Biochemical analysis of L-type calcium channels from skeletal and cardiac muscle

The development of specific pharmacological agents that modulate different types of ion channels has prompted an extensive effort to elucidate the molecular structure of these important molecules. The calcium channel blockers that specifically modulate the L-type calcium channel activity have aided in the purification and reconstitution of this channel from skeletal muscle transverse tubules. The L-type calcium channel from skeletal muscle is composed of five subunits designated alpha 1, alpha 2, beta, gamma, and sigma. The alpha 1-subunit is the pore-forming polypeptide and contains the ligand binding and phosphorylation sites through which channel activity can be modulated. The role of the other subunits in channel function remains to be studied. The calcium channel components have also been partially purified from cardiac muscle. The channel consists of at least three subunits that have properties related to the subunits of the calcium channel from skeletal muscle. A core polypeptide that can form a channel and contains ligand binding and phosphorylation sites has been identified in cardiac preparations. Here we summarize recent biochemical and molecular studies describing the structural features of these important ion channels.     

Stimulation of cardiac L-type calcium channels by extracellular ATP

The co-release of ATP with norepinephrine from sympatheticnerve terminals in the heart may augment adrenergic stimulation ofcardiac Ca²⁺ channel activity. To test for a possibledirect effect of extracellular ATP on L-type Ca²⁺ channels,single channels were reconstituted from porcine sarcolemma into planarlipid bilayers so that intracellular signaling pathways could becontrolled. Extracellular ATP (2-100 µM) increased the openprobability of the reconstituted channels, with a maximal increase of~2.6-fold and an EC₅₀ of 3.9 µM. The increase in open probability was due to an increase in channel availability and adecrease in channel inactivation rate. Other nucleotides displayed arank order of effectiveness of ATP > ,-methylene-ATP > 2-methylthio-ATP > UTP > adenosine5'-O-(3-thiotriphosphate) >> ADP; adenosine had no effect.Several antagonists of P2 receptors had no impact on the ATP-dependentincrease in open probability, indicating that receptor activation wasnot required. These results suggest that extracellular ATP and othernucleotides can stimulate the activity of cardiac L-typeCa²⁺ channels via a direct interaction with the channels.

     

Comparative analysis of cell replacement in hibernators.

1. Cell renewal in hibernators undergoes seasonal rhythm independent of the hibernation state. 2. We propose that seasonal depression of cell renewal in tissues of hibernators is caused by seasonal involution of thymus in these animals. 3. The latter is known to be involved in the control of cell proliferation. 4. The state of hibernation per se has also an effect on cellular proliferation. 5. It induces the block of cells in the permitotic phase. It is suggested that the blockage of cells in renewing tissues of hibernators under natural deep hypothermia throughout a period of torpidity represents the adaptive reaction of the organism.     

Role of gelsolin in the actin filament regulation of cardiac L-type calcium channels

The actin cytoskeleton is an important contributor to themodulation of the cell function. However, little is known about theregulatory role of this supermolecular structure in the membrane eventsthat take place in the heart. In this report, the regulation of cardiacmyocyte function by actin filament organization was investigated inneonatal mouse cardiac myocytes (NMCM) from both wild-type mice andmice genetically devoid of the actin filament severing protein gelsolin(Gsn/). Cardiac L-type calcium channel currents(I_Ca) wereassessed using the whole cell voltage-clamp technique. Addition of theactin filament stabilizer phalloidin to wild-type NMCM increasedI_Ca by 227% overcontrol conditions. The basalI_Ca ofGsn/ NMCM was 300% higher than wild-type controls. Thisincrease was completely reversed by intracellular perfusion of theGsn/ NMCM with exogenous gelsolin. Further, cytoskeletal disruption of either Gsn/ or phalloidin-dialyzedwild-type NMCM with cytochalasin D (CD) decreased the enhancedI_Ca by 84% and 87%, respectively. The data indicate that actin filament stabilization by either a lack of gelsolin or intracellular dialysis with phalloidin increase I_Ca,whereas actin filament disruption with CD or dialysis ofGsn/ NMCM with gelsolin decreaseI_Ca. We concludethat cardiac L-type calcium channel regulation is tightly controlled byactin filament organization. Actin filament rearrangement mediated by gelsolin may contribute to calcium channel inactivation.

