Combined oxygen and glucose sensing in the carotid body.
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Animals↗
Biomedical subjects
J López-Barneo
Publications and source records attributed to J López-Barneo.
Tyrosine phosphorylation of the inactivating peptide of the shaker B potassium channel: a structural-functional correlate.
A synthetic peptide patterned after the sequence of the inactivating "ball" domain of the Shaker B K(+) channel restores fast (N-type) inactivation in mutant deletion channels lacking their constitutive ball domains, as well as in K(+) channels that do not normally inactivate. We now report on the effect of phosphorylation at a single tyrosine in position 8 of the inactivating peptide both on its ability to restore fast channel inactivation in deletion mutant channels and on the conformation adopted by the phosphorylated peptide when challenged by anionic lipid vesicles, a model target mimicking features of the inactivation site in the channel protein. We find that the inactivating peptide phosphorylated at Y8 behaves functionally as well as structurally as the noninactivating mutant carrying the mutation L7E. Moreover, it is observed that the inactivating peptide can be phosphorylated by the Src tyrosine kinase either as a free peptide in solution or when forming part of the membrane-bound protein channel as the constitutive inactivating domain. These findings suggest that tyrosine phosphorylation-dephosphorylation of this inactivating ball domain could be of physiological relevance to rapidly interconvert fast-inactivating channels into delayed rectifiers and vice versa.
Amino Acid Sequence↗
Reduction of Ca(2+) channel activity by hypoxia in human and porcine coronary myocytes.
OBJECTIVE: Oxygen (O(2)) tension is a major regulator of blood flow in the coronary circulation. Hypoxia can produce vasodilation through activation of ATP regulated K(+) (K(ATP)) channels in the myocyte membrane, which leads to hyperpolarization and closure of voltage-gated Ca(2+) channels. However, there are other O(2)-sensitive mechanisms intrinsic to the vascular smooth muscle since hypoxia can relax vessels precontracted with high extracellular K(+), a condition that prevents hyperpolarization following opening of K(+) channels. The objective of the present study was to determine whether inhibition of Ca(2+) influx through voltage-dependent channels participates in the response of coronary myocytes to hypoxia. METHODS: Experiments were performed on porcine anterior descendent coronary arterial rings and on enzymatically dispersed human and porcine myocytes of the same artery. Cytosolic [Ca(2+)] was measured by microfluorimetry and whole-cell currents were recorded with the patch clamp technique. RESULTS: Hypoxia (O(2) tension approximately 20 mmHg) dilated endothelium-denuded porcine coronary arterial rings precontracted with high K(+) in the presence of glibenclamide (5 microM), a blocker of K(ATP) channels. In dispersed human and porcine myocytes, low O(2) tension decreased basal cytosolic [Ca(2+)] and transmembrane Ca(2+) influx independently of K(+) channel activation. In patch clamped cells, hypoxia reversibly inhibited L-type Ca(2+) channels. RT-PCR indicated that rHT is the predominant mRNA variant of the alpha(1C) Ca(2+) channel subunit in human coronary myocytes. CONCLUSION: Our study demonstrates, for the first time in a human preparation, that voltage-gated Ca(2+)channels in coronary myocytes are under control of O(2) tension.
Animals↗
Differential segmental activation of Ca2+ -dependent CI-and K+ channels in pulmonary arterial myocytes.
Differential segmental distribution of electrophysiologically distinct myocytes helps to explain the variability of the pulmonary arteries to vasoactive agents. We have studied whether Ca2+ -dependent CI- (CICa) and K+ (KCa) channels are activated differentially in enzymatically dispersed conduit and resistance myocytes. We measured cytosolic [Ca2+] and the changes of membrane current and potential elicited by spontaneous or agonist-induced Ca2+ oscillations. Conduit arteries contained a heterogeneous cell population with a variable mixture of KCa and CICa conductances. Resistance arteries contained a more homogeneous cell population with predominance of CICa channel activation. The relation between KCa and CICa conductances in a given conduit myocyte determines the size of the V(m)change in response to a rise of cytosolic [Ca2+]. Conduit myocytes tend to hyperpolarize towards the K+ equilibrium potential (approximately - 90 m V). In resistance myocytes, release of Ca2+ from stores activates CI Cachannels and brings Vm to a value close to the chloride equilibrium potential (approximately - 20 or - 30 m V) thus favouring opening of Ca2+ channels and Ca2+ influx. In resistance vessels CICachannels contribute to link agonist-induced Ca2+ release from stores and membrane depolarization, thus permitting protracted vasoconstriction.
