The regulation of L-type Ca2+ currents in rat cardiac myocytes

The regulation of L-type Ca2+ current in isolated rat cardiac cells was studied using the perforated patch-clamp technique. A dual effect of the cAMP-dependent phosphorylation activator, isoproterenol, at different holding potentials (V(h)) was shown. The currents increased at V(h) = -50 mV and decreased at V(h) = -30 mV. A dihydropyridine agonist, BAY K 8644, and isoproterenol had an additive effect on the activation of Ca2+ channels at holding potentials close to the resting potential. The additivity was disturbed at more positive V(h). The activating effect of BAY K 8644 did not virtually change in the presence of a protein kinase blocker, H8, and a phosphatase activator, acetylcholine. The results were interpreted within the framework of a two-site phosphorylation model with two independent pathways of Ca2+ current regulation.     

The regulation of L-type Ca2+ currents in cardiac myocytes of hibernating animals

The action of isoproterenol and BAY K 8644 on voltage-dependent Ca2+ currents in isolated ground squirrel cardiac myocytes was studied in two (active and hibernating) states of the animal. In cardiac myocytes of active animals the effect of both drugs was shown to depend on the holding potential. At Vh of about -50 mV both isoproterenol and BAY K 8644 increased the Ca2+ current and their action was additive. At Vh of about -20 mV, both drugs inhibited the Ca2+ current. In cardiac myocytes from hibernating animals, isoproterenol increased the Ca2+ current at any holding potentials, while the effect of BAY K 8644 did not differ significantly from its effect on active animals. The combined action of the two drugs caused the inhibition of the Ca2+ current at high holding potentials. In terms of the two-site Ca2+ channel model, this means that one of the two pathways of channel phosphorylation is blocked in hibernating animal cardiac cells, and BAY K 8644 restores this pathway.     

RhoA GTPase regulates L-type Ca2+ currents in cardiac myocytes

Regulation of ionic channels plays a pivotal role in controlling cardiac function. Here we show that the Rho family of small G proteins regulates L-type Ca2+ currents in ventricular cardiomyocytes. Ventricular myocytes isolated from transgenic (TG) mice that overexpress the specific GDP dissociation inhibitor Rho GDI-alpha exhibited significantly decreased basal L-type Ca2+ current density (approximately 40%) compared with myocytes from nontransgenic (NTG) mice. The Ca2+ channel agonist BAY K 8644 and the beta-adrenergic agonist isoproterenol increased Ca2+ currents in both NTG and TG myocytes to a similar maximal level, and no changes in mRNA or protein levels were observed in the Ca2+ channel alpha1-subunits. These results suggest that the channel activity but not the expression level was altered in TG myocytes. In addition, the densities of inward rectifier and transient outward K+ currents were unchanged in TG myocytes. The amplitudes and rates of basal twitches and Ca2+ transients were also similar between the two groups. When the protein was delivered directly into adult ventricular myocytes via TAT-mediated protein transduction, Rho GDI-alpha significantly decreased Ca2+ current density, which supports the idea that the defective Ca2+ channel activity in TG myocytes was a primary effect of the transgene. In addition, expression of a dominant-negative RhoA but not a dominant-negative Rac-1 or Cdc42 also significantly decreased Ca2+ current density, which indicates that inhibition of Ca2+ channel activity by overexpression of Rho GDI-alpha is mediated by inhibition of RhoA. This study points to the L-type Ca2+ channel activity as a novel downstream target of the RhoA signaling pathway.     

Ca2+ entry-independent effects of L-type Ca2+ channel modulators on Ca2+ sparks in ventricular myocytes