     

Regulation of cloned cardiac L-type calcium channels by cGMP-dependent protein kinase

We have studied the effect of 8-bromo-cyclic GMP (8-Br-cGMP) on cloned cardiac L-type calcium channel currents to determine the site and mechanism of action underlying the functional effect. Rabbit cardiac alpha(1C) subunit, in the presence or absence of beta(1) subunit (rabbit skeletal muscle) or beta(2) subunit (rat cardiac/brain), was expressed in Xenopus oocytes, and two-electrode voltage-clamp recordings were made 2 or 3 days later. Application of 8-Br-cGMP caused decreases in calcium channel currents in cells expressing the alpha(1C) subunit, whether or not a beta subunit was co-expressed. No inhibition of currents by 8-Br-cGMP was observed in the presence of the protein kinase G inhibitor KT5823. Substitutions of serine residues by alanine were made at residues Ser(533) and Ser(1371) on the alpha(1C) subunit. As for wild type, the mutant S1371A exhibited inhibition of calcium channel currents by 8-Br-cGMP, whereas no effect of 8-Br-cGMP was observed for mutant S533A. Inhibition of calcium currents by 8-Br-cGMP was also observed in the additional presence of the alpha(2)delta subunit for wild type channels but not for the mutant S533A. These results indicate that cGMP causes inhibition of L-type calcium channel currents by phosphorylation of the alpha(1C) subunit at position Ser(533) via the action of protein kinase G.     

cAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca²⁺ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac ₁ and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the ₁ subunit, resulted in Ca²⁺ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the ₁ subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca²⁺ channel functions suggesting that cardiac Ca²⁺ channels expressed in these cells behave, in terms of lack of PKA control, like Ca²⁺ channels of smooth muscle cells.     

Functional properties of cardiac L-type calcium channels transiently expressed in HEK293 cells. Roles of alpha 1 and beta subunits

The cardiac dihydropyridine-sensitive calcium channel was transiently expressed in HEK293 cells by transfecting the rabbit cardiac calcium channel alpha 1 subunit (alpha 1C) alone or in combination with the rabbit calcium channel beta subunit cloned from skeletal muscle. Transfection with alpha 1C alone leads to the expression of inward, voltage-activated, calcium or barium currents that exhibit dihydropyridine sensitivity and voltage- as well as calcium-dependent inactivation. Coexpression of the skeletal muscle beta subunit increases current density and the number of high-affinity dihydropyridine binding sites and also affects the macroscopic kinetics of the current. Recombinant alpha 1C beta channels exhibit a slowing of activation and a faster inactivation rate when either calcium or barium carries the charge. Our data suggest that both an increase in the number of channels as well as modulatory effects on gating underlie the modifications observed upon beta subunit coexpression.     

L-type calcium channels are preferentially coupled to endocytosis in bovine chromaffin cells

Exocytosis and endocytosis are Ca(2+)-dependent processes. The contribution of high-voltage activated Ca(2+) channels subtypes to exocytosis has been thoroughly studied in chromaffin cells. However, similar reports concerning endocytosis are unavailable. Thus, we studied here the effects of blockers of L (nifedipine), N (omega-conotoxin GVIA) and P/Q (omega-agatoxin IVA) Ca(2+) channel on Ca(2+) currents (I(Ca)), Ca(2+) entry (Q(Ca)), as well as on the changes in membrane capacitance (C(m)) in perforated-patch voltage-clamped bovine adrenal chromaffin cells. Using 500-ms pulses to 0 or +10 mV, given from a holding potential of -80 mV and 2 mM Ca(2+) we found that omega-conotoxin GVIA affected little the exo-endocytotic responses while omega-agatoxin IVA markedly blocked those responses. However, nifedipine blocked little exocytosis but almost completely inhibited endocytosis. We conclude that L-type Ca(2+) channels seem to be selectively coupled to endocytosis.     

