SUMMARY1. 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°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 (r = 5-4 s at 23 '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. IIK(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. I],, the inward rectifier K+ current, is much larger in ventricle than in atrium.The current-voltage relationship for IK, in ventricle exhibits a negative slope conductance between -50 and 0 mV. IK, 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.

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.

Electrical events and intracellular calcium concentration ([Ca2+]) imaged using fluo‐3 and laser scanning confocal microscopy were simultaneously monitored in single smooth muscle cells freshly isolated from guinea‐pig vas deferens or urinary bladder. Images obtained every 8 ms, during stepping from ‐60 to 0 or +10 mV for 50 ms under voltage clamp, showed that a rise in [Ca2+] could be detected within 20 ms of depolarization in five to twenty small (< 2 μm diameter) ‘hot spots’, over 95 % of which were located within 1.5 μm of the cell membrane. Depolarization at 30 s intervals activated hot spots at the same places. Cd2+ or verapamil abolished both hot spots and Ca2+‐activated K+ current (IK,Ca). Caffeine almost abolished hot spots and markedly reduced IK,Ca. Cyclopiazonic acid, which raised basal global [Ca2+], decreased the rise in hot spot [Ca2+] and IK,Ca amplitude during depolarization. These results suggest that Ca2+ entry caused Ca2+‐induced Ca2+ release (CICR). Under voltage clamp, hot spot [Ca2+] closely paralleled the rise in IK,Ca and reached a peak within 20 ms of the start of depolarization, but the rise in global [Ca2+] over the whole cell area was much slower. Step depolarization to potentials positive to ‐20 mV caused hot spots to grow in size and coalesce, leading to a rise in global [Ca2+] and contraction. Ca2+ hot spots also occurred during the up‐stroke of an evoked action potential under current clamp. It is concluded that the entry of Ca2+ in the early stages of an action potential evokes CICR from discrete subplasmalemma Ca2+ storage sites and generates hot spots that spread to initiate a contraction. The activation of Ca2+‐dependent K+ channels in the plasmalemma over hot spots initiates IK,Ca and action potential repolarization.

SUMMARY1. 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 IuM-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 /LM-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 (T = 1 S), in addition to another faster component (T = 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+ 5-2 Y. IMAIZUMI, K. MURAKI AND M. WATANABE current during the train of pulses was smaller than the decay of Ca2+ current during a long pulse.8. These results indicate that (1) the repetitive spikes on the plateau phase of the action potential are attributable to the slow inactivation and rapid reactivation of I4a and repetitive activation of IK(Ca) due to Ca2+-induced Ca2+ release from stores, and (2) the long plateau phase in the action potential may be due to a slowly inactivating Iba, a Na+-dependent late inward current, and a small delayed rectifier outward current.

Presenilin (PS) genes linked to early-onset familialAlzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of ␥-secretase, forming a high molecular weight complex with other proteins. During our attempts to identify binding partners of PS2, we cloned CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/ KChIP protein family that interacts with the C-terminal region of PS. Upon co-expression in cultured cells, CALP was directly bound to and co-localized with PS2 in endoplasmic reticulum. Alzheimer's disease (AD)1 is a progressive dementing neurodegenerative disorder characterized by a massive deposition of ␤-amyloid and tau-rich neurofibrillary lesions in the brains (reviewed in Ref. 1 and references therein). A subset of AD is inherited as an autosomal dominant trait, and mutations in three different genes have thus far been linked to early-onset autosomal dominant forms of familial AD (FAD). Among these, presenilin 1 (PS1) and PS2 account for the majority of the early onset FAD (1). PS1 and PS2 genes encode polytopic integral membrane proteins that are predominantly localized in intracellular membranes and span the membrane six to eight times.PS proteins undergo endoproteolysis to give rise to N-and C-terminal fragments, which are the preponderant forms of endogenous PS in vivo (2). These fragments form a heterodimer and are incorporated into high molecular weight (HMW) protein complexes (2-5) that are highly stabilized (t1 ⁄2 ϭ ϳ20 h; Ref.6), whereas holoproteins of PS are rapidly degraded (t1 ⁄2 ϭ ϳ2 h) (6, 7). The steady-state levels of PS fragments seem to be tightly regulated by competition for shared, but limiting, cellular factors, because overexpression of PS in transfected cells does not increase the overall level of PS fragments and replaces endogenous PS (8).PS plays an important role in the generation of amyloid ␤ peptides (A␤) by facilitating intramembranous ␥-cleavage of ␤-amyloid protein precursor (␤APP), as evidenced by the lack of A␤ production and accumulation of ␤APP C-terminal stubs in cells established from PS-null mice (9 -11). In contrast, FADlinked mutations in PS increase the production of highly fibrillogenic A␤42 (12-15), which is the initial and predominantly deposited A␤ species in AD brains (16, 17) and normally consists of only ϳ10% of total secreted A␤ (18). Moreover, genetic studies in invertebrates and PS-null mice suggested that ␥-cleavage-like proteolytic cleavage at site 3 to release Notch intracellular domain (NICD), which is the prerequisite for Notch signaling (reviewed in Ref. 19), also is facilitated by PS. Furthermore, recent findings that the two intramembranous aspartates within the 6th and 7th transmembrane (TM) domains of PS are required for ␥-secretase activities (20) and that transition state analogue ␥-secretase inhibitors specifically label PS fragments (21-24) strongly support the notion that the PS-containing macroprotein complex catalyzes ␥-cleavage and that PS may represent the catalytic ...

