Local Ca2+ entry through L-type Ca2+ channels activates Ca2+-dependent K+ channels in rabbit coronary myocytes.

Large-conductance Ca2+-dependent K+ channels (KCa), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca2+ entry through L-type Ca2+ channels (ICaL). We hypothesize that local accumulation of subsarcolemmal Ca2+ during ICaL openings amplifies this feedback. Our goal was to demonstrate that Ca2+ entry through voltage-gated ICaL channels can stimulate adjacent KCa channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward KCa current that is activated by a subsarcolemmal Ca2+ pool dissociated from bulk cytosolic Ca2+ pool (measured with indo 1) and is dependent on L-type Ca2+ channel activity. Transient activation of unitary KCa channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, -40 mV; 219 pS in near-symmetrical K+). This local interaction between the channels required the presence of Ca2+ in the pipette solution, was enhanced by the ICaL agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 micromol/L ryanodine and 30 micromol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single KCa (67 pS) and ICaL (3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle.     

Ca2+ influx through T- and L-type Ca2+ channels have different effects on myocyte contractility and induce unique cardiac phenotypes

T-type Ca(2+) channels (TTCCs) are expressed in the developing heart, are not present in the adult ventricle, and are reexpressed in cardiac diseases involving cardiac dysfunction and premature, arrhythmogenic death. The goal of this study was to determine the functional role of increased Ca(2+) influx through reexpressed TTCCs in the adult heart. A mouse line with cardiac-specific, conditional expression of the alpha1G-TTCC was used to increase Ca(2+) influx through TTCCs. alpha1G hearts had mild increases in contractility but no cardiac histopathology or premature death. This contrasts with the pathological phenotype of a previously studied mouse with increased Ca(2+) influx through the L-type Ca(2+) channel (LTCC) secondary to overexpression of its beta2a subunit. Although alpha1G and beta2a myocytes had similar increases in Ca(2+) influx, alpha1G myocytes had smaller increases in contraction magnitude, and, unlike beta2a myocytes, there were no increases in sarcoplasmic reticulum Ca(2+) loading. Ca(2+) influx through TTCCs also did not induce normal sarcoplasmic reticulum Ca(2+) release. alpha1G myocytes had changes in LTCC, SERCA2a, and phospholamban abundance, which appear to be adaptations that help maintain Ca(2+) homeostasis. Immunostaining suggested that the majority of alpha1G-TTCCs were on the surface membrane. Osmotic shock, which selectively eliminates T-tubules, induced a greater reduction in L- versus TTCC currents. These studies suggest that T- and LTCCs are in different portions of the sarcolemma (surface membrane versus T-tubules) and that Ca(2+) influx through these channels induce different effects on myocyte contractility and lead to distinct cardiac phenotypes.     

Molecular coupling of a Ca2+-activated K+ channel to L-type Ca2+ channels via alpha-actinin2

Cytoskeletal proteins are known to sculpt the structural architecture of cells. However, their role as bridges linking the functional crosstalk of different ion channels is unknown. Here, we demonstrate that a small conductance Ca(2+)-activated K(+) channels (SK2 channel), present in a variety of cells, where they integrate changes in intracellular Ca(2+) concentration [Ca(2+)(i)] with changes in K(+) conductance and membrane potential, associate with L-type Ca(2+) channels; Ca(v)1.3 and Ca(v)1.2 through a physical bridge, alpha-actinin2 in cardiac myocytes. SK2 channels do not physically interact with L-type Ca(2+) channels, instead, the 2 channels colocalize via their interaction with alpha-actinin2 cytoskeletal protein. The association of SK2 channel with alpha-actinin2 localizes the channel to the entry of external Ca(2+) source, which regulate the channel function. Furthermore, we demonstrated that the functions of SK2 channels in atrial myocytes are critically dependent on the normal expression of Ca(v)1.3 Ca(2+) channels. Null deletion of Ca(v)1.3 channel results in abnormal function of SK2 channel and prolongation of repolarization and atrial arrhythmias. Our study provides insight into the molecular mechanisms of the coupling of SK2 channel with voltage-gated Ca(2+) channel, and represents the first report linking the coupling of 2 different types of ion channels via cytoskeletal proteins.     

