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The Journal of Neuroscience, November 1, 1999, 19(21):9235-9241

An R-Type Ca²⁺ Current in Neurohypophysial Terminals Preferentially Regulates Oxytocin Secretion

Gang Wang¹, Govindan Dayanithi², Robert Newcomb³, and José R. Lemos¹

¹ Department of Physiology and Neuroscience Program, University of Massachusetts Medical School, Worcester, Massachusetts 01655, ² UPR9055-CNRS, Biologie des Neurones Endocrines, Montpellier, Cedex 5, France, and ³ Elan Pharmaceuticals, Menlo Park, California 94025

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Multiple types of voltage-dependent Ca²⁺ channels are involved in the regulation of neurotransmitter release (Tsien et al., 1991; Dunlap et al., 1995). In the nerve terminals of the neurohypophysis, the roles of L-, N-, and P/Q-type Ca²⁺ channels in neuropeptide release have been identified previously (Wang et al., 1997a). Although the L- and N-type Ca²⁺ currents play equivalent roles in both vasopressin and oxytocin release, the P/Q-type Ca²⁺ current only regulates vasopressin release. An oxytocin-release and Ca²⁺ current component is resistant to the L-, N-, and P/Q-type Ca²⁺ channel blockers but is inhibited by Ni²⁺. A new polypeptide toxin, SNX-482, which is a specific _1E-type Ca²⁺ channel blocker (Newcomb et al., 1998), was used to characterize the biophysical properties of this resistant Ca²⁺ current component and its role in neuropeptide release. This resistant component was dose dependently inhibited by SNX-482, with an IC₅₀ of 4.1 nM. Furthermore, SNX-482 did not affect the other Ca²⁺ current types in these CNS terminals. Like the N- and P/Q-type Ca²⁺ currents, this SNX-482-sensitive transient Ca²⁺ current is high-threshold activated and shows moderate steady-state inactivation. At the same concentrations, SNX-482 blocked the component of oxytocin, but not of vasopressin, release that was resistant to the other channel blockers, indicating a preferential role for this type of Ca²⁺ current in oxytocin release from neurohypophysial terminals. Our results suggest that an _1E or "R"-type Ca²⁺ channel exists in oxytocinergic nerve terminals and, thus, functions in controlling only oxytocin release from the rat neurohypophysis.

Key words: class E (_1E) Ca²⁺ channel; secretion; SNX-482; vasopressin; posterior pituitary; oxytocin

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Voltage-dependent channels are responsible for the Ca²⁺ that enters nerve terminals and elicits vesicular release of neurotransmitters (Augustine et al., 1987). Neurotransmitter release in the CNS is regulated by multiple types of Ca²⁺ channels (Dunlap et al., 1995). A number of studies have defined several electrophysiologically distinct Ca²⁺ channels on neuronal cell bodies: L-, N-, T-, and P-types (Fox et al., 1987; Bean, 1989; Tsien et al., 1991; Llinas et al., 1992). Other classes of channels, such as the Q- and R-types, have been revealed by molecular cloning (Snutch and Reiner, 1992; Ellinor et al., 1993; Sather et al., 1993; Perez-Reyes et al., 1998) and the use of Ca²⁺ antagonists (Olivera et al., 1984; Hillyard et al., 1992; Ramachandran et al., 1993; Newcomb et al., 1998). The specific role at CNS terminals of these different types of Ca²⁺ channels, however, is still unclear.

The N-type channel seems to be involved in classical neurotransmission (Hirning et al., 1988), whereas the L-type is known to regulate the secretion of certain peptides (Cazalis et al., 1987; Dunlap et al., 1995). The class E (_1E) and G (_1G) Ca²⁺ channels have been localized recently to the CNS (Westenbroek et al., 1995; Perez-Reyes et al., 1998). However, the phenotype of the expressed _1E channel is controversial (Snutch and Reiner, 1992; Randall and Tsien, 1995), and the biophysical properties of the class E current in CNS terminals remain to be determined.

