescent image analyses suggest that green fluorescent protein (GFP)-tagged BKα subunit (BKα-GFP) expressed in vascular smooth muscle cells (VSMCs), at low density, preferentially formed a heterotetrameric molecular assembly with native BKα subunits, rather than homotetrameric BKα-GFP. Movement of BKα-YFP in VSMCs (0.29 × 10(3) nm(2)/s) was far more restricted than BKα-YFP/BKβ1-CFP in HEK cells (2.5 × 10(3) nm(2)/s). Actin disruption by pretreatment with cytochalasin D in VSMCs appeared to increase the mobile behavior of BKα-YFP, which was then significantly reduced by addition of jasplakinolide. Most BKα-YFP colocalized with caveolin 1 (Cav1)-CFP in VSMCs, but unexpectedly not frequently in HEK cells. Fluorescence resonance energy transfer analyses showed the direct interaction between BKα-YFP and Cav1-CFP, particularly in VSMCs. These results, obtained by single molecule imaging in living cells, indicate that the dynamics of BKα molecules on the membrane surface are strongly restricted or regulated by its auxiliary β-subunit, cytoskeleton, and direct interaction with Cav1 in VSMCs.

Levcromakalim causes indirect endothelial hyperpolarization via a myo-endothelial pathway

Effects of K+ channel opener, levcromakalim, on vascular endothelial cells were examined. Under voltage‐ and current‐clamp conditions, application of acetylcholine to dispersed endothelial cells isolated from rabbit superior mesenteric artery (dispersed RMAECs) produced hyperpolarization and outward currents. On the other hand, dispersed RMAECs did not respond to levcromakalim. When membrane potential was recorded from endothelium in a mesenteric arterial segment, exposure to levcromakalim in a concentration range of 0.1 to 3 μM caused concentration‐dependent hyperpolarization. The hyperpolarization was observed in the absence of external Ca2+ and was inhibited by 10 μM glibenclamide. The presence of 1 mM heptanol did not affect the levcromakalin‐induced hyperpolarization, whereas treatment of the mesenteric arterial segment with 20 μM 18 β‐glycyrrhetinic acid significantly reduced the hyperpolarization. The response to acetylcholine of RMAECs in an arterial segment with 18 β‐glycyrrhetinic acid was, however, similar to that without 18 β‐glycyrrhetinic acid. These suggest that although RMAECs themselves are functionally insensitive to levcromakalim, those in an arterial segment are hyperpolarized by levcromakalim via myo‐endothelial electrical communication.

EFFECTS OF NORADRENALINE ON MEMBRANE CURRENTS AND ACTION POTENTIAL SHAPE IN SMOOTH MUSCLE CELLS FROM GUINEA-PIG URETER

1. The effects of noradrenaline (NA) on action potential shape and underlying membrane currents were examined in single smooth muscle cells freshly isolated from the ureter of the guinea‐pig. 2. The voltage‐dependent Ca2+ current (ICa) elicited upon depolarization from ‐50 to 0 mV was reduced by 27% upon application of 10 microM NA. This reduction was inhibited or converted to potentiation by internal application of low molecular weight heparin or 5 mM EGTA, indicating that it may be mediated by Ca(2+)‐dependent Ca2+ channel inactivation via inositol 1,4,5‐trisphosphate production and subsequent Ca2+ release from intracellular Ca2+ storage sites. 3. In contrast, Ba2+ current (IBa) through Ca2+ channels was potentiated by 36% in the presence of 10 microM NA. Internal application of GTP gamma S made it difficult to remove potentiation of IBa by wash‐out; internal application of GDP beta S abolished potentiation. 4. NA caused a greater reduction in the transient Ca(2+)‐dependent K+ current (IK(Ca)) upon depolarization than it did in ICa. This reduction was inhibited by internally applied heparin, suggesting that the amount of releasable Ca2+ in the storage sites was markedly reduced in the presence of NA. The sustained component of IK(Ca) which gradually increased during depolarization was also reduced by NA. 5. Action potential duration, which was recorded in a standard solution containing Ca2+, was prolonged by the application of NA. 6. It can be concluded that Ca2+ channel activity in ureter smooth muscle cells is regulated by a dual mechanism: Ca(2+)‐dependent inhibition and GTP‐binding protein‐mediated potentiation. Under physiological conditions, both ICa and IK(Ca) were reduced by NA but the reduction of IK(Ca) was much larger than that of ICa; this results in an increase in net inward current during the action potential plateau and prolongs the action potential.