Agonist-induced Receptor Activation Research Articles

Mutagenesis of important amino acid reveals unconventional homologous internalization of β 1-adrenergic receptor

Aims The study was designed to examine the internalization of Asp104Lys mutant of β 1-adrenergic receptor (β 1-AR) and compared to other mutant (Asp104Ala) and wild type receptors. Moreover, this study needs to perform the role of GRK2 (βARK1) and β-arrestin1 on this internalization of Asp104Lys mutant of β 1-AR. Main methods Binding affinity, functional potency of agonist and agonist-induced internalization were determined for wild type and both mutants of β 1-ARs stably expressed in HEK 293 cells as assessed by [ 3H] CGP12177 radioligand. We have performed GRK2 and β-arrestin1 expression levels by western blot analysis and also performed internalization of this mutant receptor after over expression and deletion of β-arrestin1 gene. Key findings In the present study, the binding affinity of (−)-isoproterenol for both mutants were significantly decreased compared to wild type. Though the mutant Asp104Ala showed agonist-induced receptor activation, interestingly this mutant was not internalized. However, the mutant Asp104Lys, which showed uncoupling with G protein, was internalized 31.77 ± 3.13% from cell surface. Asp104Lys mutant produced the same level of GRK2 expression in (–)-isoproterenol induced stimulation of wild type receptor and addition of (–)-isoproterenol further increased GRK2 expression in mutant receptors. In addition, overexpression of β-arrestin1 in mutant Asp104Lys promoted (39.75 ± 2.19%) and knockdown of β-arrestin1 by siRNA decreased (3.55 ± 1.75%) internalization compared to Asp104Lys mutant of β 1-ARs. Significance The present studies suggest that Asp104Lys mutant β 1-ARs triggers unconventional homologous internalization induced by G protein independent signals, where GRK2 and β-arrestin1 play an important role for β 1-AR internalization.

Multiple Residues in the Second Extracellular Loop Are Critical for M3 Muscarinic Acetylcholine Receptor Activation

Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys(220) residue, Gln(207), Gly(211), Arg(213), Gly(218), Ile(222), Phe(224), Leu(225), and Pro(228) were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance.

Phylogenetic appearance of neuropeptide S precursor proteins in tetrapods

Sleep and emotional behavior are two hallmarks of vertebrate animal behavior, implying that specialized neuronal circuits and dedicated neurochemical messengers may have been developed during evolution to regulate such complex behaviors. Neuropeptide S (NPS) is a newly identified peptide transmitter that activates a typical G protein-coupled receptor. Central administration of NPS produces profound arousal, enhances wakefulness and suppresses all stages of sleep. In addition, NPS can alleviate behavioral responses to stress by producing anxiolytic-like effects. A bioinformatic analysis of current genome databases revealed that the NPS peptide precursor gene is present in all vertebrates with the exception of fish. A high level of sequence conservation, especially of aminoterminal structures was detected, indicating stringent requirements for agonist-induced receptor activation. Duplication of the NPS precursor gene was only found in one out of two marsupial species with sufficient genome coverage ( Monodelphis domestica; opossum), indicating that the duplicated opossum NPS sequence might have arisen as an isolated event. Pharmacological analysis of both Monodelphis NPS peptides revealed that only the closely related NPS peptide retained agonistic activity at NPS receptors. The duplicated precursor might be either a pseudogene or could have evolved different receptor selectivity. Together, these data show that NPS is a relatively recent gene in vertebrate evolution whose appearance might coincide with its specialized physiological functions in terrestrial vertebrates.

Agonist activity of naloxone benzoylhydrazone at recombinant and native opioid receptors

