Highly mechanosensitive ion channels from graphene-embedded crown ethers.

The ability to tune ionic permeation across nanoscale pores profoundly impacts diverse fields from nanofluidic computing to drug delivery. Here, we take advantage of complex formation between crown ethers and dissolved metal ions to demonstrate graphene-based ion channels highly sensitive to externally applied lattice strain. We perform extensive room-temperature molecular dynamics simulations of the effects of tensile lattice strain on ion permeation across graphene-embedded crown ether pores. Our findings suggest the first instance of solid-state ion channels with an exponential permeation sensitivity to strain, yielding an order of magnitude ion current increase for 2% of isotropic lattice strain. Significant permeation tuning is also shown to be achievable with anisotropic strains. Finally, we demonstrate strain-controllable ion sieving in salt mixtures. The observed high mechanosensitivity is shown to arise from strain-induced control over the competition between ion-crown and ion-solvent interactions, mediated by the atomic thinness of graphene.

The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

Comparative expression of the extracellular calcium-sensing receptor in the mouse, rat, and human kidney.

The calcium-sensing receptor (CaSR) was cloned over 20 years ago and functionally demonstrated to regulate circulating levels of parathyroid hormone by maintaining physiological serum ionized calcium concentration ([Ca(2+)]). The receptor is highly expressed in the kidney; however, intrarenal and intraspecies distribution remains controversial. Recently, additional functions of the CaSR receptor in the kidney have emerged, including parathyroid hormone-independent effects. It is therefore critical to establish unequivocally the localization of the CaSR in the kidney to relate this to its proposed physiological roles. In this study, we determined CaSR expression in mouse, rat, and human kidneys using in situ hybridization, immunohistochemistry (using 8 different commercially available and custom-made antibodies), and proximity ligation assays. Negative results in mice with kidney-specific CaSR ablation confirmed the specificity of the immunohistochemistry signal. Both in situ hybridization and immunohistochemistry showed CaSR expression in the thick ascending limb, distal tubule, and collecting duct of all species, with the thick ascending limb showing the highest levels. Within the collecting ducts, there was significant heterogeneity of expression between cell types. In the proximal tubule, lower levels of immunoreactivity were detected by immunohistochemistry and proximity ligation assays. Proximity ligation assays were the only technique to demonstrate expression within glomeruli. This study demonstrated CaSR expression throughout the kidney with minimal discrepancy between species but with significant variation in the levels of expression between cell and tubule types. These findings clarify the intrarenal distribution of the CaSR and enable elucidation of the full physiological roles of the receptor within this organ.

Surgical versus conservative treatment of distal radius fractures in elderly.

The distal radius fractures (DRFs) are the second most common fracture in the elderly population. Despite their frequency, the optimal treatment of these fractures remains controversial. Several dogmatic myths on DRFs management may adversely affect their outcome and despite a strong trend versus surgical options, systematic reviews suggest that conservative treatment remains the safest option for DRFs in most cases.

Septic arthritis in children.

Osteoarticular infections are a form of diagnostic and therapeutic emergency in infants and children, even if relatively rare. Despite decades of experience with different protocols, and multiple clinical trials, today it is still difficult to determine what kind of antibiotics is really effective, what kind of associations are required, which is the optimal time range of a treatment, when and on which subjects to base the transition from a parenteral treatment to an oral one. Current philosophy aims more and more at reducing hospitalization and costs, and wants to decrease the discomfort in the family. The purpose of these guidelines is to promote a reasoned clinical and therapeutic approach, in a context of diagnostic probabilities that offer the best chance of success in reducing hospitalization with a rapid transition to an oral treatment, and then outpatient, and thus educing totally the processing time.

Effects of the polyamine spermine on arterial chemoreception.

