The inhibition of the voltage-dependent, K+ M-current (1~) following receptor-independent C prote... more The inhibition of the voltage-dependent, K+ M-current (1~) following receptor-independent C protein activation with controlled intracellular perfusion of nonhydrolyzable GTP analogs had an exponential time course, with rates hyperbolically dependent on CTP analog concentration, and a limiting value of 0.53 min-'. The inhibitory agonist muscarine caused a concentration-dependent acceleration of the rate of nucleotide-induced inhibition, with a plateau of about 20 min-l and an exponential time course. In neurons not treated with nucleotide analogs the IM recovery rate following agonist removal was 3-7 min-l. It is proposed that the overall kinetics of the transduction pathway for IM modulation is governed by the agonist-dependent kinetics of nucleotide interaction with G proteins. A simple model of IM modulation based on G proteins' kinetics has been developed. These data suggest a possible cellular process responsible for the time course of slow synaptic potentials caused by IM inhibition in sympathetic neurons.
The inhibition of the voltage-dependent, K+ M-current (1~) following receptor-independent C prote... more The inhibition of the voltage-dependent, K+ M-current (1~) following receptor-independent C protein activation with controlled intracellular perfusion of nonhydrolyzable GTP analogs had an exponential time course, with rates hyperbolically dependent on CTP analog concentration, and a limiting value of 0.53 min-'. The inhibitory agonist muscarine caused a concentration-dependent acceleration of the rate of nucleotide-induced inhibition, with a plateau of about 20 min-l and an exponential time course. In neurons not treated with nucleotide analogs the IM recovery rate following agonist removal was 3-7 min-l. It is proposed that the overall kinetics of the transduction pathway for IM modulation is governed by the agonist-dependent kinetics of nucleotide interaction with G proteins. A simple model of IM modulation based on G proteins' kinetics has been developed. These data suggest a possible cellular process responsible for the time course of slow synaptic potentials caused by IM inhibition in sympathetic neurons.
Endogenous enkephalins and ␦ opiates affect sensory function and pain sensation by inhibiting syn... more Endogenous enkephalins and ␦ opiates affect sensory function and pain sensation by inhibiting synaptic transmission in sensory circuits via delta opioid receptors (DORs). DORs have long been suspected of mediating these effects by modulating voltage-dependent Ca 2ϩ entry in primary sensory neurons. However, not only has this hypothesis never been validated in these cells, but in fact several previous studies have only turned up negative results. By using whole-cell current recordings, we show that the ␦ enkephalin analog [D-Ala 2 , D-Leu 5 ]-enkephalin (DADLE) inhibits, via DORs, L-, N-, P-, and Q-high voltageactivated Ca 2ϩ channel currents in cultured rat dorsal root ganglion (DRG) neurons. The percentage of responding cells was remarkably high (75%) within a novel subpopulation of substance P-containing neurons compared with the other cells (18-35%). DADLE (1 M) inhibited 32% of the total barium current through calcium channels (I Ba ). A ␦ (naltrindole, 1 M), but not a (-funaltrexamine, 5 M), antagonist prevented the DADLE response, whereas a DOR-2 subtype (deltorphin-II, 100 nM), but not a DOR-1 (DPDPE, 1 M), agonist mimicked the response. L-, N-, P-, and Q-type currents contributed, on average, 18, 48, 14, and 16% to the total I Ba and 19, 50, 26, and 20% to the DADLE-sensitive current, respectively. The druginsensitive R-type current component was not affected by the agonist. This work represents the first demonstration that DORs modulate Ca 2ϩ entry in sensory neurons and suggests that ␦ opioids could affect diverse Ca 2ϩ -dependent processes linked to Ca 2ϩ influx through different high-voltage-activated channel types.