During the cardiac action potential, Ca²⁺ entry through dyhidropyridine receptor L-type Ca²⁺ channels (DHPRs) activates ryanodine receptors (RyRs) Ca²⁺-release channels, resulting in massive Ca²⁺ mobilization from the sarcoplasmic reticulum (SR). This global Ca²⁺ release arises from spatiotemporal summation of many localized elementary Ca²⁺-release events, Ca²⁺ sparks. We tested whether DHPRs modulate Ca²⁺sparks in a Ca²⁺ entry-independent manner. Negative modulation by DHPR of RyRs via physical interactions is accepted in resting skeletal muscle but remains controversial in the heart. Ca²⁺ sparks were studied in cat cardiac myocytes permeabilized with saponin or internally perfused via a patch pipette. Bathing and pipette solutions contained low Ca²⁺ (100 nM). Under these conditions, Ca²⁺ sparks were detected with a stable frequency of 3–5 sparks·s^–1·100 µm^–1. The DHPR blockers nifedipine, nimodipine, FS-2, and calciseptine decreased spark frequency, whereas the DHPR agonists Bay-K8644 and FPL-64176 increased it. None of these agents altered the spatiotemporal characteristics of Ca²⁺ sparks. The DHPR modulators were also without effect on SR Ca²⁺ load (caffeine-induced Ca²⁺ transients) or sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) activity (Ca²⁺ loading rates of isolated SR microsomes) and did not change cardiac RyR channel gating (planar lipid bilayer experiments). In summary, DHPR modulators affected spark frequency in the absence of DHPR-mediated Ca²⁺ entry. This action could not be attributed to a direct action of DHPR modulators on SERCA or RyRs. Our results suggest that the activity of RyR Ca²⁺-release units in ventricular myocytes is modulated by Ca²⁺ entry-independent conformational changes in neighboring DHPRs. exitation-contraction coupling; ryanodine receptor; sarco(endo)plasmic reticulum Ca²⁺-ATPase; dihydropyridine receptor; sarcoplasmic reticulum     

Temperature-sensitive intracellular Mg2+ block of L-type Ca2+ channels in cardiac myocytes

We examined the concentration-dependent blocking effects of intracellular Mg2+ on L-type Ca2+ channels in cardiac myocytes using the whole cell patch-clamp technique. The increase of L-type Ca2+ channel current (I(Ca)) (due to relief of Mg2+ block) occurred in two temporal phases. The rapid phase (runup) transiently appeared early (<5 min) in dialysis of the low-Mg2+ solution; the slow phase began later in dialysis (>10 min). Runup was not blocked by intracellular GTP (GTP(i)). The late phase of the I(Ca) increase (late I(Ca)) was suppressed by GTP(i) (0.4 mM) and was observed in myocytes of the guinea pig or frog at higher (32 or 24 degrees C, respectively) rather than lower temperatures (24 or 17.5 degrees C, respectively). At pMg = 6.0, raising the temperature from 24 to 32 degrees C evoked late I(Ca) with a Q10 of 14.5. Restoring the temperature to 24 degrees C decreased I(Ca) with a Q10 of only 2.4. The marked difference in the Q10 values indicated that late I(Ca) (pMg = 5-6) is an irreversible phenomenon. Phosphorylation suppressed the intracellular [Mg2+] dependency of late I(Ca). This effect of phosphorylation together with the inhibitory action of GTP(i) on Mg2+-dependent blocking of I(Ca) are common properties of mammalian and amphibian cardiomyocytes.     

Differential effects of pertussis toxin on the muscarinic regulation of Ca2+ and K+ currents in frog cardiac myocytes

The ability of acetylcholine (ACh) to inhibit beta-agonist stimulated calcium current was compared to its ability to activate the inwardly rectifying potassium current IK(ACh) in frog atrial myocytes. As suggested by previous studies, ACh inhibited the calcium current at concentrations (EC50 = 8 nM) significantly lower than those required for the activation of IK(ACh) (EC50 = 101 nM). The pharmacological profiles of the two responses suggest that despite the differences in agonist sensitivity, both are mediated by the same (m2) type of muscarinic receptors. Intracellular application of GDP beta S, an inhibitor of G protein function, completely abolished both responses, implying that both actions of ACh are coupled to effectors by G proteins. In contrast, intracellular application of pertussis toxin (PTX) shifted to higher concentrations (EC50 = 170 nM) but did not abolish inhibition of the calcium current by ACh even though the block of the IK(ACh) response was complete. Increasingly large PTX concentrations and/or prolonged PTX treatments revealed a limiting, PTX- resistant inhibitory component that appears to be mediated by a PTX- insensitive G protein distinct from that mediating IK(ACh). For the PTX- sensitive components, the different agonist dependencies of IK(ACh) activation and calcium current inhibition may imply that different G proteins mediate each response although alternate possibilities involving the same G protein either functionally sequestered and/or differentially affected by interactions with effectors, can not be ruled out.     