Polyamines modulate many biological functions. Here we report a novel inhibitory modulation by spermine of catecholamine release by the rat carotid body and have identified the molecular mechanism underpinning it. We used molecular (RT-PCR and confocal microscopy) and functional (i.e., neurotransmitter release, patch clamp recording and calcium imaging) approaches to test the involvement of: (i) voltage-dependent calcium channels, and; (ii) the extracellular calcium-sensing receptor, CaR, a G protein-coupled receptor which is also activated by polyamines. RT-PCR and immunohistochemistry of isolated carotid bodies revealed that only Ca(v)1.2 and Ca(v)2.2 were expressed in type 1 cells while Ca(v)1.3, Ca(v)1.4, Ca(v)2.1, Ca(v)2.3 and Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3, could not be detected. CaR expression was detected exclusively in the nerve endings. In isolated carotid bodies, the hypoxia-dependent (7% O(2) for 10 minutes) and depolarization-evoked catecholamine release were partially suppressed by pre- (and co)-incubation with 500microM spermine. In dissociated type 1 glomus cells intracellular calcium concentration did not change following spermine treatment, but this polyamine did inhibit the depolarisation-evoked calcium influx. Whole-cell patch clamp recordings of HEK293 cells stably transfected with Ca(v)1.2 demonstrated that spermine inhibits this calcium channel. Interestingly, this inhibition was not apparent if the extracellular solution contained a concentration of Ba(2) above 2 mM as the charge carrier. In conclusion, spermine attenuates catecholamine release by the carotid body principally via inhibition of Ca(v)1.2. This mechanism may represent a negative feedback, which limits transmitter release during hypoxia.

Cysteine residues in the C-terminal tail of the human BK(Ca)alpha subunit are important for channel sensitivity to carbon monoxide.

In the presence of oxygen (O(2)), carbon monoxide (CO) is synthesised from heme by endogenous hemeoxygenases, and is a powerful activator of BK(Ca) channels. This transduction pathway has been proposed to contribute to cellular O(2) sensing in rat carotid body. In the present study we have explored the role that four cysteine residues (C820, C911, C995 and C1028), located in the vicinity of the "calcium bowl" of C-terminal of human BK(Ca)-alphasubunit, have on channel CO sensitivity. Mutant BK(Ca)-alphasubunits were generated by site-directed mutagenesis (single, double and triple cysteine residue substitutions with glycine residues) and were transiently transfected into HEK 293 cells before subsequent analysis in inside-out membrane patches. Potassium cyanide (KCN) completely abolished activation of wild type BK(Ca) channels by the CO donor, tricarbonyldichlororuthenium (II) dimer, at 100microM. In the absence of KCN the CO donor increased wild-type channel activity in a concentration-dependent manner, with an EC(50) of ca. 50microM. Single cysteine point mutations of residues C820, C995 and C1028 affected neither channel characteristics nor CO EC(50) values. In contrast, the CO sensitivity of the C911G mutation was significantly decreased (EC(50) ca. 100 M). Furthermore, all double and triple mutants which contained the C911G substitution exhibited reduced CO sensitivity, whilst those which did not contain this mutation displayed essentially unaltered CO EC(50) values. These data highlight that a single cysteine residue is crucial to the activation of BK(Ca) by CO. We suggest that CO may bind to this channel subunit in a manner similar to the transition metal-dependent co-ordination which is characteristic of several enzymes, such as CO dehydrogenase.

Hydrogen sulfide inhibits human BK(Ca) channels.