The involvement of G proteins in the transduction mechanism of M current (Im) inhibition by extra... more The involvement of G proteins in the transduction mechanism of M current (Im) inhibition by extracellular ligands in bullfrog sympathetic neurons was examined using the hydrolysis resistant nucleotide analogues GTPγS and GDPβS. Im was recorded in large (40–60 μm) isolated neurons using the patch-clamp technique in the whole-cell configuration, as well as in neurons from the intact ganglion impaled with conventional microelectrodes. In whole-cell recordings Im could be recorded without significant loss for 1 h or more provided ATP was present in the patch pipette. Muscarine, D-Ala6-LHRH, substance P and UTP reversibly inhibited Im in isolated control neurons, with full and rapid recovery of the current following agonist washout. Dialysis of isolated neurons with various concentrations of GTPγS (1–100 μM) affected, in a dose-dependent manner, the recovery of Im after its inhibition by brief agonist application. With 50 μM GTPμS, Im inhibition became completely irreversible. Similarly, the reversibility of Im inhibition by muscarine was reduced or abolished by the iontophoretic injection of GTPμS through a second microelectrode into neurons of the intact ganglion. GTPμS by itself caused a slow, agonist-independent suppression of Im in dialysed neurons, thus mimicking agonist action. Dialysis of isolated neurons with GDPβS (100—500 μM) attenuated by half or more the magnitude of Im inhibition by agonist as compared to control neurons. In addition, GDPβS attenuated the response of a given neuron to muscarine and D-Ala6-LHRH, and caused slow increase of Im, as a function of dialysis time. Incubation (2–72 h, 4–36°C) of isolated neurons or intact ganglions with activated pertussis toxin had no effect on the response to muscarine. Toxin injections to experimental animals were equally ineffective. In contrast to Im, the additional inward current with increase in conductance induced by muscarine and D-Ala6-LHRH reversed with agonist washout in GTPγS-dialysed neurons, although more slowly than in control neurons. The results in this study indicate that a G protein, possibly pertussis toxin-insensitive, provides a common coupling step linking muscarinic, substance P, D-Ala6-LHRH and UTP receptors to the inhibition of M current.
Membrane depolarization relieves the G protein-mediated inhibition or block of high threshold Ca2... more Membrane depolarization relieves the G protein-mediated inhibition or block of high threshold Ca2+ channel currents. We found that the net rate of reblocking depended on the extent of G protein activation. With low intracellular concentrations of GTP gamma S reblocking rates resembled inactivation rates; with higher concentrations reblocking rates increased progressively. Reblocking kinetics were fit with a sum of two exponential functions having time constants (in ms) tau F greater than or equal to 10 and tau S greater than or equal to 30. Unblock during depolarization was fit by a single exponential function with time constant tau A similar to tau F. A model was developed in which unblocking followed dissociation of a blocking molecule, possibly the G protein itself, from Ca2+ channels, and reblocking occurred at rates that depended on the concentration of the blocking molecule. The time course of Ca2+ entry and thus presynaptic Ca2+ levels can be regulated by both the concentration of the G-protein-dependent blocking particle and membrane potential.
This report further characterizes associative long-term synaptic modification of the ipsilateral ... more This report further characterizes associative long-term synaptic modification of the ipsilateral and contralateral synapses formed by the bilateral entorhinal cortical (EC) projection to the dentate gyrus (DG). The experimental model is the anesthetized hooded rat. The quatntitative results qualify this system as a model for studying the rules of associative synaptic modification formulated in terms of individual synapses. Bilateral DG microelectrodes recorded both ipsilateral and contralateral EC-DG responses before and after brief, high-frequency EC conditioning stimulatin. The weak contralateral pathway receivewd high-frequency coditioning before, during, or after similar conditioning of the strong, converging ipsilateral pathway. Statistical analyses revealed two types of significant, dissociated synaptic modifications, which depend on the relationship of the ipsilateral and contralateral afferents. First, contralateral EC-DG responses potentiated, depressed, or showed no change when the collateral ipsilateral responses concurrently either potentiated or remained unchanged. Correlation and contingency table analysis indicated that changes in the contralateral synaptic responses are not well predicted by changes at either neighboring synapses of the converging ipsilateral pathway or at synapses of the collateral ipsilateral pathway. The contingencies of associated pre- and postsynaptic activation determined by the conditioning paradigm, however accurately predicted the altered synaptic responses of both ipsilateral and contralateral rately predicted the altered synaptic responses of both ipsilateral and contralateral EC-DG pathways. The results imply that associative synaptic modification in the EC-DG system is specific to individual synapses and requires both appropriate presynaptic and postsynaptic activation. Because this system provides suitable controls for nonspecific dissociable, the EC-DG system can be used to study further those rules of activity-dependent associative modification that are formulated in terms of individual synapses. The discussion briefly considers published rules modification, pointing out several rules that are not consistent with the ecperimental observations and one that agrees with the present results.
Abstract: The Shaw-type K+ channel Kv3.1 was stably transfected in human embryonic kidney cells. ... more Abstract: The Shaw-type K+ channel Kv3.1 was stably transfected in human embryonic kidney cells. Voltage dependence of activation, K+ permeability, sensitivity to external tetraethylammonium, and unitary conductance were similar to Kv 3.1 channels expressed transiently in Xenopus oocytes. Kv 3.1 channels appear to be regulated because the protein kinase C activator phorbol 12,13-dibutyrate decreased Kv 3.1 currents. Based on these results, we find that the stable expression of voltage-gated K+ channels in human embryonic kidney cells appears to be well suited for analysis of both biophysical and biochemical regulatory processes.
