The biochemical activation of T-type Ca2+ channels in HEK293 cells stably expressing alpha1G and Kir2.1 subunits

In order to investigate the currently unknown cellular signaling pathways of T-type Ca(2+) channels, we decided to construct a new cell line which would stably express alpha(1G) and Kir2.1 subunits in HEK293 cells (HEK293/alpha(1G)/Kir2.1). Compared to cells which only expressed alpha(1G) (HEK293/alpha(1G)), HEK293/alpha(1G)/Kir2.1 cells produced an enormous inward rectifying current which was blocked by external Ba(2+) and Cs(+) in a concentration-dependent manner. The expression of Kir2.1 channels contributed significantly to the shift of membrane potential from -12.2+/-2.8 to -57.3+/-3.7mV. However, biophysical and pharmacological properties of alpha(1G)-mediated Ca(2+) channels remained unaffected by the expression of Kir2.1 subunits, except for the enlarging of the window current region. Biochemical activation of alpha(1G) channels using 150mM KCl brought about an increase in [Ca(2+)](i), which was blocked by mibefradil, the T-type Ca(2+) channel blocker. These data suggest that the HEK293/alpha(1G)/Kir2.1 cell line would have potential uses in the study of T-type Ca(2)(+) channel-mediated signaling pathways and possibly useful in the development of new therapeutic drugs associated with T-type Ca(2)(+) channels.     

ZD7288 inhibits low-threshold Ca(2+) channel activity and regulates sperm function

In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, has been found to inhibit the mouse sperm acrosome reaction (AR). HCN channels have not yet been either recorded or implicated in mouse sperm AR, but low-threshold (T-type) Ca(2+) channels have. Interestingly, ZD7288 blocked native T-type Ca(2+) currents in mouse spermatogenic cells with an IC(50) of about 100 microM. This blockade was more effective at voltages producing low levels of inactivation, suggesting a differential affinity of ZD7288 for different channel conformations. Furthermore, ZD7288 inhibited all cloned T-type but not high-threshold N-type channels heterologously expressed in HEK-293 cells. Our results further support the role of T-type Ca(2+) channels in the mouse sperm AR.     

Scorpion toxins that block T-type Ca2+ channels in spermatogenic cells inhibit the sperm acrosome reaction

The acrosome reaction (AR) is a Ca(2+)-dependent event required for sperm to fertilize the egg. The activation of T-type voltage-gated Ca(2+) channels plays a key role in the induction of this process. This report describes the actions of two toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively) on sperm Ca(2+) channels. Both toxins decrease T-type Ca(2+) channel activity in mouse spermatogenic cells and inhibit the AR in mature sperm. Saturating concentrations of the toxins inhibited at most approximately 70% of the whole-cell Ca(2+) current, suggesting the presence of a toxin-resistant component. In addition, both toxins inhibited approximately 60% of the AR, which is consistent with the participation of T-type Ca(2+) channels in the sperm AR.     

Prenatal stress on the kinetic properties of Ca2+ and K+ channels in offspring hippocampal CA3 pyramidal neurons

Prenatal stress is known to cause neuronal loss and oxidative damage in the hippocampus of offspring rats. To further understand the mechanisms, the present study was undertaken to investigate the effects of prenatal stress on the kinetic properties of high-voltage-activated (HVA) Ca(2+) and K(+) channels in freshly isolated hippocampal CA3 pyramidal neurons of offspring rats. Pregnant rats in the prenatal stress group were exposed to restraint stress on days 14-20 of pregnancy three times daily for 45 min. The patch clamp technique was employed to record HVA Ca(2+) and K(+) channel currents. Prenatal stress significantly increased HVA Ca(2+) channel disturbance including the maximal average HVA calcium peak current amplitude (-576.52+/-7.03 pA in control group and -702.05+/-6.82 pA in prenatal stress group, p<0.01), the maximal average HVA Ca(2+) current density (-40.89+/-0.31 pA/pF in control group and -49.44+/-0.37 pA/pF in prenatal stress group, p<0.01), and the maximal average integral current of the HVA Ca(2+) channel (106.81+/-4.20 nA ms in control group and 133.49+/-4.59 nA ms in prenatal stress group, p<0.01). The current-voltage relationship and conductance--voltage relationship of HVA Ca(2+) channels and potassium channels in offspring CA3 neurons were not affected by prenatal stress. These data suggest that exposure of animals to stressful experience during pregnancy can exert effects on calcium ion channels of offspring hippocampal neurons and that the calcium channel disturbance may play a role in prenatal stress-induced neuronal loss and oxidative damage in offspring brain.     