1. In the present study, we examined the pharmacological activity of the putative kappa3-opioid receptor agonist naloxone benzoylhydrazone (NalBzoH) at recombinant human opioid receptors individually expressed in Chinese hamster ovary (CHO) cells and native opioid receptors present in rat striatum. 2. At the mu-opioid receptor (MOR), NalBzoH stimulated guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding (pEC50=8.59) and inhibited cyclic AMP accumulation (pEC50=8.74) with maximal effects (Emax) corresponding to 55 and 65% of those obtained with the MOR agonist DAMGO, respectively. The MOR antagonist CTAP blocked the stimulatory effects of NalBzoH and DAMGO with similar potencies. 3. At the kappa-opioid receptor (KOR), NalBzoH stimulated [35S]GTPgammaS binding (pEC50=9.70) and inhibited cyclic AMP formation (pEC50=9.45) as effectively as the selective KOR agonist (-)-U-50,488. The NalBzoH effect was blocked by the KOR antagonist nor-binaltorphimine (nor-BNI) (pKi=10.30). 4. In CHO cells expressing the delta-opioid receptor (DOR), NalBzoH increased [35S]GTPgammaS binding (pEC50=8.49) and inhibited cyclic AMP formation (pEC50=8.61) almost as effectively as the DOR agonist DPDPE. Naltrindole (NTI), a selective DOR antagonist, completely blocked the response to NalBzoH (pKi of 10.40). 5. In CHO cells expressing the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), NalBzoH failed to exert agonist effects and antagonized the agonist-induced receptor activation. 6. When compared to other opioid receptor ligands, NalBzoH showed an efficacy that was lower than that of morphine at MOR, but higher at KOR and DOR. 7. In rat striatum, NalBzoH enhanced [35S]GTPgammaS binding and inhibited adenylyl cyclase activity. These effects were antagonized by either CTAP, nor-BNI or NTI, each antagonist blocking a fraction of the NalBzoH response. 8. These data demonstrate that NalBzoH displays agonist activity at MOR, DOR and KOR expressed either in a heterologous cell system or in a native environment.

Contribution of the extracellular domain to agonist-induced and constitutive LH receptor activation

Glycoprotein hormone receptors like the LH and FSH receptor are G-protein-coupled receptors characterized by a large N-terminally extracellular domain. While it is well established that the ectodomain is responsible for specific hormone binding, the activation mechanism is still not understood. To investigate the functional role of the ectodomain, we N-terminally deleted various portions of the LH receptor ectodomain. Upon addition of amino acids 1–39 of the V2 vasopressin receptor to the N-termini of the truncated LHRs, these mutants transiently expressed in COS-7 cells revealed cell surface targeting comparable to the wildtype receptor. However, mutant LHRs did not display constitutive cAMP formation, nor revealed activation by hCG. To exclude that this lack to hCG response was due to negligible agonist affinity, single-chain hCG was fused to the wildtype or truncated receptors. While the wildtype LHR fused to hCG exhibited constitutive cAMP formation after transient expression, and could further be stimulated by exogenous hormone, none of the truncated receptors displayed elevated basal activity indicating that hCG does not functionally interact with the heptahelical LHR portion and that the entire intact ectodomain is required for hormone-induced receptor activation. To examine if truncated LHRs are fundamentally misfolded and principally unable to interact with Gs, several naturally occurring activating mutations were introduced into the transmembrane helices of a truncated LHR. While all mutations induced constitutive activity in the wildtype LHR, only one mutation evoked agonist-independent cAMP formation via the truncation mutant. The activity of most non-active mutations could be recovered by N-terminal linking of the TSHR or FSHR ectodomain to the transmembrane helices of the LHR, whereas linking of the hLGR7 ectodomain was ineffective. These data highlight an as yet unappreciated structural role of the LHR ectodomain for the stabilization of a distinct conformation of the heptahelical portion poised for activation by agonist or activating mutations.

Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation.

Platelet fibrinogen receptor activation is a critical step in platelet plug formation. The fibrinogen receptor (integrin alphaIIbbeta3) is activated by agonist-mediated G(q) stimulation and resultant phospholipase C activation. We investigated the role of downstream signalling events from phospholipase C, namely the activation of protein kinase C (PKC) and rise in intracellular calcium, in agonist-induced fibrinogen receptor activation using Ro 31-8220 (a PKC inhibitor) or dimethyl BAPTA [5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid], a high-affinity calcium chelator. All the experiments were performed with human platelets treated with aspirin, to avoid positive feedback from thromboxane A2. In the presence of Ro 31-8220, platelet aggregation caused by U46619 was completely inhibited while no effect or partial inhibition was seen with ADP and the thrombin-receptor-activating peptide SFLLRN, respectively. In the presence of intracellular dimethyl BAPTA, ADP- and U46619-induced aggregation and anti-alphaIIbbeta3 antibody PAC-1 binding were completely abolished. However, similar to the effects of Ro 31-8220, dimethyl BAPTA only partially inhibited SFLLRN-induced aggregation, and was accompanied by diminished dense-granule secretion. When either PKC activation or intracellular calcium release was abrogated, aggregation and fibrinogen receptor activation with U46619 or SFLLRN was partially restored by additional selective activation of the G(i) signalling pathway. In contrast, when both PKC activity and intracellular calcium increase were simultaneously inhibited, the complete inhibition of aggregation that occurred in response to either U46619 or SFLLRN could not be restored with concomitant G(i) signalling. We conclude that, while the PKC- and calcium-regulated signalling pathways are capable of inducing activating fibrinogen receptor independently and that each can synergize with G(i) signalling to cause irreversible fibrinogen receptor activation, both pathways act synergistically to effect irreversible fibrinogen receptor activation.