The inhibition of the voltage-dependent, K+ M-current (1~) following receptor-independent C prote... more The inhibition of the voltage-dependent, K+ M-current (1~) following receptor-independent C protein activation with controlled intracellular perfusion of nonhydrolyzable GTP analogs had an exponential time course, with rates hyperbolically dependent on CTP analog concentration, and a limiting value of 0.53 min-'. The inhibitory agonist muscarine caused a concentration-dependent acceleration of the rate of nucleotide-induced inhibition, with a plateau of about 20 min-l and an exponential time course. In neurons not treated with nucleotide analogs the IM recovery rate following agonist removal was 3-7 min-l. It is proposed that the overall kinetics of the transduction pathway for IM modulation is governed by the agonist-dependent kinetics of nucleotide interaction with G proteins. A simple model of IM modulation based on G proteins' kinetics has been developed. These data suggest a possible cellular process responsible for the time course of slow synaptic potentials caused by IM inhibition in sympathetic neurons.
The inhibition of the voltage-dependent, K+ M-current (1~) following receptor-independent C prote... more The inhibition of the voltage-dependent, K+ M-current (1~) following receptor-independent C protein activation with controlled intracellular perfusion of nonhydrolyzable GTP analogs had an exponential time course, with rates hyperbolically dependent on CTP analog concentration, and a limiting value of 0.53 min-'. The inhibitory agonist muscarine caused a concentration-dependent acceleration of the rate of nucleotide-induced inhibition, with a plateau of about 20 min-l and an exponential time course. In neurons not treated with nucleotide analogs the IM recovery rate following agonist removal was 3-7 min-l. It is proposed that the overall kinetics of the transduction pathway for IM modulation is governed by the agonist-dependent kinetics of nucleotide interaction with G proteins. A simple model of IM modulation based on G proteins' kinetics has been developed. These data suggest a possible cellular process responsible for the time course of slow synaptic potentials caused by IM inhibition in sympathetic neurons.
Endogenous enkephalins and ␦ opiates affect sensory function and pain sensation by inhibiting syn... more Endogenous enkephalins and ␦ opiates affect sensory function and pain sensation by inhibiting synaptic transmission in sensory circuits via delta opioid receptors (DORs). DORs have long been suspected of mediating these effects by modulating voltage-dependent Ca 2ϩ entry in primary sensory neurons. However, not only has this hypothesis never been validated in these cells, but in fact several previous studies have only turned up negative results. By using whole-cell current recordings, we show that the ␦ enkephalin analog [D-Ala 2 , D-Leu 5 ]-enkephalin (DADLE) inhibits, via DORs, L-, N-, P-, and Q-high voltageactivated Ca 2ϩ channel currents in cultured rat dorsal root ganglion (DRG) neurons. The percentage of responding cells was remarkably high (75%) within a novel subpopulation of substance P-containing neurons compared with the other cells (18-35%). DADLE (1 M) inhibited 32% of the total barium current through calcium channels (I Ba ). A ␦ (naltrindole, 1 M), but not a (-funaltrexamine, 5 M), antagonist prevented the DADLE response, whereas a DOR-2 subtype (deltorphin-II, 100 nM), but not a DOR-1 (DPDPE, 1 M), agonist mimicked the response. L-, N-, P-, and Q-type currents contributed, on average, 18, 48, 14, and 16% to the total I Ba and 19, 50, 26, and 20% to the DADLE-sensitive current, respectively. The druginsensitive R-type current component was not affected by the agonist. This work represents the first demonstration that DORs modulate Ca 2ϩ entry in sensory neurons and suggests that ␦ opioids could affect diverse Ca 2ϩ -dependent processes linked to Ca 2ϩ influx through different high-voltage-activated channel types.