Crosstalk between voltage-independent Ca2+ channels and L-type Ca2+ channels in A7r5 vascular smooth muscle cells at elevated intracellular pH: evidence for functional coupling between L-type Ca2+ channels and a 2-APB-sensitive cation channel

This study was designed to investigate the role of voltage-independent and voltage-dependent Ca2+ channels in the Ca2+ signaling associated with intracellular alkalinization in A7r5 vascular smooth muscle cells. Extracellular administration of ammonium chloride (20 mmol/L) resulted in elevation of intracellular pH and activation of a sustained Ca2+ entry that was inhibited by 2-amino-ethoxydiphenyl borate (2-APB, 200 micromol/L) but not by verapamil (10 micro;mol/L). Alkalosis-induced Ca2+ entry was mediated by a voltage-independent cation conductance that allowed permeation of Ca2+ (PCa/PNa approximately 6), and was associated with inhibition of L-type Ca2+ currents. Alkalosis-induced inhibition of L-type Ca2+ currents was dependent on the presence of extracellular Ca2+ and was prevented by expression of a dominant-negative mutant of calmodulin. In the absence of extracellular Ca2+, with Ba2+ or Na+ as charge carrier, intracellular alkalosis failed to inhibit but potentiated L-type Ca2+ channel currents. Inhibition of Ca2+ currents through voltage-independent cation channels by 2-APB prevented alkalosis-induced inhibition of L-type Ca2+ currents. Similarly, 2-APB prevented vasopressin-induced activation of nonselective cation channels and inhibition of L-type Ca2+ currents. We suggest the existence of a pH-controlled Ca2+ entry pathway that governs the activity of smooth muscle L-type Ca2+ channels due to control of Ca2+/calmodulin-dependent negative feedback regulation. This Ca2+ entry pathway exhibits striking similarity with the pathway activated by stimulation of phospholipase-C-coupled receptors, and may involve a similar type of cation channel. We demonstrate for the first time the tight functional coupling between these voltage-independent Ca2+ channels and classical voltage-gated L-type Ca2+ channels.     

L- and T-type Ca2+ channels in canine cardiac Purkinje cells. Single-channel demonstration of L-type Ca2+ window current.

Canine cardiac Purkinje cells contain both L- and T-type calcium currents, yet the single Ca2+ channels have not been characterized from these cells. Additionally, previous studies have shown an overlap between the steady-state inactivation and activations curves for L-type Ca2+ currents, suggesting the presence of L-type Ca2+ "window" current. We used the on-cell, patch-clamp technique to study Ca2+ channels from isolated cardiac Purkinje cells. Patches contained one or more Ca2+ channels 75% of the time. L-type channels were seen in 69% and T-type channels in 73% of these patches. With 110 mM Ba2+ as the charge carrier, the conductances of the L- and T-type Ca2+ channels were 24.2 +/- 0.8 pS (n = 9) and 9.0 +/- 0.5 pS (n = 8), respectively (mean +/- SEM). With 110 mM Ca2+ as the charge carrier, the conductance of the L-type Ca2+ channel decreased to 9.7 +/- 1.2 pS (n = 4), whereas the T-type Ca2+ channel conductance was unchanged. Voltage-dependent inactivation was shown for both L- and T-type Ca2+ channels, although for L-type Ca2+ channel with Ba2+ as the charge carrier, inactivation took at least 30 seconds at a potential of +40 mV. After channel inactivation was complete, L-type Ca2+ channel reopenings were observed following repolarizing steps into the window voltage range. Thus, our data identify both L- and T-type Ca2+ channels in cardiac Purkinje cells and demonstrate, at the single-channel level, L-type channel transitions expected for a window current. Window current may play an important role in shaping the action potential and in arrhythmogenesis.     

A glutamate receptor regulates Ca2+ mobilization in hippocampal neurons.