Animals↗
Collapse of conductance is prevented by a glutamate residue conserved in voltage-dependent K(+) channels.
Voltage-dependent K(+) channel gating is influenced by the permeating ions. Extracellular K(+) determines the occupation of sites in the channels where the cation interferes with the motion of the gates. When external [K(+)] decreases, some K(+) channels open too briefly to allow the conduction of measurable current. Given that extracellular K(+) is normally low, we have studied if negatively charged amino acids in the extracellular loops of Shaker K(+) channels contribute to increase the local [K(+)]. Surprisingly, neutralization of the charge of most acidic residues has minor effects on gating. However, a glutamate residue (E418) located at the external end of the membrane spanning segment S5 is absolutely required for keeping channels active at the normal external [K(+)]. E418 is conserved in all families of voltage-dependent K(+) channels. Although the channel mutant E418Q has kinetic properties resembling those produced by removal of K(+) from the pore, it seems that E418 is not simply concentrating cations near the channel mouth, but has a direct and critical role in gating. Our data suggest that E418 contributes to stabilize the S4 voltage sensor in the depolarized position, thus permitting maintenance of the channel open conformation.
Amino Acid Sequence↗
A small domain in the N terminus of the regulatory alpha-subunit Kv2. 3 modulates Kv2.1 potassium channel gating.
Recent work has demonstrated the existence of regulatory K(+) channel alpha-subunits that are electrically silent but capable of forming heterotetramers with other pore-forming subunits to modify their function. We have investigated the molecular determinant of the modulatory effects of Kv2.3, a silent K(+) channel alpha-subunit specific of brain. This subunit induces on Kv2.1 channels a marked deceleration of activation, inactivation, and closing kinetics. We constructed chimeras of the Kv2.1 and Kv2.3 proteins and analyzed the K(+) currents resulting from the coexpression of the chimeras with Kv2.1. The data indicate that a region of 59 amino acids in the N terminus, adjacent to the first transmembrane segment, is the major structural element responsible for the regulatory function of Kv2.3. The sequence of this domain of Kv2.3 is highly divergent compared with the same region in the other channels of the Kv2 family. Replacement of the regulatory fragment of Kv2.3 by the equivalent of Kv2.1 leads to loss of modulatory function, whereas gain of modulatory function is observed when the Kv2.3 fragment is transferred to Kv2.1. Thus, this study identifies a N-terminus domain involved in Kv2.1 channel gating and in the modulation of this channel by a regulatory alpha-subunit.
Amino Acid Sequence↗
K+ and Ca2+ channel activity and cytosolic [Ca2+] in oxygen-sensing tissues.
Ion channels are known to participate in the secretory or mechanical responses of chemoreceptor cells to changes in oxygen tension (P(O2)). We review here the modifications of K+ and Ca2+ channel activity and the resulting changes in cytosolic [Ca2+] induced by low P(O2) in glomus cells and arterial smooth muscle which are well known examples of O2-sensitive cells. Glomus cells of the carotid body behave as presynaptic-like elements where hypoxia produces a reduction of K+ conductance leading to enhanced membrane excitability, Ca2+ entry and release of dopamine and other neurotransmitters. In arterial myocytes, hypoxia can inhibit or potentiate Ca2+ channel activity, thus regulating cytosolic [Ca2+] and contraction. Ca2+ channel inhibition is observed in systemic myocytes and most conduit pulmonary myocytes, whereas potentiation is seen in a population of resistance pulmonary myocytes. The mechanism whereby O2 modulates ion channel activity could depend on either the direct allosteric modulation by O2-sensing molecules or redox modification by reactive chemical species.
Animals↗
Recovery of chronic parkinsonian monkeys by autotransplants of carotid body cell aggregates into putamen.
We have studied the effect of unilateral autografts of carotid body cell aggregates into the putamen of MPTP-treated monkeys with chronic parkinsonism. Two to four weeks after transplantation, the monkeys initiated a progressive recovery of mobility with reduction of tremor and bradykinesia and restoration of fine motor abilities on the contralateral side. Apomorphine injections induced rotations toward the side of the transplant. Functional recovery was accompanied by the survival of tyrosine hydroxylase-positive (TH-positive) grafted glomus cells. A high density of TH-immunoreactive fibers was seen reinnervating broad regions of the ipsilateral putamen and caudate nucleus. The nongrafted, contralateral striatum remained deafferented. Intrastriatal autografting of carotid body tissue is a feasible technique with beneficial effects on parkinsonian monkeys; thus, this therapeutic approach could also be applied to treat patients with Parkinson's disease.