To determine any role for class E channels in CNS secretion (see Wu et al., 1998, 1999), we studied the nerve terminals of the rat neurohypophysis. This is a population of relatively homogeneous peptidergic nerve endings that allows comparative study by a number of different techniques. This has been a very useful model system for characterization of nerve terminal Ca²⁺ channels (Lemos and Nowycky, 1989; Wang et al., 1992, 1997a; Wang and Lemos, 1994; Fisher and Bourque, 1995) and of mechanisms underlying depolarization-secretion coupling (Cazalis et al., 1987; Lim et al., 1990; Lindau et al., 1992; Wang et al., 1993b, 1997a; Branchaw et al., 1998). We have shown previously that "L"- and "N"-type Ca²⁺ channels exist in nerve terminals of the neurohypophysis (Lemos and Nowycky, 1989; Wang et al., 1992) and that they control both vasopressin (AVP) and oxytocin (OT) release, except for a significant resistant component (Cazalis et al., 1987; Dayanithi et al., 1988; Wang et al., 1993b). More recently we have shown that a "Q"-type Ca²⁺ current component also exists in approximately one-half of these CNS terminals (Wang et al., 1997a). Moreover, when blockers of the Q-type Ca²⁺ current were added to the terminals, the resistant component of AVP release was essentially abolished. In contrast, a similar resistant component of OT release was unchanged by the same concentrations of the Q-type channel blockers.

Most recently we have shown that purified native SNX-482, a specific _1E channel blocker, could inhibit the neurohypophysial Ca²⁺ current (Newcomb et al., 1998). This led us to examine, using a combination of pharmacological and biophysical techniques, whether class E or R-type Ca²⁺ channels might also exist on these CNS terminals and functionally contribute to neurosecretion.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Electrophysiological recordings. As we have described previously (Wang et al., 1997a), after sedation by CO₂ the rats were killed by decapitation using a guillotine. The neurohypophysis was then excised, following previous protocols, and homogenized in a solution containing (in mM): sucrose, 270; HEPES-Tris, 10; and K-EGTA, 0.01, pH 7.25 (Cazalis et al., 1987). All chemicals were obtained from Sigma (St. Louis, MO). The isolated neurohypophysial nerve terminals could be identified under an inverted microscope (Nordmann et al., 1987). Normal Locke's solution [containing (in mM), NaCl, 145; KCl, 5; CaCl₂, 2.2; MgCl₂, 1; Na-HEPES, 10; and glucose, 15, pH 7.35] was then used to perfuse the terminals. Before patch-clamp recordings, the terminals (usually 5-8 µm in diameter) were perfused with the 5 mM Ba²⁺ (replacing CaCl₂) Locke's solution, which also contained 1 µM TTX with 0.02% BSA. To obtain perforated-patch (Rae et al., 1991) recordings in the "whole-terminal" configuration (Hamill et al., 1981), freshly made amphotericin B (240-300 µg/ml) was mixed with the pipette solution that contained (in mM): Cs-glutamate, 135; HEPES, 10; glucose, 5; CaCl₂, 2; MgCl₂, 1; and TEA, 20, pH 7.25.

As reported previously (Wang et al., 1997a), the perforated-patch recording configuration enables us to overcome problems with the rundown of Ca²⁺ currents that complicated former studies (Lemos and Nowycky, 1989; Wang et al., 1992; Wang and Lemos, 1994; Fisher and Bourque, 1995; Branchaw et al., 1998). Only terminals with perforated-patch access resistances of <10 M were chosen for further recordings. The Ba²⁺ current (I_Ba), which was activated by depolarizing from 80 to +10 mV and demonstrated both transient and long-lasting components (see, e.g., Fig. 1A), could be maintained for >1 hr without appreciable rundown. The I_Ba was filtered at 3 kHz and sampled at 10 kHz. pClamp (Axon Instruments, Burlingame, CA) was used for acquisition and analysis of data.

Peptide release. Rat neurohypophyses (see Electrophysiological recordings) were homogenized as described previously (Cazalis et al., 1987). The homogenate was centrifuged at 2400 × g for 6 min. The resulting pellet contains highly purified nerve terminals. The nerve endings were loaded onto filters (0.45 µm Acro disk; Gelman Sciences, Ann Arbor, MI) and perfused at 37°C with normal Locke's solution. Four minute fractions of perfusate were collected, and the evoked release was triggered by an 8-min-duration pulse of a depolarizing concentration (50 mM) of K⁺. The results are given as AVP or OT release per fraction using specific radioimmunoassays (Wang et al., 1997a). The medium before and after the depolarizing period contained (in mM): NaCl, 40; KHCO₃, 5; N-methyl-D-glucamine (NMG)-Cl, 100; MgCl₂, 1; CaCl₂, 2; glucose, 10; and Tris-HEPES, 10, with 0.02% BSA, pH 7.25. Depolarization medium contained 50 mM K⁺, in which the NMG was reduced to maintain the osmolarity (300-310 mOsm).