Testosterone is a potent inhibitor of L-type Ca(2+) channels

Testosterone administration is beneficial in alleviating myocardial ischaemia in men with significant coronary artery disease (CAD), a condition which is associated with hypotestosteronaemia. Infusion of physiological concentrations of testosterone into coronary arteries at angiography results in rapid vasodilatation in patients with CAD. Whilst the cardiovascular benefits of testosterone have long been documented, the underlying mechanism(s) have not yet been revealed. Here, we have investigated whether testosterone might act like widely prescribed antihypertensive dihydropyridines, as an endogenous Ca(2+) channel antagonist. To do this, we used the whole-cell patch-clamp technique to record Ca(2+) currents from the A7r5 smooth muscle cell line and HEK 293 cells stably expressing either L- or T-type Ca(2+) channels. We demonstrate that testosterone directly inhibited both native and human recombinant vascular L-type Ca(2+) channels in a manner that was voltage-independent and, crucially, displayed an IC(50) value of 38 nM, a value within the physiological range. At higher (supraphysiological) concentrations both native and human recombinant T-type channels were also inhibited by testosterone. Our data indicate that testosterone acts like widely prescribed antihypertensive dihydropyridines to reduce Ca(2+) influx into vascular smooth muscle and so promote vasodilation. This effect is likely to account for its beneficial cardiovascular actions.     

Modulation of T-type Ca2+ channels by corticotropin-releasing factor through protein kinase C pathway in MN9D dopaminergic cells

Corticotrophin-releasing factor (CRF) is the main regulator of the body's stress axis and its signal is translated through G-protein-coupled CRF receptors (CRF-R1, CRF-R2). Even though CRF receptors are present in the midbrain dopamine neurons, the cellular mechanism of CRF action is not clear yet. Since voltage-dependent Ca(2+) channels are highly expressed and important in dopamine neuronal functions, we tested the effect of CRF on voltage-dependent Ca(2+) channels in MN9D cells, a model of dopamine neurons. The application of CRF-related peptide, urocortin 1, reversibly inhibited T-type Ca(2+) currents, which was a major Ca(2+) channel in the cells. The effect of urocortin was abolished by specific CRF-R1 antagonist and was mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate. PKC inhibitors abolished the effect of urocortin. These results suggest that urocortin modulates T-type Ca(2+) channel by interacting with CRF-R1 via the activation of PKC signal pathway in MN9D cells.     

Interactions between inositol 1,4,5-trisphosphate receptors and ryanodine receptors in smooth muscle: one store or two?

This short review proposes a system of simplified functional models describing possible interactions between Ca(2+)-release channels associated with IP(3)Rs and RyRs in smooth muscle, and considers each of these models in the light of the available experimental evidence. Complete separation of IP(3)R- and RyR-gated stores seems to be unusual. Where both receptors release Ca(2+) from a common pool, simple interactions can occur since changes in the activation of one receptor type affects the availability of Ca(2+) for release through the other. Alterations in [Ca(2+)] within the sarcoplasmic reticulum can also affect the open probability of the release channels, and not just the Ca(2+)-flux through the channels when open, e.g., Ca(2+)-release through tonically active IP(3)Rs appears to limit SR Ca(2+)-content in some myocytes, and this modulates RyR activity, as indicated by changes in Ca(2+)-spark frequency. There is also evidence that intracellular release channels may co-operate, leading to positive feedback during activation. In particular, agonist-dependent activation of IP(3)Rs can promote activation of RyRs, amplifying and shaping the resulting Ca(2+)-signal. While there is little direct evidence as to the mechanism responsible for this interaction, some form of Ca(2+)-induced Ca(2+)-release in response to local increases in [Ca(2+)](c) seems likely.     

