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R I Hume

Publications and source records attributed to R I Hume.

At least 19 recordsLinked to original sources
PubMed2003

Expression level dependent changes in the properties of P2X2 receptors.

The currents of P2X(2) receptors expressed in Xenopus oocytes or HEK293 cells show significant cell-to-cell variation in many properties including the rate of desensitization and the magnitude of potentiation by zinc or acidic pH. In this study, we examined whether differences in expression levels underlie this variability. We injected Xenopus oocytes with different concentrations of RNA encoding rat P2X(2) to give a wide range of maximum current amplitudes, and then measured the potentiation of responses to 10 micro M adenosine 5'-triphosphate (ATP) by zinc or acidic pH. Individual oocytes showed potentiation ratios that ranged from 1.4- to 25-fold. Oocytes with small amplitude responses to a saturating concentration of ATP tended to have larger potentiation ratios than oocytes with large amplitude responses. This phenomenon was explained by an inverse correlation between the EC(50) for ATP and the maximum current amplitude, with the EC(50) decreasing from about 37 to 7 micro M as expression level increased. In contrast, the Hill coefficient was not correlated with the maximum current amplitude. Truncated receptors lacking the last 76 amino acids also showed an inverse correlation between the EC(50) and the maximum current amplitude. Thus, the interactions that cause expression-dependent changes in P2X(2) receptor properties must involve domains proximal to position H397.

Adenosine Triphosphate↗
PubMed2000

Generation of neurons by transient expression of neural bHLH proteins in mammalian cells.

Basic helix-loop-helix (bHLH) transcription factors are known to function during mammalian neurogenesis. Here we show that transient transfection of vectors expressing neuroD2, MASH1, ngn1 or related neural bHLH proteins, with their putative dimerization partner E12, can convert mouse P19 embryonal carcinoma cells into differentiated neurons. Transfected cells express numerous neuron-specific proteins, adopt a neuronal morphology and are electrically excitable. Thus, the expression of neural bHLH proteins is sufficient to confer a neuronal fate on uncommitted mammalian cells. Neuronal differentiation of transfected cells is preceded by elevated expression of the cyclin-dependent kinase inhibitor p27(Kip1) and cell cycle withdrawal. This demonstrates that the bHLH proteins can link neuronal differentiation to withdrawal from the cell cycle, possibly by activating the expression of p27(Kip1). The ability to generate mammalian neurons by transient expression of neural bHLH proteins should create new opportunities for studying neurogenesis and devising neural repair strategies.

Animals↗
PubMed1998

Identification of a site that modifies desensitization of P2X2 receptors.

The time course of desensitization of P2X2 receptors expressed in Xenopus oocytes and HEK 293 cells was examined. We found that there was virtually no desensitization in response to 5 microM ATP, but that responses to 50 microM ATP desensitized with a highly variable time course. The time constant of desensitization varied 250fold among oocytes (from 4 to over 1000 seconds) and 10 fold among HEK cells (from 4 to 40 seconds). Mutation of D349, which lies at the cytoplasmic end of the second transmembrane domain (to either A, N, or R), resulted in a dramatic acceleration of the median rate of desensitization, and eliminated variation in the rate of desensitization. In contrast, mutation of four other charged residues in putative transmembrane domains had no effect on desensitization. These results suggest that P2X2 receptor desensitization is strongly modulated by the intracellular environment, and that D349 is essential for this modulation.

Adenosine Triphosphate↗
PubMed1998

Two mechanisms for inward rectification of current flow through the purinoceptor P2X2 class of ATP-gated channels.