Decrease in the density of t-tubular L-type Ca2+ channel currents in failing ventricular myocytes

In some forms of cardiac hypertrophy and failure, the gain of Ca(2+)-induced Ca(2+) release [CICR; i.e., the amount of Ca(2+) released from the sarcoplasmic reticulum normalized to Ca(2+) influx through L-type Ca(2+) channels (LTCCs)] decreases despite the normal whole cell LTCC current density, ryanodine receptor number, and sarcoplasmic reticulum Ca(2+) content. This decrease in CICR gain has been proposed to arise from a change in dyad architecture or derangement of the t-tubular (TT) structure. However, the activity of surface sarcolemmal LTCCs has been reported to increase despite the unaltered whole cell LTCC current density in failing human ventricular myocytes, indicating that the "decreased CICR gain" may reflect a decrease in the TT LTCC current density in heart failure. Thus, we analyzed LTCC currents of failing ventricular myocytes of mice chronically treated with isoproterenol (Iso). Although Iso-treated mice exhibited intact t-tubules and normal LTCC subunit expression, acute occlusion of t-tubules of isolated ventricular myocytes with osmotic shock (detubulation) revealed that the TT LTCC current density was halved in Iso-treated versus control myocytes. Pharmacological analysis indicated that kinases other than PKA or Ca(2+)/calmodulin-dependent protein kinase II insufficiently activated, whereas protein phosphatase 1/2A excessively suppressed, TT LTCCs in Iso-treated versus control myocytes. These results indicate that excessive β-adrenergic stimulation causes the decrease in TT LTCC current density by altering the regulation of TT LTCCs by protein kinases and phosphatases in heart failure. This phenomenon might underlie the decreased CICR gain in heart failure.     

Functional coupling of voltage-dependent L-type Ca2+ current to Ca2+-activated K+ current in pituitary GH3 cells

Ca2+-activated K+ currents (I(K(Ca)) can contribute to action potential repolarization and after-hyperpolarization in GH3 cells. In this study, we examined how the activation of I(K(Ca) at the cellular level could be functionally coupled to Ca2+ influx through L-type Ca2+ channels. A 30-msec Ca2+ influx step to 0 mV was found to exhibit substantial contribution of Ca2+ influx through the activation of I(Ca,L) to the activation of I(K(Ca)). A bell-shaped relationship between the conditioning potentials and the integrated I(K(Ca)) was observed, suggesting that the magnitude of integrated I(Ca,L) correlates well with that of integrated I(K(Ca)) in the same cell. A linear relationship of integrated I(Ca,L) and integrated I(K(Ca)) was found with a coupling ratio of 69+/-7. The value of the coupling ratio was unaffected by the presence of Bay K 8644 or nimodipine, although these compounds could effectively affect the amplitudes of both I(K(Ca)) and I(Ca,L). However, tetrandrine could decrease the coupling ratio. Paxilline or intracellular Ca2+ buffer with EGTA decreased the coupling ratio, while apamin had no effect on it. Interestingly, phorbol 12-myristate 13-acetate also reduced the coupling ratio significantly, whereas thapsigargin increased this value. Thus, the present study indicates that the activation of I(K(Ca)) during brief Ca2+ influx, which is inhibited by paxilline, is coupled to Ca2+ influx primarily through the L-type channels. The selective modulation of I(K(Ca)) by second messengers or Ca2+ release from internal stores may affect the coupling efficiency and hence cellular excitability.     