Polypeptide toxins. The polypeptide toxins used in this study were synthetic versions prepared by Neurex Pharmaceutical Corporation (Ramachandran et al., 1993). These were termed SNX-482, the synthetic version of a novel 41 amino acid peptide isolated from the venom of the West African tarantula Hysterocrates gigas (Newcomb et al., 1998), SNX-111, the synthetic version of -conopeptide MVIIA (Olivera et al., 1994), SNX-194, the methionine-12 to norleucin-12 derivative of SNX-111, and SNX-230, the synthetic version of MVIIC (Hillyard et al., 1992). The synthetic version of -AgaIVA (Mintz et al., 1992) was purchased from Peptides International (Louisville, KY) or synthesized as described by Gaur et al., (1994). In the text we refer to the synthetic peptides by their original names or by the Neurex terms.

Data analysis. All results are given as means ± SEM, and the statistical significance of differences in groups was analyzed using SigmaStat (Jandel Scientific, San Rafael, CA) with Tukey's t tests.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ca²⁺ channel currents

In the isolated neurohypophysial terminals, the peak I_Ba, which was activated by depolarizing from 80 to +10 mV, demonstrates both transient and long-lasting components (Fig. 1A). As we have reported previously (Wang et al., 1997a), the use of the dihydropyridine (DHP) Ca²⁺ channel antagonist nicardipine (2.5 µM) selectively inhibits the long-lasting (L-) component of the Ba²⁺ currents (Fig. 1). Subsequent addition of the N-type Ca²⁺ channel blocker MVIIA (3000 nM) led to rapid inhibition of a large portion of the isolated transient component (and, to a lesser extent, the long-lasting component) of the Ba²⁺ current. This concentration has been shown previously to block the N-type component maximally (Wang et al., 1997a).

View larger version (21K): [in this window] [in a new window]   Figure 1.   A toxin-resistant Ba2+ current in neurohypophysial terminals is blocked by Ni2+. Subtypes of the macroscopic Ba2+ current (IBa) in nerve terminals can be pharmacologically dissected by applying different Ca2+ channel blockers. A, In an isolated rat neurohypophysial terminal, the IBa was elicited by depolarizations (see template above) and first recorded under control conditions (5 mM Ba2+ Locke's solution) and then after subsequent applications (shown by horizontal bars in B) of the L-type blocker nicardipine (2.5 µM), the N-type Ca2+ channel blocker MVIIA (3 µM), and the P/Q-type blockers MVIIC (100 nM) and AgaIVA (450 nM). There was a resistant Ba2+ current component that could only be dose dependently (86-258 µM) inhibited by Ni2+. B, The corresponding time-response plot of the peak values of the macroscopic IBa is shown.

In ~50% of the neurohypophysial terminals investigated, subsequent addition of low (36 nM) concentrations of MVIIC inhibited this remaining component, and higher (150 nM) concentrations almost completely abolished it (Wang et al., 1997a). In another group of terminals (~46%), however, the non-L- and -N-types of Ca²⁺ currents could not be blocked by the P/Q-type Ca²⁺ channel blockers MVIIC or AgaIVA (Fig. 1). This resistant part of the transient Ca²⁺ current appeared to be analogous to an R-type Ca²⁺ channel current (Randall and Tsien, 1995).

Pharmacology of resistant Ca²⁺ channel currents

To test whether this resistant component of the Ba²⁺ current could indeed be classified as an R-type Ca²⁺ channel current, Ni²⁺, a T- and R-type Ca²⁺ channel blocker, was applied to this terminal. Low concentrations (86µM) of Ni²⁺ inhibited the resistant current (Fig. 1). Because of the low selectivity of Ni²⁺ between Ca²⁺ channels, however, the identity of the resistant component of the Ca²⁺ current in the nerve terminal was still unclear.