The relationship between Ca2+ sparks spontaneously occurring at rest and local Ca2+ transients elicited by depolarization was analysed using two‐dimensional confocal Ca2+ images of single smooth muscle cells isolated from guinea‐pig vas deferens and urinary bladder. The current activation by these Ca2+ events was also recorded simultaneously under whole‐cell voltage clamp. Spontaneous transient outward currents (STOCs) and Ca2+ sparks were simultaneously detected at ‐40 mV in approximately 50 % of myocytes of either type. Ca2+ sparks and corresponding STOCs occurred repetitively in several discrete sites in the subplasmalemmal area. Large conductance Ca2+‐dependent K+ (BK) channel density in the plasmalemma near the Ca2+ spark sites generating STOCs was calculated to be 21 channels μm−2. When myocytes were depolarized from ‐60 to 0 mV, several local Ca2+ transients were elicited within 20 ms in exactly the same peripheral sites where sparks occurred at rest. The local Ca2+ transients often lasted over 300 ms and spread into other areas. The appearance of local Ca2+ transients occurred synchronously with the activation of Ca2+‐dependent K+ current (IK,Ca). Immunofluorescence staining of the BK channel α‐subunit (BKα) revealed a spot‐like pattern on the plasmalemma, in contrast to the uniform staining of voltage‐dependent Ca2+ channel α1C subunits along the plasmalemma. Ryanodine receptor (RyR) immunostaining also suggested punctate localization predominantly in the periphery. Double staining of BKα and RyRs revealed spot‐like co‐localization on/beneath the plasmalemma. Using pipettes of relatively low resistance, inside‐out patches that included both clustered BK channels at a density of over 20 channels μm−2 and functional Ca2+ storage sites were obtained at a low probability of ≈5 %. The averaged BK channel density was 3‐4 channels μm−2 in both types of myocyte. These results support the idea that a limited number of discrete sarcoplasmic reticulum (SR) fragments in the subplasmalemmal area play key roles in the control of BK channel activity in two ways: (i) by generating Ca2+ sparks at rest to activate STOCs and (ii) by generating Ca2+ transients presumably triggered by sparks during an action potential to activate a large IK,Ca and also induce a contraction. BK channels and RyRs may co‐localize densely at the junctional areas of plasmalemma and SR fragments, where Ca2+ sparks occur to elicit STOCs.

1 Effects of cyclopiazonic acid (CPA), a specific inhibitor of the Ca2+-ATPase in sarcoplasmic reticulum (SR) of skeletal and cardiac muscles, on contractile responses induced by Ca2"-release from intracellular storage sites were examined in the longitudinal smooth muscle strip of the guinea-pig ileum skinned with J3-escin.2 Ca2+-loading of storage sites (Ca2'-uptake) was performed in pCa 6.3 solution. The amount of Cat aken up was monitored by use of the amplitude of contraction following application of 25 mM caffeine or 25 gM inositol 1,4,5-trisphosphate (IP3). 3 Contractile responses to caffeine or IP3 were reduced or abolished when the preceding Ca2+-uptake was performed in the presence of 0.1-10g M CPA. The dose of CPA required to inhibit the contraction induced by caffeine or IP3 by 50% was approximately 0.6 gM. The CPA-sensitive Ca2"-uptake completely depended upon the presence of ATP in the solution during Ca2+-uptake.4 When 1 gM CPA was added after Ca2"-uptake, the subsequent caffeine-or IP3-induced contraction was not significantly affected by the presence of CPA. 5 Acetylcholine-induced contraction was also almost abolished when the preceding Ca2+-uptake was performed in the presence of 10gM CPA. 6 The relationship between pCa and contraction was not affected by the presence of 10gM CPA in skinned fibres where Ca2+ storage sites had been destroyed by treatment with A23187. The enhancement of contraction in pCa 6.0 solution by calmodulin was not affected by 1O gM CPA.7 These results suggest that CPA selectively inhibits ATP-dependent Ca2"-uptake into intracellular storage sites in skinned ileal smooth muscle strips. CPA appears to be a potent, reversible, and very specific inhibitor of the Ca2+-pump in the storage sites of smooth muscle, and is an extremely valuable pharmacological tool.

We describe here (1) the heterogeneous expression of Ca 2+ -independent transient (A-type) K + channel K K-subunits (Kv1.4, Kv3.3, Kv3.4, Kv4.2 and Kv4.3) in rat smooth muscle, heart and brain, (2) the molecular cloning and tissue distribution of a novel alternatively spliced variant of an A-type K + channel K K-subunit, Kv4.3, and (3) The longer splice variant is very weakly expressed in brain, but is the major product in heart. z 1997 Federation of European Biochemical Societies.