L-type and T-type Ca2+ current in cultured ventricular guinea pig myocytes

The aim of this investigation was to study L-type and T-type Ca(2+) current (I(CaL) and I(CaT)) in short-term cultured adult guinea pig ventricular myocytes. The isolated myocytes were suspended in serum-supplemented medium up to 5 days. Using whole-cell patch clamp techniques ICaL and ICaT were studied by applying voltage protocols from different holding potentials (-40 and -90 mV). After 5 days in culture the myocytes still showed their typical rod shaped morphology but a decline in cell membrane capacitance (26 %). The peak density of ICaT was reduced significantly between day 0 (-1.6+/-0.37 pA/pF, n=9) and day 5 (-0.4+/-0.13 pA/pF, n=11), whereas peak ICaL density revealed no significant differences during culturing. The I(CaT)/I(CaL) ratio dropped from 0.13 at day 0 to 0.05 at day 5. Compared with day 0 I(CaL) the steady state inactivation curve of day 1, day 3 and day 5 myocytes was slightly shifted to more negative potentials. Our data indicate that guinea pig ventricular L-type and T-type Ca(2+) channels are differently regulated in culture.     

Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

Ca2+ sparks are the elementary events of intracellular Ca2+ release from the sar-coplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca2+ channel activation contributes to the genesis of spontaneous Ca2+ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca2+ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI2 which inhibits spontaneous L-type Ca2+ channel activation, the rate of occurrence of spontaneous Ca2+ sparks was halved from 4.20 to 2.04 events/(100 μm·s), with temporal and spatial properties of individual Ca2+ sparks unchanged. Analysis of the Cd2+-sensitive spark production revealed an open probability of-10-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca2+ channels in resting heart cells contribute significantly to the production of spontaneous Ca2+ sparks.     

L-type Ca2+ channels access multiple open states to produce two components of Bay K 8644-dependent current in GH3 cells

To determine the number of L-channel populations responsible for producing the two components of whole-cell L-type Ca2+ channel current revealed by Bay K 8644 (Fass, D.M., and E.S. Levitan. 1996. J. Gen. Physiol. 108:1-11), L-type Ca2+ channel activity was recorded in cell- attached patches. Ensemble tail currents from most (six out of nine) single-channel patches had double-exponential time courses, with time constants that were similar to whole-cell tail current decay values. Also, in single-channel patches subjected to two different levels of depolarization, ensemble tail currents exactly reproduced the voltage dependence of activation of the two whole-cell components: The slow component is activated at more negative potentials than the fast component. In addition, deactivation of Bay K 8644-modified whole-cell L-current was slower after long (100-ms) depolarizations than after short (20-ms) depolarizations, and this phenomenon was also evident in ensemble tail currents from single L-channels. Thus, a single population of L-channels can produce the two components of macroscopic L-current deactivation. To determine how individual L-channels produce multiple macroscopic tail current components, we constructed ensemble tail currents from traces that contained a single opening upon repolarization and no reopenings. These ensemble tails were biexponential. This type of analysis also revealed that reopenings do not contribute to the slowing of tail current deactivation after long depolarizations. Thus, individual L-channels must have access to several open states to produce multiple macroscopic current components. We also obtained evidence that access to these open states can vary over time. Use of several open states may give L-channels the flexibility to participate in many cell functions.     

Activation of L-type Ca2+ currents in cardiac myocytes by photoreleased GTP.

L-type calcium currents (ICa) were recorded from isolated ventricular myocytes by using standard patch-clamp methods. In the absence of agonist, photorelease of GTP by flash photolysis of intracellularly applied caged-GTP rapidly increased the amplitude of ICa over a wide range of membrane potentials. Control experiments clearly demonstrated that this effect was not due to either the release of photolytic by-products or to the light flash itself. The timecourse for activation of ICa by photolysis of caged-GTP was markedly altered by intracellular application of either GDP beta S or GTP gamma S. Upon maximal stimulation of ICa by intracellular dialysis with cAMP, photoreleased GTP induced a small, rapid increase in ICa followed by a gradual inhibition. The presence of Rp-cAMPS intracellularly reduced both the magnitude of the response to photoreleased GTP and its time to peak. Similar effects were observed when protein kinase inhibitor dialysed the cell interior, suggesting that both cAMP-dependent and independent processes were involved in this effect. We conclude that rapid release of GTP within ventricular myocytes, in the absence of agonist, causes rapid activation of L-type Ca2+ current. Mechanisms underlying this effect include stimulation of adenylate cyclase, together with other, as yet uncharacterized, GTP-dependent pathways for increasing ICa in the heart.     

Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes

We investigatedthe relationship between voltage-operatedCa²⁺ channel current and thecorresponding intracellular Ca²⁺concentration([Ca²⁺]_i)change (Ca²⁺ transient) in guineapig gastric myocytes. Fluorescence microspectroscopy was combined withconventional whole cell patch-clamp technique, and fura 2 (80 µM) wasadded to CsCl-rich pipette solution. Step depolarization to 0 mVinduced inward Ca²⁺ current(I_Ca) andconcomitantly raised[Ca²⁺]_i.Both responses were suppressed by nicardipine, an L-typeCa²⁺ channel blocker, and thevoltage dependence of Ca²⁺transient was similar to the current-voltage relation ofI_Ca. When pulseduration was increased by up to 900 ms, peakCa²⁺ transient increased andreached a steady state when stimulation was for longer. The calculatedfast Ca²⁺ buffering capacity(B value), determined as the ratio ofthe time integral ofI_Ca divided bythe amplitude of Ca²⁺ transient,was not significantly increased after depletion of Ca²⁺ stores by the cyclicapplication of caffeine (10 mM) in the presence of ryanodine (4 µM).The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmicreticulum Ca²⁺-ATPase inhibitor,decreased B value by ~20% in areversible manner. When KCl pipette solution was used,Ca²⁺-activatedK⁺ current[I_K(Ca)]was also recorded during step depolarization. CPA sensitivelysuppressed the initial peak and oscillations of I_K(Ca) withirregular effects on Ca²⁺transients. The above results suggest that, in guinea pig gastric myocyte, Ca²⁺ transient is tightlycoupled to I_Caduring depolarization, and global[Ca²⁺]_iis not significantly affected byCa²⁺-inducedCa²⁺ release from sarcoplasmicreticulum during depolarization.

     

Bay K8644及nifedipine对大鼠下丘脑神经元L—型Ca^2＋通道特性的影响

采用神经元急性分离和膜片箍技术以及细胞贴附式方式记录通道活动,探讨DHP类Ca^2 通道激动剂Bay K8644及拮抗剂nifedipine对下丘脑神经元L－型Ca^2 通道的影响,结果显示,在Bay K8644作用下,通道开放形式发生变化,明显可见多级开放;通道平均开放时间,平均开放概况显著增加,但单通道电导无明显变化。nifedipine的作用与Bay K8644相反。结果提示,Bay K8644对下丘脑神经元L－型Ca^2 通道有明显激动作用 nifedipine有显著抑制作用。     

Voltage-independent changes in L-type Ca(2+) current uncoupled from SR Ca(2+) release in cardiac myocytes

To determine the effect of voltage-independent alterations of L-type Ca(2+) current (I(Ca)) on the sarcoplasmic reticular (SR) Ca(2+) release in cardiac myocytes, we measured I(Ca) and cytosolic Ca(2+) transients (Ca(i)(2+); intracellular Ca(2+) concentration) in voltage-clamped rat ventricular myocytes during 1) an abrupt increase of extracellular [Ca(2+)] (Ca(o)(2+)) or 2) application of 1 microM FPL-64176, a Ca(2+) channel agonist, to selectively alter I(Ca) in the absence of changes in SR Ca(2+) loading. On the first depolarization in higher Ca(o)(2+), peak I(Ca) was increased by 46 +/- 6% (P < 0.001), but the increases in the maximal rate of rise of Ca(i)(2+) (dCa(i)(2+)/dt(max), where t is time; an index of SR Ca(2+) release flux) and the Ca(i)(2+) transient amplitude were not significant. Rapid exposure to FPL-64176 greatly slowed inactivation of I(Ca), increasing its time integral by 117 +/- 8% (P < 0.001) without significantly increasing peak I(Ca), dCa(i)(2+)/dt(max), or amplitude of the corresponding Ca(i)(2+) transient. Prolongation of exposure to higher Ca(o)(2+) or FPL-64176 did not further increase peak I(Ca) but greatly increased dCa(i)(2+)/dt(max), Ca(i)(2+) transient amplitude, and the gain of Ca(2+) release (dCa(i)(2+)/dt(max)/I(Ca)), evidently due to augmentation of the SR Ca(2+) loading. Also, the time to peak dCa(i)(2+)/dt(max) was significantly increased in the continuous presence of higher Ca(o)(2+) (by 37 +/- 5%, P < 0.001) or FPL-64176 (by 63 +/- 5%, P < 0.002). Our experiments provide the first evidence of a marked disparity between an increased peak I(Ca) and the corresponding SR Ca(2+) release. We attribute this to saturation of the SR Ca(2+) release flux as predicted by local control theory. Prolongation of the SR Ca(2+) release flux, caused by combined actions of a larger I(Ca) and maximally augmented SR Ca(2+) loading, might reflect additional Ca(2+) release from corbular SR.     