Hydrogen sulfide (H(2)S) is produced endogenously in many types of mammalian cells. Evidence is now accumulating to suggest that H(2)S is an endogenous signalling molecule, with a variety of molecular targets, including ion channels. Here, we describe the effects of H(2)S on the large conductance, calcium-sensitive potassium channel (BK(Ca)). This channel contributes to carotid body glomus cell excitability and oxygen-sensitivity. The experiments were performed on HEK 293 cells, stably expressing the human BK(Ca) channel alpha subunit, using patch-clamp in the inside-out configuration. The H(2)S donor, NaSH (100microM-10 mM), inhibited BK(Ca) channels in a concentration-dependent manner with an IC(50) of ca. 670microM. In contrast to the known effects of CO donors, the H(2)S donor maximally decreased the open state probability by over 50% and shifted the half activation voltage by more than +16mV. In addition, although 1 mM KCN completely suppressed CO-evoked channel activation, it was without effect on the H(2)S-induced channel inhibition, suggesting that the effects of CO and H(2)S were non-competitive. RT-PCR showed that mRNA for both of the H(2)S-producing enzymes, cystathionine-beta-synthase and cystathionine-gamma-lyase, were expressed in HEK 293 cells and in rat carotid body. Furthermore, immunohistochemistry was able to localise cystathionine-gamma-lyase to glomus cells, indicating that the carotid body has the endogenous capacity to produce H(2)S. In conclusion, we have shown that H(2)S and CO have opposing effects on BK(Ca)channels, suggesting that these gases have separate modes of action and that they modulate carotid body activity by binding at different motifs in the BK(Ca)alphasubunit.

Altered expression of iron transport proteins in streptozotocin-induced diabetic rat kidney.

Diabetes mellitus is associated with altered iron homeostasis in both human and animal diabetic models. Iron is a metal oxidant capable of generating reactive oxygen species (ROS) and has been postulated to contribute to diabetic nephropathy. Two proteins involved in iron metabolism that are expressed in the kidney are the divalent metal transporter, DMT1 (Slc11a2), and the Transferrin Receptor (TfR). Thus, we investigated whether renal DMT1 or TfR expression is altered in diabetes, as this could potentially affect ROS generation and contribute to diabetic nephropathy. Rats were rendered diabetic with streptozotocin (STZ-diabetes) and renal DMT1 and TfR expression studied using semi-quantitative immunoblotting and immunofluorescence. In STZ-diabetic Sprague-Dawley rats, renal DMT1 expression was significantly reduced and TfR expression increased after 2 weeks. DMT1 downregulation was observed in both proximal tubules and collecting ducts. Renal DMT1 expression was also decreased in Wistar rats following 12 weeks of STZ-diabetes, an effect that was fully corrected by insulin-replacement but not by cotreatment with the aldose reductase inhibitor, sorbinil. Increased renal TfR expression was also observed in STZ-diabetic Wistar rats together with elevated cellular iron accumulation. Together these data demonstrate renal DMT1 downregulation and TfR upregulation in STZ-diabetes. Whilst the consequence of altered DMT1 expression on renal iron handling and oxidant damage remains to be determined, the attenuation of the putative lysosomal iron exit pathway in proximal tubules could potentially explain lysosomal iron accumulation reported in human diabetes and STZ-diabetic animals.

FGF23-regulated production of Fetuin-A (AHSG) in osteocytes.

INTRODUCTION: AHSG, a serum glycoprotein with recognized anti-calcification activity, has also been suggested to modulate both bone formation and resorption. Though the bulk of AHSG is mostly synthesized in the liver, it has been claimed that also bone cells might produce it. However, the extent of the bone AHSG production and the potential controlling factors remain to be definitively proven. A relevant number of studies support the notion that FGF23, a bone-derived hormone, not only regulates the most important mineral metabolism (MM) related factors (phosphate, parathyroid hormone, vitamin D, etc.), but might be also involved in cardiovascular (CV) outcome, both in chronic kidney disease (CKD) patients and in the general population. Furthermore, in addition to some direct autocrine and paracrine effects in bone, FGF23 has been suggested to interact with AHSG. In this study we investigated if AHSG is really produced by bone cells, and if its bone production is related and/or controlled by FGF23, using cultured bone cells, according to a new method recently published by our group. RESULTS: Our data show that AHSG is consistently produced in osteocytes and to a far lesser extent in osteoblasts. Both FGF23 addition to the culture medium and its over-expression in osteocytes were associated with a consistent increase of both AHSG mRNA and protein, while FGF23 silencing was followed by opposite effects. Though most of these results were largely affected by the blockage of FGF23 receptors, the role of these receptors in the different experimental sets is still not completely clarified. In addition, we found that FGF23 and AHSG proteins co-localized both in cytoplasm and nucleus, which suggests a possible reciprocal interactivity. CONCLUSIONS: Our data not only confirm that AHSG is produced in bone, mainly in osteocytes, but show for the first time that its production is modulated by FGF23. Since both proteins play important roles in the bone and cardiovascular pathology, these results add new pieces to the puzzling relationship between bone and vascular pathology, in particular in CKD patients, prompting future investigations in this field.