1. The ATP receptor subunit P2X2 was expressed in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. ATP-activated currents were studied with two-electrode voltage clamp recordings from oocytes, whole-cell recordings from HEK 293 cells, and outside-out patch clamp recordings from both cell types. The steady-state current-voltage (I-V) relation showed profound inward rectification in all recording configurations. 2. Recordings from outside-out patches demonstrated that inward rectification does not require intracellular Mg2+ or polyamines, and that inward rectification was present when the same solution was used on both sides of the patch. 3. Voltage jump experiments were performed to evaluate the voltage dependence of channel gating. After fast voltage jumps, instantaneous current jumps were followed by substantial relaxations to the steady state. The time course of the current relaxations could be fitted by single exponential functions. The instantaneous I-V relation was less inwardly rectifying than the steady-state I-V relation; however, it was not linear. 4. Single channel recordings indicated that the single channel conductance became smaller when the membrane potential became more positive. This decrease could quantitatively account for inward rectification of the instantaneous I-V relation. 5. We conclude that inward rectification of P2X2 is due to two mechanisms: voltage-dependent gating and voltage dependence of the single channel conductance.

Adenosine Triphosphate↗
PubMed1995

Changes in responsiveness to extracellular ATP in chick skeletal muscle during development and upon denervation.

Skeletal muscles in developing chick embryos were tested for responsiveness to adenosine 5'-triphosphate (ATP), a substance known to depolarize chick skeletal muscle in culture. The sensitivity to extracellular ATP was tested at various stages of development in five different muscles; pectoralis superficia, anterior latissimus dorsi, posterior latissimus dorsi, sartorious, and gastrocnemius. At the earliest time that muscles were tested (Embryonic Day 6, stage 30 of Hamburger and Hamilton, 1951) application of ATP(50-100 microM) elicited vigorous contractions in all five muscles, but within a few days (Embryonic Day 17, stage 43) none of the muscles contracted in response to ATP. Sensitivity declined at approximately the same time in all five of these muscles. Intracellular recordings made from muscle fibers near the time of hatching (Embryonic Days 18-21 or Postnatal Days 1-2) indicated that the loss of the ability to contract in response to ATP was due to the total loss of responsiveness to ATP. Surgical denervation of the anterior latissimus dorsi and posterior latissimus dorsi was performed in a series of chicks 1-2 days after hatching, and the ability of these muscles to contract in response to ATP was tested 3-10 days after the surgery. Contractions in response to ATP were present in many of the muscles. Thus denervation of muscles in newly hatched chicks led to the reappearance of sensitivity to ATP. The disappearance of ATP responsiveness shortly after muscles become innervated and the reappearance of ATP responsiveness following denervation suggest that the expression of ATP responsiveness is regulated by motor neurons.

Adenosine Triphosphate↗
PubMed1995

Dihydropyridines and verapamil inhibit voltage-dependent K+ current in isolated outer hair cells of the guinea pig.

Dihydropyridines and verapamil are widely used as blockers of voltage-dependent Ca++ channels. In this work we show that these compounds can have a direct blocking action on a class of voltage-activated potassium channels. Voltage-dependent whole-cell currents were recorded from isolated guinea-pig outer hair cells (OHCs) under conditions such that the free Ca++ concentration in both the internal and external solutions was minimized. A substantial Ca(++)-independent K+ current was revealed by this procedure. Both conventional K+ and Ca++ channel ligands inhibited this current. The order of potency (in terms of the half inhibitory concentrations (IC50) of channel inhibitors) was: nimodipine (6 microM) > Bay K 8644 (8 microM) > verapamil (11 microM) > 4-aminopyridine (22 microM) > nifedipine (32 microM) > quinine (49 microM) > TEA (10236 microM). Except for verapamil, these channel ligands reduced the size of the K+ currents without much alteration of the time course of the currents. In contrast, verapamil caused a more than 10-fold increase in the apparent inactivation rate of the K+ currents without significantly altering the activation of the currents. The observation that relatively low concentrations of calcium channel ligands can directly inhibit potassium currents in isolated OHCs indicates that caution should be taken when these pharmacological agents are used as tools for studying cochlear hair cell physiology.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Contact with myelin evokes a release of calcium from internal stores in neonatal rat oligodendrocytes in vitro.