A newly discovered polypeptide toxin, SNX-482, was found to be a specific blocker of the class E (_1E) Ca²⁺ channel (Newcomb et al., 1998). This toxin made it possible for us to identify the Ni²⁺-sensitive type of Ca²⁺ current and to probe its function in neurohypophysial nerve terminals (Wang et al., 1997b; Dayanithi et al., 1999).

First, the effects of SNX-482 on the long-lasting and transient components of the Ba²⁺ current of the neurohypophysial terminals were examined (Fig. 2A). The isolated, transient component of the Ba²⁺ current usually includes an N-type and either a P/Q-type or a resistant component of Ca²⁺ channel currents (Wang et al., 1997a). The IC₅₀ for the undifferentiated transient I_Ba, obtained from the equation I = I_max{1  [x/(IC₅₀ + x)]}, is 226 nM (Fig. 2B). This is similar to that for SNX-482 to inhibit the cloned _1B (N-type) Ca²⁺ channel current but much higher than that (IC₅₀ = 10 nM) to block the heterologously expressed _1E-type currents (Newcomb et al., 1998). The toxin does not affect the DHP-sensitive or L-type current in these terminals. These results indicate that, at high concentrations, SNX-482 could block some combination of N-, P/Q-, and/or class E-type Ca²⁺ channels in the terminals.

View larger version (15K): [in this window] [in a new window]   Figure 2.   SNX-482 inhibits only the transient IBa in nerve terminals. Dose-dependent inhibition by SNX-482 of the total macroscopic IBa of neurohypophysial terminals is shown. A, A representative time-response plot (see, e.g., Fig. 1B) of the effect of 1-30 nM SNX-482 on the total macroscopic IBa current. Note that the remaining current in the nerve terminal was sensitive to MVIIA and nicardipine. B, Dose-response curve for the effect of SNX-482 on the undifferentiated transient macroscopic IBa (n = 3). The solid line was obtained from fitting with the equation I = Imax{1  [x/(IC50 + x)]}, where I is the current amplitude at a given voltage, Imax is the maximum current, and x is the blocker's concentration. ctrl, Control.

Application of a combination of DHP, MVIIA, and MVIIC or of high concentrations of MVIIA/SNX-194 and MVIIC/AgaIVA allowed us to obtain isolated "resistant" Ba²⁺ or Ca²⁺ currents (Fig. 3A). SNX-482, in a dose-dependent manner (in a total of seven terminals), inhibited the isolated resistant currents (Fig. 3A,B) with an IC₅₀ of 4.1 nM (Fig. 3C), similar to that found for the _1E Ca²⁺ currents expressed in human embryonic kidney (HEK) cells (Newcomb et al., 1998). The inhibition by SNX-482 of the resistant-type Ba²⁺ current is reversible (Fig. 3D). Furthermore, both SNX-482 and Ni²⁺ inhibited the same previously resistant component of the Ba²⁺ current (Fig. 3D).

View larger version (27K): [in this window] [in a new window]   Figure 3.   SNX-482 blocks the previously resistant neurohypophysial IBa. The IBa was elicited by depolarizations to 0 mV. A, Representative traces of resistant macroscopic IBa inhibited by SNX-482 in a dose-dependent manner after an application of high concentrations of SNX-194 (3 µM) and MVIIC (2 µM) to block the other components. B, The I-V relation of the macroscopic IBa under control conditions () and in the presence of SNX-194 and MVIIC () and 3 nM (), 6 nM (), and 24 nM () SNX-482. C, The dose-response curve of the effects of SNX-482 on the isolated resistant IBa in neurohypophysial terminals. The dotted line fit was obtained from the equation I = Imax{1  [x/(IC50 + x)]}. D, Reversibility of the effects of SNX-482 and Ni2+ on the isolated resistant (to nicardipine + MVIIA + MVIIC) IBa. Ni2+ and SNX-482 appear to inhibit the same component of the current.

Any sensitivity of P/Q-type currents in the nerve terminals to SNX-482 was then examined. As shown in Figure 4A, in the presence of the L-type blocker nicardipine and the N-type blocker MVIIA, the remaining Ba²⁺ component was not affected by SNX-482, although it was inhibited by the P/Q-type blocker AgaIVA. This confirmed that SNX-482 is not a P/Q-type or class A channel blocker (Newcomb et al., 1998). The inhibition of the resistant Ba²⁺ current component, in ~46% of the neurohypo-physial terminals investigated, by both Ni²⁺ and SNX-482 lead us to conclude that this channel current most closely resembles that of the _1E Ca²⁺ channel subunit expressed in HEK cells (Newcomb et al., 1998).

