Whole-cell patch-clamp recordings were used to characterize calcium channel types that are modulated by mu-opioid receptor activation in rat dorsal root ganglion (DRG) neurons. Five distinct components of high-threshold calcium current were isolated on the basis of their sensitivity to the selective channel blockers omega-conotoxin GVIA, nifedipine, omega-conotoxin MVIIC, or omega-agatoxin IVA. The mu-opioid selective agonist Tyr-Pro-NMePhe-D-Pro-NH2 (PLO17) routinely suppressed high-threshold currents and this effect was always reduced by omega-conotoxin GVIA. A fraction of PLO17-sensitive current remained after omega-conotoxin GVIA that was eliminated by application of omega-agatoxin IVA alone or in combination with omega-conotoxin MVIIC. Nifedipine had no effect on mu-opioid responses nor did PLO17 affect the slow component of tail current induced by Bay K 8644. These data suggest that mu-opioid receptors are negatively coupled to three types of calcium channels in rat DRG neurons, including an omega-conotoxin GVIA-sensitive (N-type) channel, an omega-agatoxin IVA-sensitive (P-type) channel and an omega-conotoxin MVIIC-sensitive, nifedipine/GVIA/omega-Aga IVA-resistant (presumptive Q-type) channel.

Whole-cell patch-clamp recordings were used to examine the regulation of voltage-dependent calcium channels by mu- and kappa-opioid receptors in acutely isolated rat dorsal root ganglion (DRG) sensory neurons. Agonists selective for either mu- (Tyr-Pro-NMePhe-D-Pro-NH2, PLO17) or kappa-opioid receptors (dynorphin A, U69,593) inhibited high-threshold calcium currents in a reversible and naloxone-sensitive manner, whereas administration of D-Pen2,5-enkephalin, a delta-selective agonist, was without effect. However, none of the opioids reduced low-threshold T- type currents. The inhibitory effects of PLO17 were blocked by the irreversible mu-opioid antagonist beta-funaltrexamine but not the kappa- opioid antagonist nor-binaltorphimine, while responses to kappa-opioid agonists showed the opposite pattern of antagonist sensitivity. In addition, many cells responded to both PLO17 and dynorphin A (or U69,593), and in these neurons the inhibitory response to one agonist was occluded when tested in the presence of the other. These data suggest that mu- and kappa-opioid receptors are coexpressed on at least some DRG neurons and appear to be functionally coupled to a common pool of calcium channels. Both rapidly inactivating (transient) and sustained components of high-threshold current, arising from pharmacologically distinct types of calcium channels, were identified in our neurons. Activation of mu-opioid receptors selectively reduced the transient component of currents evoked at +10 mV from Vh = -80 mV, while sparing the sustained component. The transient component was irreversibly blocked by the N-type channel antagonist omega-conotoxin GVIA (omega-CgTx), and in one-half of the neurons there was a concomitant loss of the response to PLO17. In the remaining neurons, PLO17 continued to reduce a small fraction of omega-CgTx-insensitive current and subsequent administration of the L-type channel blocker nifedipine in saturating concentrations failed to reduce the opioid- induced inhibitory effect. These data demonstrate that mu-opioid receptors are negatively coupled to several pharmacologically distinct types of calcium channels in DRG sensory neurons, one that was blocked by omega-CgTx and thus likely to be N-type, and a second that was resistant to blockade by N- and L-type channel blockers.

Whole-cell patch-clamp recordings were performed together with time-resolved measurements of membrane capacitance (C m ) in nerve terminals acutely dissociated from neurohypophysis of adult rats to investigate modulation of Ca 2ϩ currents and secretion by activation of opioid receptors. Bath superfusion of the -opioid agonists U69,593 (0.3-1 M), dynorphin A (1 M), or U50,488H (1-3 M) reversibly suppressed the peak amplitude of Ca 2ϩ currents 32.7 Ϯ 2.7% (in 41 of 56 terminals), 37.4 Ϯ 5.3% (in 5 of 8 terminals), and 33.5 Ϯ 8.1% (in 5 of 10 terminals), respectively. In contrast, tests in 11 terminals revealed no effect of the -opioid agonist [D-Pen 2,5 ]-enkephalin (1-3 M; n ϭ 7) or of the ␦-agonist Tyr-D-Ala-Gly-N-Me-PheGly-ol (1 M; n ϭ 4) on Ca 2ϩ currents. Three components of high-threshold current were distinguished on the basis of their sensitivity to blockade by -conotoxin GVIA, nicardipine, and -conotoxin MVIIC: N-, L-, and P/Q-type current, respectively.Administration of U69,593 inhibited N-type current in these nerve terminals on average 32%, whereas L-type current was reduced 64%, and P/Q-type current was inhibited 28%. Monitoring of changes in C m in response to brief depolarizing steps revealed that the -opioid-induced reductions in N-, L-, or P/Q-type currents were accompanied by attenuations in two kinetically distinct components of Ca 2ϩ -dependent exocytotic release. These data provide strong evidence of a functional linkage between blockade of Ca 2ϩ influx through voltagedependent Ca 2ϩ channels and inhibitory modulation of release by presynaptic opioid receptors in mammalian central nerve endings.

It has recently been shown that the activation of mu-opioid receptors inhibits several components of calcium channel current in rat DRG sensory neurons. mu-Opioid receptors, acting through the pertussis toxin (PTX)-sensitive substrate Gi, also reduce the activity of neuronal adenylate cyclase, but the relationship of this effect to changes in calcium channel activity has yet to be determined. Using whole-cell recordings from acutely isolated rat DRG neurons, we examined the ability of the mu-opioid-selective agonist Tyr-Pro-NMe-Phe-D-Pro-NH2 (PLO17) to reduce calcium current after treatment with PTX and in the presence of the nonhydrolyzable GTP analog guanosine 5'-[-thio]triphosphate (GTP gamma S), to assess the role of G-proteins in the coupling of mu-opioid receptors to calcium channels. Inhibition of current by PLO17 was mimicked or rendered irreversible by intracellular administration of GTP gamma S, an activator of G-proteins, and was blocked by pretreatment of neurons with PTX. In contrast, when the catalytic subunit of cAMP-dependent protein kinase was included in the recording pipette, calcium currents increased in magnitude throughout the recording without attenuation of responses to PLO17. Thus, the mu-opioid-induced inhibition of calcium current occurs through activation of a Gi- or G(o)-type G-protein, but independent of changes in adenylate cyclase activity. As a first step in identifying this G-protein, we compared the ability of several antisera directed against specific regions of Gi and G(o)alpha subunits to block the inhibition in current by PLO17. Intracellular dialysis with an antiserum specific for G(o) (GC/2) attenuated calcium current inhibition by PLO17 in five of six neurons by an average of 75%. In contrast, there was no attenuation in the response to PLO17 when neurons were dialyzed with an anti-Gi1 alpha/Gi2 alpha antiserum (AS/7) or antibodies specific for alpha subunits of Gi proteins (Gi1/Gi2 or Gi3) in an identical manner. These results suggest that in rat DRG neurons mu-opioid receptors couple to calcium channels via the PTX-sensitive G(o) subclass of GTP-binding proteins.

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