Calcium-induced transitions between the spontaneous miniature outward and the transient outward currents in retinal amacrine cells

Spontaneous miniature outward currents (SMOCs) occur in a subset of retinal amacrine cells at membrane potentials between -60 and -40 mV. At more depolarized potentials, a transient outward current (I(to)) appears and SMOCs disappear. Both SMOCs and the I(to) are K(+) currents carried by BK channels. They both arise from Ca(2+) influx through high voltage-activated (HVA) Ca(2+) channels, which stimulates release of internal Ca(2+) from caffeine- and ryanodine-sensitive stores. An increase in Ca(2+) influx resulted in an increase in SMOC frequency, but also led to a decline in SMOC mean amplitude. This reduction showed a temporal dependence: the effect being greater in the latter part of a voltage step. Thus, Ca(2+) influx, although required to generate SMOCs, also produced a negative modulation of their amplitudes. Increasing Ca(2+) influx also led to a decline in the first latency to SMOC occurrence. A combination of these effects resulted in the disappearance of SMOCs, along with the concomitant appearance of the I(to) at high levels of Ca(2+) influx. Therefore, low levels of Ca(2+) influx, arising from low levels of activation of the HVA Ca(2+) channels, produce randomly occurring SMOCs within the range of -60 to -40 mV. Further depolarization leads to greater activation of the HVA Ca(2+) channels, larger Ca(2+) influx, and the disappearance of discontinuous SMOCs, along with the appearance of the I(to). Based on their characteristics, SMOCs in retinal neurons may function as synaptic noise suppressors at quiescent glutamatergic synapses.     

Allosteric voltage gating of potassium channels I. Mslo ionic currents in the absence of Ca(2+).

Activation of large conductance Ca(2+)-activated K(+) channels is controlled by both cytoplasmic Ca(2+) and membrane potential. To study the mechanism of voltage-dependent gating, we examined mSlo Ca(2+)-activated K(+) currents in excised macropatches from Xenopus oocytes in the virtual absence of Ca(2+) (<1 nM). In response to a voltage step, I(K) activates with an exponential time course, following a brief delay. The delay suggests that rapid transitions precede channel opening. The later exponential time course suggests that activation also involves a slower rate-limiting step. However, the time constant of I(K) relaxation [tau(I(K))] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. tau(I(K)) increases weakly with voltage from -500 to -20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = -0.29 e). Similarly, the steady state G(K)-V relationship exhibits a maximum voltage dependence (z = 2 e) from 0 to +100 mV, and is weakly voltage dependent (z congruent with 0.4 e) at more negative voltages, where P(o) = 10(-5)-10(-6). These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors. In the absence of Ca(2+), this allosteric mechanism results in a gating scheme with five closed (C) and five open (O) states, where the majority of the channel's voltage dependence results from rapid C-C and O-O transitions, whereas the C-O transitions are rate limiting and weakly voltage dependent. These conclusions not only provide a framework for interpreting studies of large conductance Ca(2+)-activated K(+) channel voltage gating, but also have important implications for understanding the mechanism of Ca(2+) sensitivity.     

R-Type Ca<Superscript>2+</Superscript>-channel Activity Is Associated with Chromogranin A Secretion in Human Neuroendocrine Tumor BON Cells

This electrophysiological study was undertaken to investigate the role of voltage-operated Ca(2+) channels (VOCCs) in cultivated human neuroendocrine tumor (NET) cells. Patch-clamp techniques, measurements of intracellular Ca(2+) ([Ca(2+)](i)), and secretion analysis were performed using cultured human NET BON cells. Ba(2+) inward currents through R-type channels (Ca(V)2.3) were measured and identified by SNX-482 (10 n M), a novel voltage-sensitive R-type Ca(2+) channel antagonist. In the presence of nifedipine (5 micro M), omega-Conotoxin GVIA (100 n M) and omega-Agatoxin IVA (20 n M), R-type channel currents were also detectable. Release of Ca(2+) from intracellular Ca(2+) stores by intracellular application of inositol-1,4,5-trisphosphate (InsP(3); 10 micro M) via the patch pipette during whole-cell configuration as well as induction of capacitative Ca(2+) entry (CCE), a passive maneuver to release Ca(2+) from intracellular Ca(2+) stores, led to an increase in [Ca(2+)](i). This effect could be reduced by SNX-482 (20 n M). In addition, SNX-482 (25 n M) also decreased chromogranin A (CgA) secretion, whereas omega-Conotoxin GVIA (500 n M) and nifedipine (5 micro M) failed to reduce CgA secretion. We conclude that these data reveal neuronal R-type channel activity (Ca(V)2.3), for the first time associated with CgA secretion in BON cells. Influx of Ca(2+) by activation of R-type channels may lead to an increase of intracellular Ca(2+), which stimulates CgA secretion. Thus, R-type channels could play an important role in certain clinical characteristics of NETs, such as the hypersecretion syndrome.     