The involvement of G proteins in the transduction mechanism of M current (Im) inhibition by extra... more The involvement of G proteins in the transduction mechanism of M current (Im) inhibition by extracellular ligands in bullfrog sympathetic neurons was examined using the hydrolysis resistant nucleotide analogues GTPγS and GDPβS. Im was recorded in large (40–60 μm) isolated neurons using the patch-clamp technique in the whole-cell configuration, as well as in neurons from the intact ganglion impaled with conventional microelectrodes. In whole-cell recordings Im could be recorded without significant loss for 1 h or more provided ATP was present in the patch pipette. Muscarine, D-Ala6-LHRH, substance P and UTP reversibly inhibited Im in isolated control neurons, with full and rapid recovery of the current following agonist washout. Dialysis of isolated neurons with various concentrations of GTPγS (1–100 μM) affected, in a dose-dependent manner, the recovery of Im after its inhibition by brief agonist application. With 50 μM GTPμS, Im inhibition became completely irreversible. Similarly, the reversibility of Im inhibition by muscarine was reduced or abolished by the iontophoretic injection of GTPμS through a second microelectrode into neurons of the intact ganglion. GTPμS by itself caused a slow, agonist-independent suppression of Im in dialysed neurons, thus mimicking agonist action. Dialysis of isolated neurons with GDPβS (100—500 μM) attenuated by half or more the magnitude of Im inhibition by agonist as compared to control neurons. In addition, GDPβS attenuated the response of a given neuron to muscarine and D-Ala6-LHRH, and caused slow increase of Im, as a function of dialysis time. Incubation (2–72 h, 4–36°C) of isolated neurons or intact ganglions with activated pertussis toxin had no effect on the response to muscarine. Toxin injections to experimental animals were equally ineffective. In contrast to Im, the additional inward current with increase in conductance induced by muscarine and D-Ala6-LHRH reversed with agonist washout in GTPγS-dialysed neurons, although more slowly than in control neurons. The results in this study indicate that a G protein, possibly pertussis toxin-insensitive, provides a common coupling step linking muscarinic, substance P, D-Ala6-LHRH and UTP receptors to the inhibition of M current.
Membrane depolarization relieves the G protein-mediated inhibition or block of high threshold Ca2... more Membrane depolarization relieves the G protein-mediated inhibition or block of high threshold Ca2+ channel currents. We found that the net rate of reblocking depended on the extent of G protein activation. With low intracellular concentrations of GTP gamma S reblocking rates resembled inactivation rates; with higher concentrations reblocking rates increased progressively. Reblocking kinetics were fit with a sum of two exponential functions having time constants (in ms) tau F greater than or equal to 10 and tau S greater than or equal to 30. Unblock during depolarization was fit by a single exponential function with time constant tau A similar to tau F. A model was developed in which unblocking followed dissociation of a blocking molecule, possibly the G protein itself, from Ca2+ channels, and reblocking occurred at rates that depended on the concentration of the blocking molecule. The time course of Ca2+ entry and thus presynaptic Ca2+ levels can be regulated by both the concentration of the G-protein-dependent blocking particle and membrane potential.
This report further characterizes associative long-term synaptic modification of the ipsilateral ... more This report further characterizes associative long-term synaptic modification of the ipsilateral and contralateral synapses formed by the bilateral entorhinal cortical (EC) projection to the dentate gyrus (DG). The experimental model is the anesthetized hooded rat. The quatntitative results qualify this system as a model for studying the rules of associative synaptic modification formulated in terms of individual synapses. Bilateral DG microelectrodes recorded both ipsilateral and contralateral EC-DG responses before and after brief, high-frequency EC conditioning stimulatin. The weak contralateral pathway receivewd high-frequency coditioning before, during, or after similar conditioning of the strong, converging ipsilateral pathway. Statistical analyses revealed two types of significant, dissociated synaptic modifications, which depend on the relationship of the ipsilateral and contralateral afferents. First, contralateral EC-DG responses potentiated, depressed, or showed no change when the collateral ipsilateral responses concurrently either potentiated or remained unchanged. Correlation and contingency table analysis indicated that changes in the contralateral synaptic responses are not well predicted by changes at either neighboring synapses of the converging ipsilateral pathway or at synapses of the collateral ipsilateral pathway. The contingencies of associated pre- and postsynaptic activation determined by the conditioning paradigm, however accurately predicted the altered synaptic responses of both ipsilateral and contralateral rately predicted the altered synaptic responses of both ipsilateral and contralateral EC-DG pathways. The results imply that associative synaptic modification in the EC-DG system is specific to individual synapses and requires both appropriate presynaptic and postsynaptic activation. Because this system provides suitable controls for nonspecific dissociable, the EC-DG system can be used to study further those rules of activity-dependent associative modification that are formulated in terms of individual synapses. The discussion briefly considers published rules modification, pointing out several rules that are not consistent with the ecperimental observations and one that agrees with the present results.
Abstract: The Shaw-type K+ channel Kv3.1 was stably transfected in human embryonic kidney cells. ... more Abstract: The Shaw-type K+ channel Kv3.1 was stably transfected in human embryonic kidney cells. Voltage dependence of activation, K+ permeability, sensitivity to external tetraethylammonium, and unitary conductance were similar to Kv 3.1 channels expressed transiently in Xenopus oocytes. Kv 3.1 channels appear to be regulated because the protein kinase C activator phorbol 12,13-dibutyrate decreased Kv 3.1 currents. Based on these results, we find that the stable expression of voltage-gated K+ channels in human embryonic kidney cells appears to be well suited for analysis of both biophysical and biochemical regulatory processes.
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Papers by Hector Lopez