Animals↗
Cellular and functional recovery of Parkinsonian rats after intrastriatal transplantation of carotid body cell aggregates.
We have tested the suitability of chromaffin-like carotid body glomus cells for dopamine cell replacement in Parkinsonian rats. Intrastriatal grafting of cell aggregates resulted in almost optimal abolishment of motor asymmetries and deficits of sensorimotor orientation. Recovery of transplanted animals was apparent 10 days after surgery and progressed throughout the 3 months of the study. The behavioral effects were correlated with the long survival of glomus cells in the host brain. In host tissue, glomus cells were organized into glomerulus-like structures and retained the ability to secrete dopamine. Several weeks after transplantation, dopaminergic fibers emerged from the graft, reinnervating the striatal gray matter. The special durability of grafted glomus cells in the conditions of brain parenchyma could be related to their sensitivity to hypoxia, which is known to induce cell growth, excitability, and dopamine synthesis. This work should stimulate research on the clinical applicability of carotid body autotransplants in Parkinson's disease.
Amphetamine↗
NMDA-glutamate receptors regulate phosphorylation of dendritic cytoskeletal proteins in the hippocampus.
Most forms of synaptic potentiation need the activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors which generate changes in dendritic morphology of postsynaptic neurons. Since microtubule proteins have an essential role in dendritic morphology, they may be involved and regulated during the modifications of dendritic morphology associated with synaptic potentiation. The phosphorylation of the microtubule-associated proteins (MAPs) has been analyzed in situ after activation or blockade of NMDA-glutamate receptors in hippocampal slices. The phosphorylation of MAP1B and MAP2 has been studied by using several antibodies raised against phosphorylation-sensitive epitopes. Whereas antibodies 125 and 305 recognize phosphorylated epitopes on MAP1B and MAP2, respectively, Ab 842 recognizes a phosphorylatable sequence on MAP1B only when it is dephosphorylated. NMDA treatment decreased the phosphorylation state of the epitope recognized by the antibody 305 on MAP2 and caused a slight dephosphorylation of MAP1B sequences recognized by Ab 125 and 842. Moreover, exposure to APV (an antagonist of NMDA-glutamate receptors) counteracted the effect of NMDA and induced an increase in the phosphorylation state of these sequences in MAP2. Since phosphorylation regulates the interaction of MAPs with cytoskeleton, the results suggest that the modulation of the phosphorylated state of MAP2 by NMDA-glutamate receptors may be implicated in dendritic plasticity.
2-Amino-5-phosphonovalerate↗
Identification and functional characterization of a K+ channel alpha-subunit with regulatory properties specific to brain.
The physiological diversity of K+ channels mainly depends on the expression of several genes encoding different alpha-subunits. We have cloned a new K+ channel alpha-subunit (Kv2.3r) that is unable to form functional channels on its own but that has a major regulatory function. Kv2.3r can coassemble selectively with other alpha-subunits to form functional heteromultimeric K+ channels with kinetic properties that differ from those of the parent channels. Kv2.3r is expressed exclusively in the brain, being concentrated particularly in neocortical neurons. The functional expression of this regulatory alpha-subunit represents a novel mechanism without precedents in voltage-gated channels, which might contribute to further increase the functional diversity of K+ channels necessary to specify the intrinsic electrical properties of individual neurons.
Amino Acid Sequence↗
Recombinant Ca2+ channels get O2 sensitive.
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Animals↗
Pore mutations in Shaker K+ channels distinguish between the sites of tetraethylammonium blockade and C-type inactivation.
1. We have studied the effect of external K+ and tetraethylammonium (TEA) on several mutants of Shaker B K+ channels with amino acid substitutions in the pore which alter TEA affinity and the rate of C-type inactivation. In all channels studied high external K+ makes C-type inactivation slower. 2. In the wild-type channel, TEA blockade is voltage dependent and produces slowing of the inactivation time course. However, in the double mutant channel (T449Y, D447E) TEA blockade, although of higher affinity, is voltage independent and does not affect the rate of C-type inactivation. 3. Mutants with a charged amino acid at position 449 (T449K and T449E) are resistant to TEA block. In these channels, C-type inactivation is also unaffected by TEA. 4. These results indicate that the sites where TEA blocks and competes with C-type inactivation can be segregated. To modulate inactivation, TEA must enter deeply into the channel mouth. These results suggest that C-type inactivation is not due to a large molecular rearrangement in the outer channel vestibule, but it is essentially produced by a conformational change restricted to a local site in the pore.