Hyperpolarization, but not depolarization, increases intracellular Ca(2+) level in cultured chick myoblasts.

Ca(2+) influx appears to be important for triggering myoblast fusion. It remains, however, unclear how Ca(2+) influx rises prior to myoblast fusion. The present study examines a possible involvement of the voltage-dependent Ca(2+) influx pathways. Treatment with the L-type Ca(2+) channel blockers, diltiazem, and nifedipine did not alter cytosolic Ca(2+) levels. Depolarization with high K(+) solution and activation of Ca(2+) channel with Bay K 8644, and agonist of voltage dependent Ca(2+) channels, failed to elicit increases intracellular Ca(2+) level, indicating the absence of depolarization-operated mechanisms. In contrast, phloretin, an agonist of Ca(2+)-activated potassium (K(Ca)) channels, was able to hyperpolarize membrane potential and promoted Ca(2+) influx. These effects were completely abolished by treatment of charybdotoxin, a specific inhibitor of K(Ca) channels. In addition, gadolinium, a potent stretch-activated channel (SAC) blocker, prevented the phloretin-mediated Ca(2+) increase, indicating the involvement of SACs in Ca(2+) influx. Furthermore, phloretin stimulated precocious myoblast fusion and this effect was blocked with gadolinium or charybdotoxin. Taken together, these results suggest that induced hyperpolarization, but not depolarization increases Ca(2+) influx through stretch-activated channels, and in turn triggers myoblast fusion.     

The voltage-sensitive release mechanism of excitation contraction coupling in rabbit cardiac muscle is explained by calcium-induced calcium release

The putative voltage-sensitive release mechanism (VSRM) was investigated in rabbit cardiac myocytes at 37 degrees C with high resistance microelectrodes to minimize intracellular dialysis. When the holding potential was adjusted from -40 to -60 mV, the putative VSRM was expected to operate alongside CICR. Under these conditions however, we did not observe a plateau at positive potentials of the cell shortening versus voltage relationship. The threshold for cell shortening changed by -10 mV, but this resulted from a similar change of the threshold for activation of inward current. Cell shortening under conditions where the putative VSRM was expected to operate was blocked in a dose dependent way by nifedipine and CdCl2 and blocked completely by NiCl2. "Tail contractions" persisted in the presence of nifedipine and CdCl2 but were blocked completely by NiCl2. Block of early outward current by 4-aminopyridine and 4-acetoamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS) demonstrated persisting inward current during test depolarizations despite the presence of nifedipine and CdCl2. Inward current did not persist in the presence of NiCl2. A tonic component of cell shortening that was prominent during depolarizations to positive potentials under conditions selective for the putative VSRM was sensitive to rapidly applied changes in superfusate [Na+] and to the outward Na+/Ca2+ exchange current blocking drug KB-R7943. This component of cell shortening was thought to be the result of Na+/Ca2+ exchange-mediated excitation contraction coupling. Cell shortening recorded under conditions selective for the putative VSRM was increased by the enhanced state of phosphorylation induced by isoprenaline (1 microM) and by enhancing sarcoplasmic reticulum Ca2+ content by manipulation of the conditioning steps. Under these conditions, cell shortening at positive test depolarizations was converted from tonic to phasic. We conclude that the putative VSRM is explained by CICR with the Ca2+ "trigger" supplied by unblocked L-type Ca2+ channels and Na+/Ca2+ exchange.     

Effects of (-)-epigallocatechin-3-gallate in Ca2+ -permeable non-selective cation channels and voltage-operated Ca2+ channels in vascular smooth muscle cells