Evidence for a Model of Agonist-induced Activation of 5-Hydroxytryptamine 2A Serotonin Receptors That Involves the Disruption of a Strong Ionic Interaction between Helices 3 and 6

5-Hydroxytryptamine 2A (5-HT2A) receptors are essential for the actions of serotonin (5-hydroxytryptamine (5-HT)) on physiological processes as diverse as vascular smooth muscle contraction, platelet aggregation, perception, and emotion. In this study, we investigated the molecular mechanism(s) by which 5-HT activates 5-HT2A receptors using a combination of approaches including site-directed mutagenesis, molecular modeling, and pharmacological analysis using the sensitive, cell-based functional assay R-SAT. Alanine-scanning mutagenesis of residues close to the intracellular end of H6 of the 5-HT2A receptor implicated glutamate Glu-318(6.30) in receptor activation, as also predicted by a newly constructed molecular model of the 5-HT2A receptor, which was based on the x-ray structure of bovine rhodopsin. Close examination of the molecular model suggested that Glu-318(6.30) could form a strong ionic interaction with Arg-173(3.50) of the highly conserved "(D/E)RY motif" located at the interface between the third transmembrane segment and the second intracellular loop (i2). A direct prediction of this hypothesis, that disrupting this ionic interaction by an E318(6.30)R mutation would lead to a highly constitutively active receptor with enhanced affinity for agonist, was confirmed using R-SAT. Taken together, these results predict that the disruption of a strong ionic interaction between transmembrane helices 3 and 6 of 5-HT2A receptors is essential for agonist-induced receptor activation and, as recently predicted by ourselves (B. L. Roth and D. A. Shapiro (2001) Expert Opin. Ther. Targets 5, 685-695) and others, that this may represent a general mechanism of activation for many, but not all, G-protein-coupled receptors.

Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.

Potent desensitization of human P2X 3 receptors by diadenosine polyphosphates

In this study, the receptor desensitizing effects of diadenosine polyphosphates at recombinant human P2X 3 (hP2X 3) receptors were examined. Administration of Ap 3A, Ap 4A, Ap 5A or Ap 6A inhibited the hP2X 3 receptor-mediated response to a subsequent application of 3 μM αβ-methyleneATP (αβ-meATP), in a concentration-dependent manner, with IC 50 values 2707, 42, 59 and 46 nM, respectively. These agonists did not desensitize αβ-meATP responses mediated by the slowly desensitizing heteromeric human P2X 2/3 receptor. hP2X 3 receptor desensitization was reversible and was not observed following the increase in intracellular Ca 2+ levels produced by carbachol. A similar pattern of desensitization evoked by Ap 5A was also observed using electrophysiological recordings of Xenopus oocytes expressing hP2X 3 receptors. These data demonstrate that diadenosine polyphosphates, found endogenously in the central nervous system, can readily desensitize hP2X 3 receptors at nanomolar concentrations that are 10-fold lower than are required to produce agonist-induced receptor activation. Thus, P2X 3 receptor desensitization by diadenosine polyphosphates may provide an important modulatory mechanism of P2X 3 receptor activation in vivo.

Dependence of agonist activation on an aromatic moiety in the DPLIY motif of the gonadotropin-releasing hormone receptor.

In the GnRH receptor, the NPX2-3Y motif that is present in the seventh transmembrane helix of most G protein-coupled receptors is unusual in containing Asp instead of Asn but retains the highly conserved Tyr residue. The importance of this aromatic residue in the DPLIY sequence of the GnRH receptor function was analyzed by replacing Tyr322 with Ala or Phe residues. The Y322A mutant receptor expressed in COS-7 cells had high agonist binding affinity, but its ability to interact with G protein(s) and to activate inositol phosphate production in response to GnRH was abolished. Although functionally inactive, the Y322A mutant receptor was internalized at about 50% of the rate of the wild type receptor in agonist-treated cells. When Tyr322 was replaced with Phe to preserve its aromatic nature, the Y322F mutant receptor displayed normal G protein activation and inositol phosphate responses to GnRH and was internalized in the same manner as the wild type receptor. These findings demonstrate that the aromatic moiety of the Tyr322 component of the DPLIY motif in the GnRH receptor is a critical determinant of agonist-induced receptor activation and signal transduction.