We investigated the effect of various excitatory amino acids on intracellular free Ca2+ concentration ( [Ca2+]i) in single mouse hippocampal neurons in vitro by using the Ca2+-sensitive dye fura-2. In normal physiological solution, glutamate, kainate, N-methyl-D-aspartate, and quisqualate all produced increases in [Ca2+]i. When all extracellular Ca2+ was removed, kainate and N-methyl-D-aspartate were completely ineffective, but quisqualate and glutamate were able to produce a spike-like Ca2+ transient, presumably reflecting the release of Ca2+ from intracellular stores. Ca2+ transients of similar shape could also be produced by the alpha 1-adrenergic agonist phenylephrine. After the production of a Ca2+ transient a second addition of quisqualate was ineffective unless intracellular stores were refilled by loading the cell with Ca2+ following depolarization in Ca2+-containing medium. None of the conventional excitatory amino acid receptor antagonists inhibited the Ca2+-mobilizing effects of quisqualate. Furthermore alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was unable to produce Ca2+ mobilization in Ca2+-free medium, although it could produce Ca2+ influx in Ca2+-containing medium. Thus, glutamate can produce mobilization of Ca2+ from intracellular stores in hippocampal neurons by acting on a quisqualate-sensitive but AMPA-insensitive receptor. This receptor is therefore distinct from the quisqualate receptor that produces cell depolarization. The possibility that this Ca2+-mobilizing effect is mediated by inositol triphosphate production is discussed.     

Selective inhibition of L-type Ca2+ channels in A7r5 cells by physiological levels of testosterone

Testosterone has marked beneficial cardiovascular effects, many of which have been attributed to a vasodilatory action. However, the molecular target of testosterone underlying this effect is subject to debate. In this study, we have used microfluorimetry as a noninvasive means of examining whether testosterone could exert dilatory effects via inhibition of voltage-gated Ca2+ entry in the model vascular smooth muscle cell line, A7r5. Rises of [Ca2+]i evoked by 50 mm K+ -containing solution were suppressed in a concentration-dependent manner by testosterone (IC50, 3.1 nm) and by the nonaromatizable analog, 5beta-dihydrotestosterone (IC50, 6.9 nm). The effects of testosterone were apparent in the presence of pimozide (to block T-type Ca2+ channels) but not nifedipine (to block L-type Ca2+ channels). Testosterone did not alter Ca2+ mobilization from intracellular stores by the prostaglandin analog U46619 or capacitative Ca2+ entry in cells pretreated with thapsigargin. Our results indicate that testosterone, at physiological concentrations, can selectively suppress Ca2+ entry into A7r5 cells via L-type Ca2+ channels. We suggest this effect is a likely mechanism underlying its vasodilatory actions and beneficial cardiovascular effects.     

Calcicludine,a venom peptide of the Kunitz-type protease inhibitor family,is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons.

Calcicludine (CaC) is a 60-amino acid polypeptide from the venom of Dendroaspis angusticeps. It is structurally homologous to the Kunitz-type protease inhibitor, to dendrotoxins, which block K+ channels, and to the protease inhibitor domain of the amyloid beta protein that accumulates in Alzheimer disease. Voltage-clamp experiments on a variety of excitable cells have shown that CaC specifically blocks most of the high-threshold Ca2+ channels (L-, N-, or P-type) in the 10-100 nM range. Particularly high densities of specific 125I-labeled CaC binding sites were found in the olfactory bulb, in the molecular layer of the dentate gyrus and the stratum oriens of CA3 field in the hippocampal formation, and in the granular layer of the cerebellum. 125I-labeled CaC binds with a high affinity (Kd = 15 pM) to a single class of noninteracting sites in rat olfactory bulb microsomes. The distribution of CaC binding sites in cerebella of three mutant mice (Weaver, Reeler, and Purkinje cell degeneration) clearly shows that the specific high-affinity labeling is associated with granule cells. Electrophysiological experiments on rat cerebellar granule neurons in primary culture have shown that CaC potently blocks the L-type component of the Ca2+ current (K0.5 = 0.2 nM). Then CaC, in the nanomolar range, appears to be a highly potent blocker of an L-subtype of neuronal Ca2+ channels.     