The effects of (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of tea, on Ca(2+)-permeable non-selective cation currents (NSCC) and voltage-operated Ca(2+) channels (VOCC) have been investigated in cultured rat aortic smooth muscle cells using the whole-cell voltage-clamp technique. Under the Cs(+)/tetraethylammonium (TEA)-containing internal solution, and in the presence of nifedipine (1 microM), EGCG (30 microM) activated a long-lasting inward current, with a reversal potential (E(rev)) of approximately 0 mV. This current was not significantly altered by the replacement of [Cl(-)](i) or [Cl(-)](o), implying that the inward current was not a chloride channel, but a NSCC. SKF 96365 (30 microM) and Cd(2+) (500 microM) almost completely abolished the EGCG-induced NSCC. A higher dose of EGCG (100 microM) additionally activated a nifedipine-sensitive inward current in the absence of depolarization protocol. EGCG (100 microM) also potentiated a nifedipine-sensitive voltage-dependent Ba(2+)-current during the first 5 min of incubation. However, after > 10 min of incubation with EGCG, this current was significantly inhibited. Our results suggest that EGCG caused a Ca(2+) influx into smooth muscle cells via VOCC (probably L-type) and other SKF-96365- and Cd(2+)-sensitive Ca(2+)-permeable channels. The action described here may be responsible for the contraction induced by EGCG in rat aortic rings and for the rise of the intracellular concentration of Ca(2+) in rat aortic smooth muscle cells evoked by this catechin. On the other hand, the inhibition of VOCC after > 10 min of incubation may be, in part, responsible for the relaxation of rat aorta induced by EGCG.     

A store-operated calcium channel in Drosophila S2 cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ > Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.     

Functional coupling of Ca(2+) channels to ryanodine receptors at presynaptic terminals. Amplification of exocytosis and plasticity

Ca(2+)-induced Ca(2+) release (CICR) enhances a variety of cellular Ca(2+) signaling and functions. How CICR affects impulse-evoked transmitter release is unknown. At frog motor nerve terminals, repetitive Ca(2+) entries slowly prime and subsequently activate the mechanism of CICR via ryanodine receptors and asynchronous exocytosis of transmitters. Further Ca(2+) entry inactivates the CICR mechanism and the absence of Ca(2+) entry for >1 min results in its slow depriming. We now report here that the activation of this unique CICR markedly enhances impulse-evoked exocytosis of transmitter. The conditioning nerve stimulation (10-20 Hz, 2-10 min) that primes the CICR mechanism produced the marked enhancement of the amplitude and quantal content of end-plate potentials (EPPs) that decayed double exponentially with time constants of 1.85 and 10 min. The enhancement was blocked by inhibitors of ryanodine receptors and was accompanied by a slight prolongation of the peak times of EPP and the end-plate currents estimated from deconvolution of EPP. The conditioning nerve stimulation also enhanced single impulse- and tetanus-induced rises in intracellular Ca(2+) in the terminals with little change in time course. There was no change in the rate of growth of the amplitudes of EPPs in a short train after the conditioning stimulation. On the other hand, the augmentation and potentiation of EPP were enhanced, and then decreased in parallel with changes in intraterminal Ca(2+) during repetition of tetani. The results suggest that ryanodine receptors exist close to voltage-gated Ca(2+) channels in the presynaptic terminals and amplify the impulse-evoked exocytosis and its plasticity via CICR after Ca(2+)-dependent priming.     

RYR2 proteins contribute to the formation of Ca(2+) sparks in smooth muscle

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca(2+) release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca(2+) release events (Ca(2+) sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca(2+) release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca(2+) sparks, Ca(2+)-induced Ca(2+) release, and Ca(2+) waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6(-/-), and RYR3(-/-) mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca(2+) sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca(2+) sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca(2+) -induced Ca(2+) release was similarly augmented in FKBP12.6(-/-), but not in RYR3 null cells relative to wild-type. Finally, Ca(2+) wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca(2+) wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca(2+) sparks in smooth muscle, and the finding of normal Ca(2+) sparks and CICR in RYR3 null mice, indicate that Ca(2+) release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca(2+) sparks, and associated STOCs, in smooth muscle.     

Functional triads consisting of ryanodine receptors, Ca(2+) channels, and Ca(2+)-activated K(+) channels in bullfrog sympathetic neurons. Plastic modulation of action potential