View larger version (17K): [in this window] [in a new window]   Figure 4.   SNX-482 does not block the neurohypophysial P/Q-type Ca2+ current. A, A representative time-response curve of the peak values of the macroscopic IBa of an isolated nerve terminal insensitive to SNX-482 is shown. The IBa was recorded under control conditions and after subsequent applications of the L-type Ca2+ channel blocker nicardipine (2.5 µM) and the N-type Ca2+ channel blocker MVIIA (4 µM). SNX-482 (40 nM) did not affect the remaining Ba2+ current, which was subsequently blocked by the P/Q-type blocker AgaIVA (450 nM). B, By the use of the same protocol described in A, SNX-482 and AgaIVA each had a partial effect on the non-L and non-N-type Ba2+ current in a different nerve terminal. Therefore, some (~5%) neurohypophysial terminals have both P/Q- and R-type currents.

Interestingly, in ~5% of the terminals investigated (n = 21), in addition to the L- and N-type Ca²⁺ channel currents, there appears to exist both P/Q- and SNX-482-sensitive-type Ba²⁺ currents. Figure 4B is an example of this, showing that the non-L- and non-N-type Ba²⁺ currents were partially sensitive to both SNX-482 and AgaIVA.

Biophysical properties

Biophysical characterization of the resistant component of the neurohypophysial terminal I_Ba also favors a class E or R-type Ca²⁺ channel classification. This component of the current is a transient, high-voltage-activated Ba²⁺ current with an inactivation rate constant of 21 ± 3 msec (n = 7) during a step to 0 mV (see Fig. 3A). Figure 5A illustrates the activation (V_1/2 = 14.2 mV) and steady-state inactivation (V_1/2 = 58.8 mV) of the SNX-482-sensitive component of the neurohypophysial terminal Ba²⁺ current. The inactivating rate constant and activation and steady-state inactivation curves (Fig. 5A) of this neurohypophysial Ca²⁺ current component are most consistent with those of the R-type Ca²⁺ channel in granule cells (Randall and Tsien, 1995). Nevertheless, the other transient Ca²⁺ current components (N- and P/Q-type) appear to have biophysical properties similar to those of this "R"-type Ca²⁺ component (Wang et al., 1997a).

View larger version (17K): [in this window] [in a new window]   Figure 5.   Voltage dependence and kinetics of Ba2+ and Ca2+ currents in neurohypophysial terminals. A, Activation () and steady-state inactivation () curves for the pharmacologically isolated (see Fig. 1) R-type macroscopic Ba2+ currents are illustrated (n = 3). The peak Ba2+ currents elicited were normalized to the maximal currents (for the steady-state inactivation curve) or conductances (for the activation curve) and plotted versus the holding potentials or the step potentials, respectively. Data for activation and steady-state inactivation were fit using appropriate forms of the Boltzmann equation. For activation, C = Cmax{1 + exp[(V1/2  Vs)/k]}1, and for steady-state inactivation, I = Imax{1 + exp[(Vs V1/2)/k]}1, where C is the conductance at a given voltage, Cmax is the maximum conductance, I is the current amplitude at a given voltage, Imax is the maximum current, Vs is the voltage step, V1/2 is the midpoint potential, and k is the slope parameter. The fitting of the two curves for the isolated resistant (R)-type IBa gives V1/2 values of 14.1 mV (k = 5.0) and 58.8 mV (k = 5.7) for activation and inactivation, respectively. B, Comparison of amplitudes and inactivation rates of the total versus R-type Ca2+ and Ba2+ currents is shown. Bottom histograms, The 5 mM Ba2+ total currents (n = 5) and the isolated R-type Ba2+ currents (n = 4) were larger than the 5 mM Ca2+ total currents (n = 5) and isolated R-type Ca2+ currents (n = 3), respectively (p < 0.01). Top histograms, The inactivation time constant () was obtained from the same groups of terminals (as above). The 5 mM Ba2+ total currents had larger  values, or slower inactivation, than did the 5 mM Ca2+ current group (p < 0.01). The R-type Ba2+ currents, however, had  values similar to those of the R-type Ca2+ currents (p > 0.05).