The response of neonatal rat oligodendrocytes to contact with myelin extracts prepared from the central and peripheral nervous system was examined. Contact with either CNS myelin or PNS myelin resulted in collapse of the fine structure of the leading edge of oligodendrocytes in vitro. The collapse of the fine structure of oligodendrocyte processes was preceded by a substantial (approximately fivefold) increase in intracellular free calcium concentration. The calcium concentration increase was due, at least in part, to a release of calcium from internal stores, since it persisted when extracellular calcium was removed by chelation with EGTA. The increase in calcium concentration and the coincident morphological change suggest that oligodendrocytes might be able to recognize and react to specific molecules on the surface of other oligodendrocytes.

Animals↗
PubMed1994

Locus coeruleus neuron growth cones and spinal cord regeneration.

One of the major impediments to successful recovery of function after a spinal cord injury is thought to be the reaction of the neuronal growth cone to inhibitory influences in the local environment in or around the site of the injury. The growth cones of locus coeruleus neurons studied in vitro collapsed upon contact with an extract of CNS myelin but did not collapse on contact with an extract of PNS myelin. Coincident with the collapse of the growth cone, was an increase in internal free calcium concentration that was predominantly the result of an influx of calcium through channels in the plasma membrane. Omega-conotoxin, which specifically blocks N-type voltage-gated calcium channels, blocked both the myelin-induced calcium influx and the coincident collapse of the growth cone.

Animals↗
PubMed1993

Single potassium channel currents activated by extracellular ATP in developing chick skeletal muscle: a role for second messengers.

1. In developing chick skeletal muscle, extracellular ATP activates an early excitatory current and a delayed potassium current. Previous work had shown that the potassium current elicited by ATP is sensitive to temperature and activates with a delay of nearly 1 s, suggesting that a second messenger is involved. The existence of a second messenger was confirmed by the observation that single potassium channels were activated in cell-attached patches, when ATP was applied outside of the patch pipette. 2. Two classes of ATP-activated potassium-channel currents were observed in cell-attached patches: one had a slope conductance of 23 pS, whereas the other had a slope conductance of 51 pS. 3. Pharmacological manipulations suggested that activation of the whole-cell potassium current by ATP did not require cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositol 1,4,5-triphosphate (IP3), nitric oxide, or a rise in internal free calcium. Additional pharmacological experiments suggested that activation of the whole-cell potassium current might not require activation of a G protein and probably did not involve intracellular protein phosphorylation. 4. The ability of arachidonic acid and its metabolites to activate potassium channels in chick skeletal muscle was also tested. Arachidonic acid, several prostaglandins and several leukotrienes activated whole-cell potassium currents. However, results with several inhibitors suggested that arachidonic acid and its metabolites are not necessary for activation of the whole-cell potassium current by ATP. 5. In excised outside-out membrane patches, ATP activated a single class of potassium channels. The slope conductance of these channels indicated that they are likely to be identical to the smaller of the two classes of second messenger activated potassium channels observed in cell-attached patches. 6. The observation that the larger class of potassium channels observed in cell-attached patches was absent in excised patches suggests that activation of these channels by ATP requires a cytosolic factor that is easily dialyzed away. In contrast, the observation that the smaller class of potassium channels could still be activated by ATP in excised patches suggests that the two classes of potassium channels are activated by different mechanisms. These results also indicate that all the molecules involved in coupling ATP receptor activation to opening of the smaller class of potassium channels remain closely associated with an excised patch. One possible explanation is that there might be an intramembranous second messenger.

Adenosine Triphosphate↗
PubMed1993

Voltage-dependent block by neomycin of the ATP-induced whole cell current of guinea-pig outer hair cells.