Fluorescent ryanodine revealed the distribution of ryanodine receptors in the submembrane cytoplasm (less than a few micrometers) of cultured bullfrog sympathetic ganglion cells. Rises in cytosolic Ca(2+) ([Ca(2+)](i)) elicited by single or repetitive action potentials (APs) propagated at a high speed (150 microm/s) in constant amplitude and rate of rise in the cytoplasm bearing ryanodine receptors, and then in the slower, waning manner in the deeper region. Ryanodine (10 microM), a ryanodine receptor blocker (and/or a half opener), or thapsigargin (1-2 microM), a Ca(2+)-pump blocker, or omega-conotoxin GVIA (omega-CgTx, 1 microM), a N-type Ca(2+) channel blocker, blocked the fast propagation, but did not affect the slower spread. Ca(2+) entry thus triggered the regenerative activation of Ca(2+)-induced Ca(2+) release (CICR) in the submembrane region, followed by buffered Ca(2+) diffusion in the deeper cytoplasm. Computer simulation assuming Ca(2+) release in the submembrane region reproduced the Ca(2+) dynamics. Ryanodine or thapsigargin decreased the rate of spike repolarization of an AP to 80%, but not in the presence of iberiotoxin (IbTx, 100 nM), a BK-type Ca(2+)-activated K(+) channel blocker, or omega-CgTx, both of which decreased the rate to 50%. The spike repolarization rate and the amplitude of a single AP-induced rise in [Ca(2+)](i) gradually decreased to a plateau during repetition of APs at 50 Hz, but reduced less in the presence of ryanodine or thapsigargin. The amplitude of each of the [Ca(2+)](i) rise correlated well with the reduction in the IbTx-sensitive component of spike repolarization. The apamin-sensitive SK-type Ca(2+)-activated K(+) current, underlying the afterhyperpolarization of APs, increased during repetitive APs, decayed faster than the accompanying rise in [Ca(2+)](i), and was suppressed by CICR blockers. Thus, ryanodine receptors form a functional triad with N-type Ca(2+) channels and BK channels, and a loose coupling with SK channels in bullfrog sympathetic neurons, plastically modulating AP.     

Remodeling excitation-contraction coupling of hypertrophied ventricular myocytes is dependent on T-type calcium channels expression

We utilized Wistar rats with monocrotaline (MCT)-induced right ventricular hypertrophy (RVH) in order to evaluate the T-type Ca2+ channel current (ICaT) for myocardial contraction. RT-PCR provides that mRNA for T-type Ca2+ channel alpha1-subunits in hypertrophied myocytes was significantly higher than those in control rats (alpha1G; 264+/-36%, alpha1H; 191+/-34%; P<0.05). By whole-cell patch-clamp study, ICaT was recorded only in hypertrophied myocytes but not in control myocytes. The application of 50 nmol/L nifedipine reduced the twitch tension of the right ventricles equally in the control and RVH rats. On the other hand, 0.5 micromol/L mibefradil, a T-type Ca2+ channel blocker, strongly inhibited the twitch tension of the RVH muscle (control 6.4+/-0.8% vs. RVH 20.0+/-2.3% at 5 Hz; P<0.01). In conclusion, our results indicate the functional expression of T-type Ca2+ channels in the hypertrophied heart and their contribution to the remodeling of excitation-contraction coupling in the cardiac myocyte.     

Protein kinase A and C are "Gatekeepers" of capacitative Ca2+ entry in the prothoracic gland cells of the silkworm, Bombyx mori

Application of protein kinases A and C inhibitors to the prothoracic glands cells of the silkworm, Bombyx mori, resulted in slow and gradual increases in intracellular Ca(2+) ([Ca(2+)](i)). Pharmacological manipulation of the Ca(2+) signalling cascades in the prothoracic gland cells of B. mori suggests that these increases of [Ca(2+)](i) are mediated neither by voltage-gated Ca(2+) channels nor by intracellular Ca(2+) stores. Rather they result from slow Ca(2+) leak from plasma membrane Ca(2+) channels that are sensitive to agents that inhibit capacitative Ca(2+) entry and are abolished in the absence of extracellular Ca(2+). Okadaic acid, an inhibitor of PP1 and PP2A phosphatases, blocked the increase in [Ca(2+)](i) produced by the inhibitors of protein kinase A and C. The combined results indicate that the capacitative Ca(2+) entry channels in prothoracic gland cells of B. mori are probably modulated by protein kinases A and C.     

Location of the permeation pathway in the recombinant type 1 inositol 1,4,5-trisphosphate receptor.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) forms ligand-regulated intracellular Ca(2+) release channels in the endoplasmic reticulum of all mammalian cells. The InsP(3)R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP(3)R pore. Mutant InsP(3)Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; P(Ca)/P(Cs) = 6.3). These mutant channels bound InsP(3), but ligand occupancy did not regulate the constitutively open pore (P(o) > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398-2589) near the COOH terminus of the protein forms the InsP(3)R pore. Further, we have produced a constitutively open InsP(3)R pore mutant that is ideal for future site-directed mutagenesis studies of the structure-function relationships that define Ca(2+) permeation through the InsP(3)R channel.