Cell surface, Ca2+(cation)-sensing receptor(s): one or many?

In mammals Ca2+ concentration in the extracellular fluids ([Ca2+]o) is essential for a number of vital processes varying from bone mineralization to blood coagulation, regulation of enzymatic processes, modulation of permeability and excitability of plasma membranes. For this reason [Ca2+]o is under strict control of a complex homeostatic system that includes parathyroid glands, kidneys, bones and intestine. The extracellular Ca(2+)-sensing receptor (CaR) is an essential component of this system, regulating parathyroid hormone secretion, calcium (and magnesium) excretion by the kidney, bone remodeling and Ca2+ reabsorption by the gastrointestinal tract. Structurally, the CaR is a novel member of a growing G protein-coupled receptor superfamily, which includes metabotropic glutamate receptors (mGluRs) [1], [gamma]-aminoisobutyric acid (GABA-B) receptors [2] and vomeronasal organ receptors [3]. Initially identified from bovine parathyroid glands [4], within the 5 years following its identification CaR presence has rapidly been identified as extending to organs where the link with mineral ion metabolism has not been elucidated (i.e. brain, stomach, eye, skin and many other epithelial cells) (see [5] for review). The role of the receptor in these regions is largely unknown, but it appears to be somewhat related to phenomena such as chemotaxis, cell proliferation and programmed cell death. This review will describe the discovery of a novel class of ion-sensing receptor(s), receptor-effector coupling and the roles of the CaR inside and outside the Ca2+o homeostatic system.

Voltage- and Ca2+-dependent burst generation in neuroendocrine Dahlgren cells in the teleost Platichthys flesus.

The neuroendocrine Type 1 Dahlgren cells of the caudal neurosecretory system of the flounder display characteristic bursting activity, which may increase secretion efficiency. The firing activity pattern in these cells was voltage-dependent; when progressively depolarized, cells moved from silent (approximately -70 mV), through bursting and phasic to tonic firing (< -65 mV). Brief (10 s) evoked bursts of spikes were followed by a slow after-depolarization (ADP; amplitude up to 10 mV, duration 10-200 s), which was also voltage-dependent and could trigger a prolonged burst. The ADP was significantly reduced in the absence of external Ca(2+) ions or the presence of the L-type Ca(2+) channel blocker, nifedipine. BayK 8644 (which increases L-type channel open times) significantly increased ADP duration, whereas the Ca(2+)-activated nonselective cation channel blocker, flufenamic acid, had no effect. Pharmacological blockade of Ca(2+)-activated K(+) channels, using apamin and charybdotoxin, increased the duration of both ADP and evoked bursts. However, action potential waveform was unaffected by either apamin/charybdotoxin, nifedipine, BayK 8644 or removal of external Ca(2+). The short duration (approximately 100 ms), hyperpolarization-activated, postspike depolarizing afterpotentials (DAP), were significantly reduced by nifedipine. We propose that long duration ADPs underlie bursts and that short duration DAPs play a role in modulation of spike frequency.

Nutritional Management of Patients With Esophageal and Esophagogastric Junction Cancer.

BACKGROUND: Malnutrition is common in patients with esophageal and esophagogastric cancer. Compared to patients with other digestive and extradigestive neoplasia, the highest incidence (78.9%) was found in those with esophageal cancer. Malnutrition is associated with postoperative complications, increased morbidity, and prolonged hospital stays. METHODS: The authors review the impact and causes of malnutrition in esophageal cancer patients and present strategies that can be used to preserve or restore the nutritional status in this patient population throughout treatment. RESULTS: Patients usually are unable to sustain weight on oral intake alone and require additional means of nutritional support. Several methods can be used to provide nutritional care to the esophageal cancer patient, such as diet modification, oral supplementation, and enteral or parenteral nutrition. The enteral route is preferred due to preservation of gut integrity, reduced risk of complications, and less expense. In terminally ill patients, minimal nutritional intervention may be all that is needed to achieve patient comfort. CONCLUSIONS: In order to improve clinical outcomes and the quality of life for patients with esophageal and esophagogastric cancers, the extent of malnutrition must be identified and treated.