1. The effects of externally applied ATP and neomycin on whole cell currents of isolated guinea pig cochlear outer hair cells (OHCs) were studied using the whole cell voltage-clamp technique. In OCHs held at -70 mV, ATP activated a large inward current. In the presence of neomycin, the ATP-induced whole cell current activated along a relatively unaltered time course, but the current then decreased to a reduced steady level. The neomycin inhibition of the ATP-induced current was dose dependent. The half-inhibitory concentration (IC50) of neomycin measured at steady state was estimated to be 90 microM. 2. Neomycin inhibition of the ATP response could not be reversed by increasing the concentration of ATP, indicating that the effect was noncompetitive. The inhibition was voltage dependent and was greatly reduced when OHCs were held at positive potentials. 3. Cells treated with 100 microM ATP gave maximal current responses. Addition of neomycin substantially increased membrane current noise of the 100 microM ATP responses. When neomycin concentration was varied from 10 to 500 microM, the current noise level peaked between 50 and 100 microM. The noise increase was observed at negative holding potentials but not at positive potentials. 4. The neomycin-induced whole cell current noise was used to estimate the size of the underlying elementary current. The ATP-induced single channel current of OHCs at -70 mV was estimated to be approximately 0.3 pA. The number of ATP-activated channels in a single OHC was estimated to be in the range of a few thousand. 5. The characteristics of the neomycin inhibition of ATP-induced currents were consistent with an open channel blocking mechanism. Analysis of the voltage dependence of the steady state neomycin inhibition suggested a neomycin binding site at an electrical distance of 0.3 from the extracellular side.

Acoustic Stimulation↗
PubMed1993

Omega-conotoxin prevents myelin-evoked growth cone collapse in neonatal rat locus coeruleus neurons in vitro.

The response of neonatal rat locus coeruleus neurons to contact with myelin extracts prepared from the CNS and PNS was examined. The growth cones of these neurons collapsed following contact with central myelin, but continued to elongate on contact with peripheral myelin. Central myelin elicited an increase in the intracellular free calcium concentration in these growth cones, while peripheral myelin did not. This increase appeared to require transmembrane calcium flux, since it was blocked by extracellular EGTA, and also by extracellular cobalt. These neurons express N- and L-type calcium channels. Exposure to 5 microM omega-conotoxin GVIA, a specific blocker of N-type channels, prevented both the myelin-evoked increase in growth cone calcium concentration and the collapse of growth cones.

Animals↗
PubMed1992

Structural determinants of barium permeation and rectification in non-NMDA glutamate receptor channels.

A single site in recombinant glutamate receptor channels of the GluR1-GluR4 family has been previously identified as a key regulator of ion permeation. The natural amino acid at this position (arginine in GluR2 but glutamine in GluR1, GluR3, and GluR4) determines both the ability to pass outward current and the divalent cation permeability of kainate-activated receptor channels. By mutagenesis of GluR6, we demonstrated that the same site also controls the ability to pass outward current in another non-NMDA receptor family. Additional mutations at and near this site in GluR3 indicated that the position of the arginine is critical to function, that the ability to pass outward current is not necessarily linked to low barium permeability, and that the size as well as the charge of the side chain at this position influences barium permeation. These results provide evidence that this site forms part of the selectivity filter of glutamate receptor channels.

Amino Acid Sequence↗
PubMed1991

Identification of a site in glutamate receptor subunits that controls calcium permeability.

The neurotransmitter glutamate mediates excitatory synaptic transmission throughout the brain. A family of genes encoding subunits of the non-N-methyl-D-aspartate (non-NMDA) type of glutamate receptor has been cloned. Some combinations of these subunits assemble into receptors with a substantial permeability to calcium, whereas others do not. To investigate the structural features that control ion permeation through these ligand-gated channels, mutant receptor subunits with single-amino acid changes were constructed. Mutation of a certain amino acid that results in a net charge change (from glutamine to arginine or vice versa) alters both the current-voltage relation and the calcium permeability of non-NMDA receptors. A site has thus been identified that regulates the permeation properties of these glutamate receptors.

Amino Acid Sequence↗
PubMed1991

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons.