Characterization and localization of two ion-binding sites within the pore of cardiac L-type calcium channels

L-type Ca channels from porcine cardiac sarcolemma were incorporated into planar lipid bilayers. We characterized interactions of permeant and blocking ions with the channel's pore by (a) studying the current-voltage relationships for Ca2+ and Na+ when equal concentrations of the ions were present in both internal and external solutions, (b) testing the dose-dependent block of Ba2+ currents through the channels by internally applied cadmium, and (c) examining the dose and voltage dependence of the block of Na+ currents through the channels by internally and externally applied Ca2+. We found that the I-V relationship for Na+ appears symmetrical through the origin when equal concentrations of Na+ are present on both sides of the channel (gamma = 90 pS in 200 mM NaCl). The conductance for outward Ca2+ currents with 100 mM Ca2+ on both sides of the channel is approximately 8 pS, a value identical to that observed for inward currents when 100 mM Ca2+ was present outside only. This provides evidence that ions pass through the channel equally well regardless of the direction of net flux. In addition, we find that internal Cd2+ is as effective as external Cd2+ in blocking Ba2+ currents through the channels, again suggesting identical interactions of ions with each end of the pore. Finally, we find that micromolar Ca2+, either in the internal or in the external solution, blocks Na+ currents through the channels. The affinity for internally applied Ca2+ appears the same as that for externally applied Ca2+. The voltage dependence of the Ca(2+)-block suggests that the sites to which Ca2+ binds are located approximately 15% and approximately 85% of the electric field into the pore. Taken together, these data provide direct experimental evidence for the existence of at least two ion binding sites with high affinity for Ca2+, and support the idea that the sites are symmetrically located within the electric field across L-type Ca channels.     

Calcium-dependent inactivation of L-type calcium channels in planar lipid bilayers.

Intracellular Ca2+ can inhibit the activity of voltage-gated Ca channels by modulating the rate of channel inactivation. Ca(2+)-dependent inactivation of these channels may be a common negative feedback process important for regulating Ca2+ entry under physiological and pathological conditions. This article demonstrates that the inactivation of cardiac L-type Ca channels, reconstituted into planar lipid bilayers and studied in the presence of a dihydropyridine agonist, is sensitive to Ca2+. The rates and extents of inactivation, determined from ensemble averages of unitary Ba2+ currents, decreased when the calcium concentration facing the intracellular surface of the channel ([Ca2+]i) was lowered from approximately 10 microM to 20 nM by the addition of Ca2+ chelators. The rates and extents of Ba2+ current inactivation could also be increased by subsequent addition of Ca2+ raising the [Ca2+]i to 15 microM, thus demonstrating that the Ca2+ dependence of inactivation could be reversibly regulated by changes in [Ca2+]i. In addition, reconstituted Ca channels inactivated more quickly when the inward current was carried by Ca2+ than when it was carried by Ba2+, suggesting that local increases in [Ca2+]i could activate Ca(2+)-dependent inactivation. These data support models in which Ca2+ binds to the channel itself or to closely associated regulatory proteins to control the rate of channel inactivation, and are inconsistent with purely enzymatic models for channel inactivation.     

Regulation of the cardiac L-type calcium channel in L6 cells by arginine-vasopressin

L-type Ca2+ channel activity was measured in L6 cells as nifedipine-sensitive barium (Ba2+; 5 mM) influx in a depolarizing salt solution containing 140 mM KCl. Addition of AVP (arginine-vasopressin) during Ba2+ uptake reduced the rate of Ba2+ influx by 60-100%; this was followed by a gradual restoration of the initial rate of Ba2+ uptake. Blockade of PKC (protein kinase C) by pretreatment with 10 muM bisindolylmaleimide did not affect the initial inhibition of Ba2+ influx, but completely abolished the recovery phase. The effect of AVP was half-maximal at 10 nM AVP and was blocked by the V1a receptor antagonist d-(CH2)(5)-Tyr(Me)-AVP. Activation of G(alphas) by isoprenaline or cholera toxin antagonized the actions of AVP on Ba2+ uptake. This protection persisted in the presence of the PKA (protein kinase A) inhibitor KT5720, and was not mimicked by agents that increase cAMP. Inhibition of Ba2+ influx was also elicited by ATP and ET (endothelin 1) with an order of effectiveness ET相似文献   

Stimulation of single L-type calcium channels in rat pituitary GH3 cells by thyrotropin-releasing hormone.