Molecular aspects of adrenergic modulation of cardiac L-type Ca2+ channels

L-type Ca(2+) channels are predominantly regulated by beta-adrenergic stimulation, enhancing L-type Ca(2+) current by increasing the mean channel open time and/or the opening probability of functional Ca(2+) channels. Stimulation of beta-adrenergic receptors (ARs) results in an increased cyclic adenosine monophosphate (cAMP) production by adenylate cyclase (AC) and consequently activation of protein kinase (PK) A and phosphorylation of L-type Ca(2+) channels by this enzyme. Beta(1)-Adrenergic receptors couple exclusively to the G protein Gs, producing a widespread increase in cAMP levels in the cell, whereas beta(2)-adrenergic receptors couple to both Gs and Gi, producing a more localized activation of L-type Ca(2+) channels. Other signaling intermediates (protein kinase C, protein kinase G or protein tyrosine kinase (PTK)) either have negative effects on L-type Ca(2+) current, or they interact with the stimulatory effect of the protein kinase A pathway.     

Ca2+ sparks in rabbit ventricular myocytes evoked by action potentials: involvement of clusters of L-type Ca2+ channels

It is not clear how many L-type Ca2+ channels (LCCs) are required to ensure that a Ca2+ spark is triggered during a normal mammalian action potential (AP). We investigated this in rabbit ventricular myocytes by examining both the properties of sparks evoked by APs and the activity of LCCs. We measured Ca2+ sparks evoked by repeated APs with pipettes containing 2 mmol/L EGTA and single LCC activity in cell-attached patches depolarized to +50 mV using pipettes containing 110 mmol/L Ba2+. With 2 mmol/L Ca2+ in the external solution, we observed sparks at the beginning of every evoked AP at numerous locations. Each spark was observed repeatedly at a fixed location and began during a limited interval after the AP peak. These sparks occurred with a probability of approximately unity. However, the chance that an LCC does not open during the interval when a spark is triggered is quite high ( approximately 0.13). Therefore, because single channels open with a probability significantly lower than 1, more than one LCC must be available to ensure that sparks are triggered with a probability of approximately unity. We conclude that it is likely that a cluster of LCCs is involved in gating a cluster of ryanodine receptors at the beginning of an AP.     

Ca2+ signaling amplification by oligomerization of L-type Cav1.2 channels

Ca(2+) influx via L-type Ca(v)1.2 channels is essential for multiple physiological processes, including gene expression, excitability, and contraction. Amplification of the Ca(2+) signals produced by the opening of these channels is a hallmark of many intracellular signaling cascades, including excitation-contraction coupling in heart. Using optogenetic approaches, we discovered that Ca(v)1.2 channels form clusters of varied sizes in ventricular myocytes. Physical interaction between these channels via their C-tails renders them capable of coordinating their gating, thereby amplifying Ca(2+) influx and excitation-contraction coupling. Light-induced fusion of WT Ca(v)1.2 channels with Ca(v)1.2 channels carrying a gain-of-function mutation that causes arrhythmias and autism in humans with Timothy syndrome (Ca(v)1.2-TS) increased Ca(2+) currents, diastolic and systolic Ca(2+) levels, contractility and the frequency of arrhythmogenic Ca(2+) fluctuations in ventricular myocytes. Our data indicate that these changes in Ca(2+) signaling resulted from Ca(v)1.2-TS increasing the activity of adjoining WT Ca(v)1.2 channels. Collectively, these data support the concept that oligomerization of Ca(v)1.2 channels via their C termini can result in the amplification of Ca(2+) influx into excitable cells.     

Regulation of vascular L-type Ca2+ channels by phosphatidylinositol 3,4,5-trisphosphate