We have examined the physiological properties of transmission at newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Chick neurons were labeled with fluorescent carbocyanine dyes before they were placed into culture (Honig and Hume, 1986), and were studied by making intracellular recordings during the first 2 weeks of coculture. Evoked monosynaptic excitatory postsynaptic potentials (EPSPs) were not observed until 48 h of coculture. Beyond this time, the frequency with which connected pairs could be found did not vary greatly with time. With repetitive stimulation, the evoked monosynaptic EPSPs fluctuated in amplitude from trial to trial and showed depression at frequencies as low as 1 Hz. To gain further information about the quantitative properties of transmission at newly formed synapses, we analyzed the pattern of fluctuations of delayed release EPSPs. In mature systems, delayed release EPSPs are known to represent responses to single quanta, or to the synchronous release of a small number of quanta. For more than half of the connections we studied, the histograms of delayed release EPSPs were extremely broad. This result suggested that either quantal responses are drawn from a continuous distribution that has a large coefficient of variation or that there are several distinct size classes of quantal responses. The pattern of fluctuations of monosynaptic EPSPs was consistent with both of these possibilities, and was inconsistent with the possibility that monosynaptic EPSPs are composed of quantal subunits with very little intrinsic variation. Although variation in the size of responses to single quanta might arise in a number of ways, one attractive explanation for our results is that the density and type of acetylcholine receptors varies among the different synaptic sites on the surface of developing sympathetic ganglion neurons.

Action Potentials↗
PubMed1991

A receptor that is highly specific for extracellular ATP in developing chick skeletal muscle in vitro.

1. Extracellular adenosine 5'-triphosphate (ATP) activated an early excitatory conductance followed by a late potassium conductance in developing chick skeletal muscle. A series of ATP analogues were tested for their ability to activate these two conductances. All compounds tested were either agonists for both responses or for neither. Furthermore, the potency of agonists was similar for the two responses. 2. The order of potency for agonists was ATP approximately adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) approximately 2-methylthio-ATP (2-CH3S-ATP) greater than 2'-deoxy-ATP approximately 3'-deoxy-ATP greater than adenosine 5'-tetraphosphate (ATP-OPO3) approximately adenosine 5'-diphosphate (ADP). Many other ATP analogues were not agonists. 3. Activation of the excitatory response did not require divalent cations. Furthermore, the concentration-response relation of the excitatory response was similar when ATP was applied as the free anion of ATP (ATP4-) or complexed with a divalent cation (M.ATP2-). 4. Three antagonists of the ATP response were characterized. 8-Br-ATP was a weak antagonist, while 2',3'-dialdehyde-ATP and DIDS (4,4'-diisocyanatostilbene-2,2'-disulphonic acid) were potent irreversible inhibitors. The two conductances were equally affected by these antagonists. 5. These results suggest that both ATP responses are activated through the same receptor type, or two very similar receptors.

Adenosine Triphosphate↗
PubMed1990

Permeation of both cations and anions through a single class of ATP-activated ion channels in developing chick skeletal muscle.

Micromolar concentrations of extracellular adenosine 5'-triphosphate (ATP) elicit a rapid excitatory response in developing chick skeletal muscle. Excitation is the result of a simultaneous increase in membrane permeability to sodium, potassium, and chloride ions. In the present study we quantify the selectivity of the ATP response, and provide evidence that a single class of ATP-activated ion channels conducts both cations and anions. Experiments were performed on myoballs using the whole-cell patch-clamp technique. We estimated permeability ratios by measuring the shift in reversal potential when one ion was substituted for another. We found that monovalent cations, divalent cations, and monovalent anions all permeate the membrane during the ATP response, and that there was only moderate selectivity between many of these ions. Calcium was the most permeant ion tested. To determine if ATP activates a single class of channels that conducts both cations and anions, or if ATP activates separate classes of cation and anion channels, we analyzed the fluctuations about the mean current induced by ATP. Ionic conditions were arranged so that the reversal potential for cations was +50 mV and the reversal potential for anions was -50 mV. Under these conditions, if ATP activates a single class of channels, ATP should not evoke an increase in noise at the reversal potential of the ATP current. However, if ATP activates separate classes of cation and anion channels, ATP should evoke a significant increase in noise at the reversal potential of the ATP current. At both +40 and -50 mV ATP elicited a clear increase in noise, but at the reversal potential of the ATP current (-5 mV), no increase in noise above background was seen. These results indicate that there is only a single class of excitatory ATP-activated channels, which do not select by charge. Based on analysis of the noise spectrum, the conductance of individual channels is estimated to be 0.2-0.4 pS.

Adenosine Triphosphate↗