Hormonal stimulation of voltage-dependent Ca2+ channels in pituitary cells is thought to contribute to the sustained phase of Ca2+ entry and secretion induced by secretion stimulating hormones and has been suggested as a mechanism for refilling the Ca2+ stores. Using the cell-attached patch-clamp technique, we studied the stimulation of single Ca2+ channels by thyrotropin-releasing hormone (TRH) in rat GH3 cells. We show that TRH applied from the bath switched the activity of single L-type Ca2+ channels from a gating mode with very low open probability (po) to a gating mode with slightly smaller conductance but 10 times higher po. Interconversions between these two gating modes were also observed under basal conditions, where the equilibrium was shifted towards the low po mode. TRH applied from the pipette had no effect, indicating the involvement of a cytosolic compound in the stimulatory pathway. We show that TRH does not potentiate all the L-type Ca2+ channels in a given membrane patch and report evidence for co-expression of two functionally different L-type Ca2+ channels. Our results uncover the biophysical mechanism of hormonal stimulation of voltage-dependent Ca2+ channels in GH3 cells and are consistent with differential modulation of different subtypes of dihydropyridine-sensitive Ca2+ channels.     

Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.     

L-type calcium channels in adrenal chromaffin cells: role in pace-making and secretion

Voltage-gated L-type (Cav1.2 and Cav1.3) channels are widely expressed in cardiovascular tissues and represent the critical drug-target for the treatment of several cardiovascular diseases. The two isoforms are also abundantly expressed in neuronal and neuroendocrine tissues. In the brain, Cav1.2 and Cav1.3 channels control synaptic plasticity, somatic activity, neuronal differentiation and brain aging. In neuroendocrine cells, they are involved in the genesis of action potential generation, bursting activity and hormone secretion. Recent studies have shown that Cav1.2 and Cav1.3 are also expressed in chromaffin cells but their functional role has not yet been identified despite that L-type channels possess interesting characteristics, which confer them an important role in the control of catecholamine secretion during action potentials stimulation. In intact rat adrenal glands L-type channels are responsible for adrenaline and noradrenaline release following splanchnic nerve stimulation or nicotinic receptor activation. L-type channels can be either up- or down-modulated by membrane autoreceptors following distinct second messenger pathways. L-type channels are tightly coupled to BK channels and activate at relatively low-voltages. In this way they contribute to the action potential hyperpolarization and to the pace-maker current controlling action potential firings. L-type channels are shown also to regulate the fast secretion of the immediate readily releasable pool of vesicles with the same Ca(2+)-efficiency of other voltage-gated Ca(2+) channels. In mouse adrenal slices, repeated action potential-like stimulations drive L-type channels to a state of enhanced stimulus-secretion efficiency regulated by beta-adrenergic receptors. Here we will review all these novel findings and discuss the possible implication for a specific role of L-type channels in the control of chromaffin cells activity.     