Activation of membrane receptors

Many extracellular messengers interact with discriminate receptors on the cell surface. Some of bound ligands activate receptors whereas others fail to do so. Only activated receptors are capable of generating and transferring signal through the membrane. Recent advances in our understanding of agonist-induced and constitutive receptor activation suggest several molecular mechanisms for receptor activation, signal generation and transmembrane signal transfer.

Functional role in ligand binding and receptor activation of an asparagine residue present in the sixth transmembrane domain of all muscarinic acetylcholine receptors.

The molecular mechanisms through which muscarinic receptors are activated upon binding of the neurotransmitter acetylcholine (ACh) are still poorly understood. Classical structure-function relationship studies have previously established that the ACh ester moiety plays a key role in muscarinic receptor recognition and activation. Consistent with this notion, all recently proposed three-dimensional muscarinic receptor models predict that an asparagine residue present in transmembrane domain VI of all muscarinic receptors is critically involved in the binding of the ACh ester moiety by means of hydrogen bonding. To test the correctness of this hypothesis, we created several mutant m3 muscarinic receptors in which this residue (Asn507) was replaced with alanine, serine, or aspartic acid. Radioligand binding studies with transfected COS-7 cells showed that, in contrast to the predictions made based on molecular modeling studies, all three mutant receptors were able to bind ACh and the structurally related muscarinic agonist, carbachol, with high affinities which differed from the corresponding wild type values by less than 5-fold. However, all three mutations led to dramatic reductions (235-28,300-fold) in binding affinities for certain subclasses of muscarinic antagonists including atropine-like agents and pirenzepine. The m3(Asn507-->Ala) and m3(Asn507-->Asp) mutant receptors were able to mediate carbachol-induced phosphatidylinositol hydrolysis in a fashion similar to that of the wild type receptor. Interestingly, the m3(Asn507-->Ser) mutant receptor displayed about 2-fold increased basal inositol phosphate levels, raising the possibility that it is constitutively active. In conclusion, our data suggest that the asparagine residue present in transmembrane domain VI of all muscarinic receptors is not critical for ACh binding and agonist-induced receptor activation, but plays a key role in the binding of certain subclasses of muscarinic antagonists.

A Metabolic View of Receptor Activation in Cultured Cells Following Cryopreservation

The effect of cryopreservation on agonist-induced receptor activation in mammalian cells was investigated with the Cytosensor microphysiometer, a biosensor that monitors cellular metabolic activity by measuring changes in extracellular pH. In this study, two different cell types—nonadherent TF-1 cells (from a human erythroleukemia patient) and adherent WT3 cells (CHO-K1 cells transfected with the m 1 muscarinic acetylcholine receptor)—were cryopreserved by freezing in a disposable cell capsule used in the microphysiometer. The recovery of metabolic activity by TF-1 cells was observed over ∼1 h following thawing. Responses of the TF-1 cells to granulocyte-macrophage colony-stimulating factor (GM-CSF) and platelet activating factor (PAF) were measured before cryopreservation and 90 min after thawing. The GM-CSF and PAF responses retained 71 ± 14% and 73 ± 10% of maximum stimulation, respectively. Post-thaw cholinergic stimulation of WT3 cells was 73 ± 9% of its level in similarly treated but unfrozen cells. Cryopreservation caused no detectable difference in desensitization of the response due to repeated application of carbachol. These results demonstrate the feasibility of pharmacological studies with cryopreserved cells in the microphysiometer and further suggest that the microphysiometer may be useful in exploring the biological consequences of cryopreservation in the early post-thaw period.

Role of conserved threonine and tyrosine residues in acetylcholine binding and muscarinic receptor activation. A study with m3 muscarinic receptor point mutants.