The mitochondrial Ca2+-activated K+ channel activator, NS 1619 inhibits L-type Ca2+ channels in rat ventricular myocytes

We examined the effects of the mitochondrial Ca(2+)-activated K(+) (mitoBK(Ca)) channel activator NS 1619 on L-type Ca(2+) channels in rat ventricular myocytes. NS 1619 inhibited the Ca(2+) current in a dose-dependent manner. NS 1619 shifted the activation curve to more positive potentials, but did not have a significant effect on the inactivation curve. Pretreatment with inhibitors of membrane BK(Ca) channel, mitoBK(Ca) channel, protein kinase C, protein kinase A, and protein kinase G had little effect on the Ca(2+) current and did not alter the inhibitory effect of NS 1619 significantly. The application of additional NS 1619 in the presence of isoproterenol, a selective beta-adrenoreceptor agonist, reduced the Ca(2+) current to approximately the same level as a single application of NS 1619. In conclusion, our results suggest that NS 1619 inhibits the Ca(2+) current independent of the mitoBK(Ca) channel and protein kinases. Since NS 1619 is widely used to study mitoBK(Ca) channel function, it is essential to verify these unexpected effects of NS 1619 before experimental data can be interpreted accurately.     

Serotonin increases L-type Ca2+ current and SR Ca2+ content through 5-HT4 receptors in failing rat ventricular cardiomyocytes

Rats with congestive heart failure (CHF) develop ventricular inotropic responsiveness to serotonin (5-HT), mediated through 5-HT(2A) and 5-HT(4) receptors. Human ventricle is similarly responsive to 5-HT through 5-HT(4) receptors. We studied isolated ventricular cardiomyocytes to clarify the effects of 5-HT on intracellular Ca(2+) handling. Left-ventricular cardiomyocytes were isolated from male Wistar rats 6 wk after induction of postinfarction CHF. Contractile function and Ca(2+) transients were measured in field-stimulated cardiomyocytes, and L-type Ca(2+) current (I(Ca,L)) and sarcoplasmic reticulum (SR) Ca(2+) content were measured in voltage-clamped cells. Protein phosphorylation was measured by Western blotting or phosphoprotein gel staining. 5-HT(4)- and 5-HT(2A)-receptor stimulation induced a positive inotropic response of 33 and 18% (both P < 0.05) and also increased the Ca(2+) transient (44 and 6%, respectively; both P < 0.05). I(Ca,L) and SR Ca(2+) content increased only after 5-HT(4)-receptor stimulation (57 and 65%; both P < 0.05). Phospholamban serine(16) (PLB-Ser(16)) and troponin I phosphorylation increased by 26 and 13% after 5-HT(4)-receptor stimulation (P < 0.05). 5-HT(2A)-receptor stimulation increased the action potential duration and did not significantly change the phosphorylation of PLB-Ser(16) or troponin I, but it increased myosin light chain 2 (MLC2) phosphorylation. In conclusion, the positive inotropic response to 5-HT(4) stimulation results from increased I(Ca,L) and increased phosphorylation of PLB-Ser(16), which increases the SR Ca(2+) content. 5-HT(4) stimulation is thus, like beta-adrenoceptor stimulation, possibly energetically unfavorable in CHF. 5-HT(2A)-receptor stimulation, previously studied in acute CHF, induces a positive inotropic response also in chronic CHF, probably mediated by MLC2 phosphorylation.