The many roles of the calcium-sensing receptor in health and disease.

The physiological relevance of calcium in many vital processes requires that its concentration in extracellular fluids be kept within a narrow range. The near-constancy of this parameter emphasizes the remarkable sensitivity of cells sensing changes in extracellular calcium concentration to minimal fluctuations (< 2%) and the level of sophistication of the homeostatic system (1). The identification of a cell surface, Ca2+ (polyvalent cation)-sensing receptor (CaR), has shed considerable light on the molecular aspects of hypercalcemia on cell function (2). Activation of the receptor by calcium triggers an intracellular cascade of second messengers producing a variety of biological effects, many of which have yet to be understood. This suggests, for the first time, that Ca2+ can exert its effects in a hormone-like fashion without crossing the plasma membrane. The demonstration that inherited genetic disorders of Ca2+ homeostasis are associated with mutations that reduce or enhance responsiveness of the receptor to extracellular Ca2+ concentration clearly proposes CaR as the main regulator of divalent mineral ion excretion (3). This hypothesis is confirmed by the assessment of the presence of the receptor in all regions involved in Ca2+ homeostasis (e.g., parathyroid glands, kidney, calcitonin-secreting C cells, bone-derived cell lines, and intestine) (1,4-8). Recently, the receptor has also been found in regions not normally involved in mineral ion metabolism, such as the brain, eye, stomach, and pancreas (9-13). This clearly indicates a much broader relevance of CaR in the maintenance of local ionic homeostasis and, possibly, in the involvement in vital processes such as the regulation of cell fate.

Expression of cartilage-derived retinoic acid-sensitive protein during healing of the rat tooth-extraction socket.

Cartilage-derived retinoic acid sensitive protein (CD-RAP) is a recently described, cartilage-specific protein. During early healing of the tooth-extraction socket, cells express both chondrogenic and osteogenic cell markers, but no cartilage is formed. Cartilaginous collagen type II protein, a major constituent of hyaline cartilage, has not been detected in the healing socket, although type IX collagen, which coats these fibres, has been detected transiently in early socket healing. This study investigated the spatial and temporal expression of CD-RAP and various osteoblast cell markers, i.e., alkaline phosphatase, osteopontin, osteonectin and osteocalcin, during healing. Immunolocalization of these proteins was determined in the rat tooth socket at 3, 4, 5, 6, 7, 8, 10 and 14 days after extraction. CD-RAP was expressed by preosteoblast cells maximally at 6, 7, and 8 days after extraction. Fully differentiated osteoblasts expressed osteocalcin, a specific osteoblast marker. Preosteoblasts and fibroblasts did not express osteocalcin. On double immunofluorescent staining, some preosteoblasts coexpressed CD-RAP (indicative of chondrogenic differentiation), and either alkaline phosphatase or osteopontin (markers of osteogenic stem-cell maturation). There was no colocalization between osteopontin and osteonectin. CD-RAP was unique amongst the cell markers used in that it was expressed by preosteoblasts, but not by osteoblasts lining the newly formed trabeculae. CD-RAP may have an important role in osteoblast cell differentiation during bone healing.

Ultrastructural, histochemical and electrophysiological study of calf gallbladder epithelium.