The relative permeabilities for Ca²⁺ versus Ba²⁺ between the total channel currents and the isolated SNX-482-sensitive or R-type current were compared, as shown in Figure 5B. Ba²⁺ currents were significantly larger than the corresponding Ca²⁺ currents for both the total and the isolated components. The inactivation rate constant, however, differed. The total current showed slower inactivation with Ba²⁺ as compared with Ca²⁺ as the charge carrier, whereas the R-type currents showed no difference in their inactivation with either Ba²⁺ or Ca²⁺.

Peptide release

In a previous report (Wang et al., 1997a), we found that a significant portion of OT release could not be inhibited even by simultaneous applications of L-, N-, and P/Q-type Ca²⁺ channel blockers. To determine whether the class E or R-type Ca²⁺ channel could play a role in this secretion, we measured both OT and AVP release in the same samples collected from perfused populations of nerve terminals (Fig. 6). Capitalizing on the same pharmacological protocol used to isolate the R-type component electrophysiologically (see Fig. 3A), we revealed a similar resistant component (42.3%) of Ca²⁺-dependent OT release (Fig. 6B). In these experiments, high K⁺ alone induced OT release of 4258 ± 306 pg (n = 4), and both nicardipine and MVIIA, given in combination, reduced (by 57.7 ± 3.8%) high-K⁺-stimulated release to 1812 ± 376 pg. SNX-482 (20 nM) completely blocked the remaining stimulated OT release (to basal level, 159 ± 33 pg). In contrast, a similar resistant component (38.4%) of stimulated AVP release (406 ± 30 pg) was essentially unchanged (458 ± 63 pg; n = 4) by the same concentration of this R-type channel blocker (Fig. 6A). As shown previously (Wang et al., 1997a), this resistant component of AVP release was blocked (to basal level, 60 ± 3 pg) by the P/Q-type blocker MVIIC. These results were the same even if the order of drugs was reversed or scrambled (data not shown). Furthermore, stimulated release was stable during prolonged applications of each of the Ca²⁺ channel blockers, indicating that steady-state effects had been established.

View larger version (25K): [in this window] [in a new window]   Figure 6.   Effects of the Ca2+ channel blocker SNX-482 on AVP versus OT release from nerve terminals. A, AVP release was repeatedly stimulated (see arrows) with 50 mM K+. The isolated neurohypophysial terminals were challenged with elevated K+ either in the absence (first arrow) of any channel blockers or in the presence of 2.5 µM nicardipine (L-type Ca2+ channel blocker) and 1 µM MVIIA (N-type Ca2+ channel blocker) and then subsequently with the addition of 20 nM SNX-482 (R-type Ca2+ channel blocker) and finally plus 300 nM MVIIC (P/Q-type Ca2+ channel blocker). All these drugs were present for at least 20 min before, during, and after the K+ stimulation (see differently shaded horizontal bars above). The inhibitions were mostly reversible, as indicated by control K+ stimulation after washout (last arrow). All data points represent the mean of four to six experiments. B, OT release was repeatedly stimulated with 50 mM K+. The nerve terminals were challenged with elevated K+ either in the absence (first arrow) of any channel blockers or in the presence of 2.5 µM nicardipine (L) and 1 µM MVIIA (N), then 20 nM SNX-482 (R), and finally 300 nM MVIIC (P/Q) (see horizontal bars above). This inhibition was also mostly reversible (last arrow). Note that the same perfusate samples were assayed for both OT and AVP.