Susceptibility of insulinoma cells to cadmium and modulation by L-type calcium channels

Cadmium (Cd), a toxic metal that induces apoptosis and necrosis in a variety of cells, accumulates in pancreas and may be a cause of diabetes in humans. In the insulinoma cells line HIT-T15 (HIT), we measured internal calcium (Ca) and Cd levels by the fluorescent dye Fura-2 and confirm that L-type voltage-dependent calcium channels (VDCC) play a major role in glucose response and represent a pathway of Cd influx in these cells. Therefore we examined the role of VDCC in acute Cd poisoning by comparing its accumulation and cytotoxic effect in HIT cells and in epithelial-like VDCC-free HeLa cells. Cultures were incubated with 10–300 M Cd for 15 min–6 h. While negligible at the end of the treatment, HIT cell death was evident after 18–24 h, and it was time-, dose- and serum-dependent. Short (60 min) Cd treatments with lower doses (100 M in serum-free medium) induced delayed apoptotic cell death, as demonstrated by DNA fragmentation on agarose gels and segmentation of DAPI-stained nuclei. Longer incubations and/or higher concentrations caused mainly necrosis. The same treatments were largely harmless in HeLa cells, in which neither death nor DNA fragmentation was observed. The Ca antagonist nimodipine was capable to prevent HIT cell death at lower doses of Cd and to restore the apoptotic condition at higher doses, indicating that reduction of Cd flux through VDCC modulates Cd toxicity. These data demonstrate a specific sensitivity to Cd of insulinoma cells that can be significant for pancreatic -cell pathology.Published online: March 2005     

Inactivation of L-type calcium channels in cardiomyocytes. Experimental and theoretical approaches

The L-type calcium current (ICa) plays an important role in excitation-contraction coupling of heart cells. It is critical for forming the major trigger for Ca(2+)-induced Ca(2+) release from the sarcoplasmic reticulum and hence its feedback regulation is of fundamental biological significance. The channel inactivation sharpens the kinetics and temporal precision of the Ca(2+) signals so that it prevents longer-term increases in free intracellular Ca(2+) concentration. Cardiac L-type Ca(2+) channels are known to inactivate through voltage- and Ca(2+)-dependent mechanisms. Pure voltage-dependent inactivation has a much slower time course of development than Ca(2+)-dependent inactivation and plays minor role in inhibition of Ca(2+) influx into the cell. The major determinant of the inactivation kinetics of Ca(2+) current during depolarization is Ca(2+)-dependent mechanisms. Furthermore, it is possible to distinguish two phases in Ca(2+)-dependent inactivation of calcium current: a slow phase that depends on Ca(2+) flow through the channels (Ca(2+) current-dependent inactivation) and a fast one that depends on Ca(2+) released from the sarcoplasmic reticulum (Ca(2+) release-dependent inactivation). Although both Ca(2+) released from the SR and Ca(2+) permeating channels play a role, SR-released Ca(2+) is the most effective inactivation mechanism in inhibition of Ca(2+) entry through the channel.     

Anomalous L-type calcium channels of rat spinal motoneurons

Single channel patch-clamp recordings show that embryonic rat spinal motoneurons express anomalous L-type calcium channels, which reopen upon repolarization to resting potentials, displaying both short and long reopenings. The probability of reopening increases with increasing voltage of the preceding depolarization without any apparent correlation with inactivation during the depolarization. The probability of long with respect to short reopenings increases with increasing length of the depolarization, with little change in the total number of reopenings and in their delay. With less negative repolarization voltages, the delay increases, while the mean duration of both short and long reopenings decreases, remaining longer than that of the openings during the preceding depolarization. Open times decrease with increasing voltage in the range -60 to +40 mV. Closed times tend to increase at V > 20 mV. The open probability is low at all voltages and has an anomalous bell-shaped voltage dependence. We provide evidence that short and long reopenings of anomalous L-type channels correspond to two gating modes, whose relative probability depends on voltage. Positive voltages favor both the transition from a short-opening to a long-opening mode and the occupancy of a closed state outside the activation pathway within each mode from which the channel reopens upon repolarization. The voltage dependence of the probability of reopenings reflects the voltage dependence of the occupancy of these closed states, while the relative probability of long with respect to short reopenings reflects the voltage dependence of the equilibrium between modes. The anomalous gating persists after patch excision, and therefore our data rule out voltage-dependent block by diffusible ions as the basis for the anomalous gating and imply that a diffusible cytosolic factor is not necessary for voltage-dependent potentiation of anomalous L-type channels.