Structure-function relationship studies of the m3 muscarinic acetylcholine receptor have recently identified a series of threonine and tyrosine residues (all located within the hydrophobic receptor core) that are critically involved in acetylcholine binding (Wess, J., Gdula, D., and Brann, M.R. (1991) EMBO J. 10, 3729-3734). To gain further insight into the functional roles of these amino acids, the agonist binding properties of six rat m3 muscarinic receptor point mutants, in which the critical threonine and tyrosine residues had been individually replaced by alanine and phenylalanine, respectively, were studied in greater detail following their transient expression in COS-7 cells. The binding profiles of a series of acetylcholine derivatives suggest that the altered threonine and tyrosine residues are primarily involved in the interaction of the acetylcholine ester moiety with the receptor protein. The two m3 receptor point mutants, Thr234----Ala and Tyr506----Phe, which showed the most pronounced decreases in acetylcholine binding affinities (approximately 40-60-fold as compared with the wild-type receptor), were stably expressed in CHO cells for further functional analysis. Both mutant receptors were found to be severely impaired in their ability to stimulate agonist-dependent phosphatidylinositol hydrolysis. Consistent with this observation, acetylcholine binding to the two mutant receptors was not significantly affected by addition of the GTP analog Gpp(NH)p (5'-guanylyl imidodiphosphate). Our data suggest that Thr234 and Tyr506 (located within transmembrane domains V and VI, respectively), which are conserved among all muscarinic receptors (m1-m5), may play an important role in agonist-induced muscarinic receptor activation.

Activation of single ion channels from toad retinal rod inner segments by cyclic GMP: concentration dependence.

1. Patch-clamp recordings of single cyclic GMP-activated channels from toad rod photoreceptors were made in inside-out membrane patches containing only one such channel. Patches were obtained from the inner segment, where the density of cyclic GMP-activated channels is lower than in the outer segment, making one-channel patches possible. The dependence of channel gating on cyclic GMP concentration ([cyclic GMP]) was studied. At low [cyclic GMP] (5-10 microM), channel openings were infrequent and occurred as bursts of rapid opening and closing. As [cyclic GMP] was increased, bursts became more frequent, until at 1 mM the activity fused into long bouts of rapid flicker between open and closed states. 2. The duration of brief openings and closings (flicker) within bursts was not affected by [cyclic GMP]. This suggests that the rapid flicker within bursts results from an intrinsic channel property not associated with agonist-induced receptor activation. 3. At 10 microM-cyclic GMP, the distribution of closed times was fitted by a sum of three exponential components. The briefest, with time constant averaging 0.29 ms, corresponded to the brief closings within bursts, while the two longer components, with time constants averaging 3.5 and 32 ms, corresponded to much longer closings between bursts. At 0.5 or 1 mM-cyclic GMP (saturation), the longer components disappeared, and the distribution of closed times was fitted by a single-exponential equation with the same time constant as the briefest component observed at lower concentrations. 4. Because the channel continued to flicker even at high [cyclic GMP], the maximal probability of being in the open state (Po) did not approach 1.0, averaging 0.30 +/- 0.05 (N = 8). The relation between Po and [cyclic GMP] was fitted by the Hill equation with an exponent of 3, suggesting that binding of cyclic GMP to multiple sites is required to open the channel.

Interactions of substance P antagonists with serotonin in the mouse spinal cord

Administered intrathecally (IT) to mice, the putative substance P antagonist [D-Pro2,D-Trp7,9-substance P (DPDT) blocked substance P- and serotonin-induced reciprocal hindlimb scratching with ID50 values of 4.6 (2.9-6.9) and 3.0 (1.9-4.8) micrograms, respectively. The duration of this antagonistic effect was 90-120 min. In contrast, DPDT did not block bombesin-, somatostatin-, glycine- or glutamate-induced scratching. These data indicate that DPDT is an effective antagonist of serotonin-induced behaviors in the mouse spinal cord. Phenoxybenzamine (IT) also blocked substance P- and serotonin-induced scratching. Its onset of action was more rapid for serotonin than for DPDT implying differences in agonist-induced receptor activation. Methysergide (IT) blocked serotonin-induced scratching [ID50 = 0.7 (0.3-1.5) micrograms], but not substance P-induced scratching. Similar to DPDT, [D-Arg1,D-Trp7,9,Leu11]-substance P, [des-Arg1,D-Pro2, D-Trp7,9]-substance P(2-11) and [D-Pro4,D-Trp7,9]-substance P(4-11) blocked substance P and serotonin-induced scratching. In contrast, [D-Pro2,D-Phe7,D-Trp9]-substance P and [D-Pro4,D-Trp7,9,10]-substance P(4-11) blocked only substance P-induced scratching. Thus, some, but not all putative substance P antagonists may also be behavioral antagonists of serotonin in the mouse spinal cord.