Calf (Bos taurus) gallbladder, contrary to that of most mammalian, does not concentrate bile. It is to be ascertained whether this phenomenon is the result of a lack of fluid absorption or a balance between the latter and fluid secretion. To this end we began a characterization of bovine gallbladder epithelium by means of electron microscopy, immunocytochemistry, enzymatic activity and electrophysiological measurements. We also partially characterized bile content. The ultrastructural examination showed that surface epithelial cells have the general structure that is observed in absorptive epithelia. Mucous secretory activity is also evident. Moreover, two distinct types of secreting cells line the glands in the lamina propria and contribute to the production of an abundant secretion. The cell surface shows a marked reactivity with the anti-alkaline phosphatase serum. Most of the activity of alkaline phosphatase and L-gamma-glutamyltransferase is found at the apical side of the epithelium. Electrophysiological parameters indicate that this is a low resistance epithelium. Therefore, coexistence of features typical of absorptive epithelia and the inability of concentrating bile suggest that, in this organ, fluid absorption and secretion are both present.

Molecular and clinical analysis of a neonatal severe hyperparathyroidism case caused by a stop mutation in the calcium-sensing receptor extracellular domain representing in effect a human 'knockout'.

OBJECTIVE: Loss-of-function calcium-sensing receptor (CAR) mutations cause elevated parathyroid hormone (PTH) secretion and hypercalcaemia. Although full Car deletion is possible in mice, most human CAR mutations result from a single amino acid substitution that maintains partial function. However, here, we report a case of neonatal severe hyperparathyroidism (NSHPT) in which the truncated CaR lacks any transmembrane domain (CaR(R392X)), in effect a full CAR 'knockout'. CASE REPORT: The infant (daughter of distant cousins) presented with hypercalcaemia (5.5-6  mmol/l corrected calcium (2.15-2.65)) and elevated PTH concentrations (650-950  pmol/l (12-81)) together with skeletal demineralisation. NSHPT was confirmed by CAR gene sequencing (homozygous c.1174C-to-T mutation) requiring total parathyroidectomy during which only two glands were located and removed, resulting in normalisation of her serum PTH/calcium levels. DESIGN AND METHODS: The R392X stop codon was inserted into human CAR and the resulting mutant (CaR(R392X)) expressed transiently in HEK-293 cells. RESULTS: CaR(R392X) expressed as a 54  kDa dimeric glycoprotein that was undetectable in conditioned medium or in the patient's urine. The membrane localisation observed for wild-type CaR in parathyroid gland and transfected HEK-293 cells was absent from the proband's parathyroid gland and from CaR(R392X)-transfected cells. Expression of the mutant was localised to endoplasmic reticulum consistent with its lack of functional activity. CONCLUSIONS: Intriguingly, the patient remained normocalcaemic throughout childhood (2.5 mM corrected calcium, 11 pg/ml PTH (10-71), age 8 years) but exhibited mild asymptomatic hypocalcaemia at age 10 years, now treated with 1-hydroxycholecalciferol and Ca2+ supplementation. Despite representing a virtual CAR knockout, the patient displays no obvious pathologies beyond her calcium homeostatic dysfunction.

Spermine attenuates carotid body glomus cell oxygen sensing by inhibiting L-type Ca²(+) channels.

An increase in intracellular Ca²(+) is crucial to O2 sensing by the carotid body. Polyamines have been reported to modulate both the extracellular Ca²(+)-sensing receptor (CaR) and voltage-gated Ca²(+) channels in a number of cell types. Using RT-PCR and immunohistochemistry, the predominant voltage-gated Ca²(+) channels expressed in the adult rat carotid body were L (Ca(V)1.2) and N (Ca(V)2.2)-type. CaR mRNA could not be amplified from carotid bodies, but the protein was expressed in the nerve endings. Spermine inhibited the hypoxia-evoked catecholamine release from isolated carotid bodies and attenuated the depolarization- and hypoxia-evoked Ca²(+) influx into isolated glomus cells. In agreement with data from carotid body, recombinant Ca(V)1.2 was also inhibited by spermine. In contrast, the positive allosteric modulator of CaR, R-568, was without effect on hypoxia-induced catecholamine release from carotid bodies and depolarization-evoked Ca²(+) influx into glomus cells. These data show that spermine exerts a negative influence on carotid body O2 sensing by inhibiting L-type Ca²(+) channels.