Modulation of voltage-gated L-type Ca2+ channels by phosphoinositide 3-kinase (PI3K) regulates Ca2+ entry and plays a crucial role in vascular excitation-contraction coupling. Angiotensin II (Ang II) activates Ca2+ entry by stimulating L-type Ca2+ channels through Gbeta-sensitive PI3K in portal vein myocytes. Moreover, PI3K and Ca2+ entry activation have been reported to be necessary for receptor tyrosine kinase-coupled and G protein-coupled receptor-induced DNA synthesis in vascular cells. We have previously shown that tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks are able to modulate vascular L-type Ca2+ channels. PI3Ks display 2 enzymatic activities: a lipid-kinase activity leading to the formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3 or PIP3] and a serine-kinase activity. Here we show that exogenous PIP3 applied into the cell through the patch pipette is able to reproduce the Ca2+ channel-stimulating effect of Ang II and PI3Ks. Moreover, the Ang II-induced PI3K-mediated stimulation of Ca2+ channel and the resulting increase in cytosolic Ca2+ concentration are blocked by the anti-PIP3 antibody. Mutants of PI3K transfected into vascular myocytes also revealed the essential role of the lipid-kinase activity of PI3K in Ang II-induced Ca2+ responses. These results suggest that PIP3 is necessary and sufficient to activate a Ca2+ influx in vascular myocytes stimulated by Ang II.     

N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.     

Inhibition of N- and L-type Ca2+ channels by the spider venom toxin omega-Aga-IIIA.

omega-Aga-IIIA, an 8.5-kDa peptide toxin isolated from the venom of Agelenopsis aperta, was found to be a highly potent inhibitor of Ca channels in cardiac muscle and in peripheral and central neurons of rats and frogs. Cardiac L-type Ca channels were completely (Kd approximately 0.6 nM) blocked by omega-Aga-IIIA. In sensory neurons, the toxin inhibited most high-threshold Ca current but not T-type Ca current. omega-Aga-IIIA blocked with similar potency (Kd approximately 1.5 nM) both omega-conotoxin GVIA-sensitive and dihydropyridine-sensitive current components but left a fraction (approximately 35%) of high-threshold current that was also resistant to omega-conotoxin and dihydropyridines. The toxin blocks N- and L-type channels with equal potency and therefore may identify a high-affinity binding site common to these two Ca channel types.     

Ca2+ channels of the L-type in peripheral blood lymphocytes of essential hypertensives

Ca²⁺ channels of the L-type were assayed in human peripheral blood lymphocytes of normotensive control subjects and of essential hypertensives using radioligand binding assay techniques. The dihydropyridine Ca²⁺ channel blocker [³H](+)-PN 200-110 [isopropyl1-4-(2,1,3-benzoxadiazol-4-yl)1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3-pyridine carboxylate] was used as a ligand. [³H](+)-PN 200 110 was bound specifically to human peripheral blood lymphocytes in a manner consistent with the labeling of Ca²⁺ channels of the L-type. No significant differences in the dissociation constant (K_d), in the maximum density of binding sites (B_max) or in the pharmacological profile of [³H](+)-PN 200 110 binding were found between normotensive subjects and different degree essential hypertensives. Analysis of the intralymphocytic free Ca²⁺ concentration did not reveal differences between normotensive subjects and essential hypertensives. Although hypertension is associated with altered membrane handling of Ca²⁺, no changes in the expression of peripheral blood lymphocyte Ca²⁺ channels of the L-type or in intralymphocytic Ca²⁺ concentrations were found in essential hypertensives. Human peripheral blood lymphocytes therefore cannot represent a peripheral marker of altered Ca²⁺ handling in hypertension.     

An apamin-sensitive Ca2+-activated K+ current in hippocampal pyramidal neurons

In hippocampal and other cortical neurons, action potentials are followed by afterhyperpolarizations (AHPs) generated by the activation of small-conductance Ca2+-activated K+ channels (SK channels). By shaping the neuronal firing pattern, these AHPs contribute to the regulation of excitability and to the encoding function of neurons. Here we report that CA1 pyramidal neurons express an AHP current that is suppressed by apamin and is involved in the control of repetitive firing. This current presents distinct kinetic and pharmacological features, and it is modulated differently than the apamin-insensitive slow AHP current. Furthermore, our in situ hybridizations show that the apamin-sensitive SK subunits are expressed in CA1 pyramidal neurons, providing a potential molecular correlate to the apamin-sensitive AHP current. Altogether, these results clarify the discrepancy between the reported high density of apamin-binding sites in the CA1 region and the apparent lack of an apamin-sensitive current in CA1 pyramidal neurons, and they may explain the effects of this toxin on hippocampal synaptic plasticity and learning.