Animals↗
Oxygen sensing by ion channels.
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Animals↗
Synthesis of a photoaffinity labeling analogue of the inactivating peptide of the Shaker B potassium channel.
A photoactivatable derivative of the inactivating peptide of the Shaker B potassium channel (ShB peptide) has been synthesized from ShB peptide containing an added cysteine residue at the peptide carboxy-terminus and 1-(p-azidosalicylamido)-4-(iodoacetamido)butane. The peptide derivative restores rapid inactivation in the deletion mutant Shaker Bdelta6-46 potassium channel in a manner indistinguishable from that of the wild-type ShB peptide. Also, both peptides display similar conformational behavior when challenged in vitro by an artificial model target that partly imitates the properties of the putative receptor site for the inactivating peptide in the Shaker B potassium channel. Therefore, we conclude that both functionally and conformationally the photoreactive peptide derivative is an adequate analogue of the wild-type ShB peptide, suitable for photoaffinity labeling of its binding site in the Shaker B potassium channel. Moreover, because the ShB peptide also serves as an efficient inactivating peptide for a large variety of other potassium channels, it appears that the photoreactive analogue may be useful to explore homologous sites in many different channel proteins.
Affinity Labels↗
Contrasting effects of hypoxia on cytosolic Ca2+ spikes in conduit and resistance myocytes of the rabbit pulmonary artery.
1. The effects of hypoxia on cytosolic Ca2+ ([Ca2+]i) and spontaneous cytosolic Ca2+ spikes were examined in fura 2-loaded myocytes isolated from conduit and resistance branches of the rabbit pulmonary artery. In all myocyte classes, generation of the Ca2+ spikes was modulated by basal [Ca2+]i which, in turn, was influenced by the influx of Ca2+ through L-type Ca2+ channels of the plasmalemma. 2. Conduit and resistance myocytes responded distinctly to hypoxia. In most conduit myocytes (approximately 82% of total; n = 23) exposure to hypoxia reduced basal [Ca2+]i. This effect was often associated with the abolition of the Ca2+ spikes. Hypoxia gave rise to two main responses in resistance myocytes. In a subset of resistance myocytes (41 % of total; n = 34) hypoxia incremented basal [Ca2+]i but reduced Ca2+ spike amplitude. This response mimicked the effect of membrane depolarization with K+ and was reverted by nifedipine or the removal of extracellular Ca2+. In a second subset of resistance myocytes (59% of total; n = 34) hypoxia decreased basal [Ca2+]i and, in most cases, increased spike amplitude; a response counteracted by depolarization with K+. 3. These results indicate that hypoxia can differentially modulate [Ca2+]i in smooth muscle cells from large and small diameter pulmonary vessels through a dual effect on transmembrane Ca2+ influx. Our observations further demonstrate the longitudinal heterogeneity of myocytes along the pulmonary arterial tree and help to explain the hypoxic vasomotor responses in the pulmonary circulation.
Animals↗
Differential oxygen sensitivity of calcium channels in rabbit smooth muscle cells of conduit and resistance pulmonary arteries.
1. Calcium currents were recorded from smooth muscle cells dispersed from conduit and resistance rabbit pulmonary arteries. We tested the hypothesis that Ca2+ channel activity was regulated by environmental O2 tension. 2. Conduit (proximal) and resistance (distal) myocytes differ in their Ca2+ channel density and responses to low PO2. Ca2+ current density in distal myocytes (20.7 +/- 7.4 pA pF-1, n = 10) is almost twice the value in proximal myocytes (12.6 +/- 5.5 pA pF-1, n = 39). In proximal myocytes, the predominant response to reductions in PO2 is inhibition of the calcium current (n = 12) at membrane potentials below 0 mV, whereas potentiation of current amplitude is observed in distal myocytes (n = 24). 3. Hypoxia also produces opposite shifts in the conductance-voltage relationships along the voltage axis. The average displacements induced by low PO2 are +5.05 +/- 2.98 mV (n = 5) in proximal myocytes and -6.06 +/- 2.45 (n = 10) in distal myocytes. 4. These findings demonstrate longitudinal differences in Ca2+ channel density and O2 sensitivity in myocytes along the pulmonary arterial tree. These results may help to understand the differential reactivity to hypoxia of the pulmonary vasculature: vasodilatation in conduit arteries and vasoconstriction in resistance vessels.
Animals↗
Modulation of voltage-gated Ca2+ channels by O2 tension. Significance for arterial oxygen chemoreception.
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Animals↗