We have also performed a set of experiments to compare quantitatively the SNX- 482 block of OT release with the IC₅₀ of this toxin on R-type calcium channels. As described in the Figure 6 legend, the nerve terminals were challenged with 50 mM K⁺ either in the absence of any channel blocker (control) or in the presence of both 2.5 µM nicardipine (L-type channel blocker) and 1 µM MVIIA (N-type channel blocker) and then subsequently with varying concentrations of SNX-482 (1, 5, 10, 20, 50, or 100 nM). In this batch of experiments, K⁺ alone evoked 3678 ± 139 pg (n = 3). L- and N-type channel blockers reduced this OT release by 59.7% (to 2194 ± 100 pg). Further addition of 1, 5, 10, 20, 50, or 100 nM SNX-482 suppressed the resistant OT release by 8% (to 2018 ± 30 pg), 32.6% (to 1478 ± 54 pg), 61.4% (to 846 ± 40 pg), 93.4% (to 145 ± 7 pg), 95.6% (to 96 ± 6 pg), and 97.6% (to 52 ± 6 pg), respectively. The IC₅₀ calculated from the equation r = R_max{1  [x/(IC₅₀ + x)]} for the SNX-482 block of OT release is 6.8 nM, which is comparable with the IC₅₀ for the toxin on R-type calcium currents. Finally, the effects of SNX-482 on OT versus AVP release were significantly (p < .001) different, thus revealing the importance of the class E or R-type component in only OT release.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The isolated neurohypophysial terminals are uniquely useful for studying the pharmacological, biophysical, and functional properties of Ca²⁺ channels at the site of secretion, and they have revealed a surprising pharmacological and functional complexity in the CNS presynaptic Ca²⁺ channel family.

Four different components of Ca²⁺-dependent neuropeptide release

The regulation of neurotransmission in the mammalian CNS has been characterized by the involvement of multiple types of voltage-dependent Ca²⁺ channels, each of which might play a specific role in the regulation of neurotransmission. In the mammalian neurohypophysial system, the L- and N-type Ca²⁺ channels play an equivalent role in both AVP and OT release. This is quite different from the role of Ca²⁺ channels in classical neurotransmission, in which the N- and P/Q-type, instead of the L-type, Ca²⁺ channel current are dominant in controlling neurotransmitter release (Hirning et al., 1988; Wheeler et al., 1994; Dunlap et al., 1995). Furthermore, the P/Q-type Ca²⁺ channel has turned out to be critical for AVP release from neurohypophysial nerve terminals (Wang et al., 1997a). Finally, the present results have demonstrated that an SNX-482-sensitive Ca²⁺ current is responsible for an important part of OT release.

Identity of the resistant Ca²⁺ channel in nerve terminals

We have now shown that the SNX-482-sensitive Ca²⁺ current has similar biophysical properties to that of the class E channel. The phenotype of the expressed class E channel (Zhang et al., 1993) can resemble that of native currents described as either R- (Randall and Tsien, 1995) or T-type (Snutch and Reiner, 1992). The T-type Ca²⁺ channel in the CNS is a low-voltage-activated channel that is affected by Ni²⁺ (Tsien et al., 1991; Fisher and Bourque, 1996). The terminal SNX-482-sensitive Ca²⁺ current, although also blocked by Ni²⁺, is moderately high voltage activated and more permeable to Ba²⁺ than to Ca²⁺ (Fig. 5B). Thus, in terms of its biophysical properties, this channel appears to be different from the T-type Ca²⁺ channel.

A correspondence between cloned expressed class E calcium channels and various currents described as resistant, or R-type, is suggested by similar electrophysiological properties and resistance to selective antagonists of N, P/Q, and L-type calcium channels (Newcomb et al., 1998). In the absence of a selective antagonist of the class E calcium channel, however, the correspondence between calcium channel classes defined by cDNA sequencing and electrophysiology has been unclear, and the role of class E calcium channels in physiology has not been studied previously (but see Wu et al., 1998, 1999). Our study capitalizes on the recent discovery of a selective class E antagonist from tarantula venom, SNX-482, and it has allowed us to analyze directly the identity, function, and pharmacology of the resistant-type calcium channels in CNS terminals.

The initial studies of the in vitro actions of native SNX-482 have revealed unanticipated diversity in the response of native R-type currents to the peptide (Newcomb et al., 1998). Nevertheless, because low nanomolar concentrations of SNX-482 have no effects on other calcium channel subtypes (see Figs. 2, 4) (Newcomb et al., 1998), the potent block of the neurohypophysial R-type current demonstrates that the resistant current isolated pharmacologically is not simply residual current flowing through incompletely blocked N-, P/Q-, and L-type calcium channels. Thus, the variability of the response of native R-type currents almost certainly indicates pharmacological heterogeneity of the distinct entities, perhaps splice variants, which are responsible for these currents.

These variants could also explain the fact that class E mRNA has, so far, not been detected in the hypothalamic magnocellular somata that project to the neurohypophysis (Gainer and Chin, 1998). In contrast, preliminary evidence (G. Dayanithi, unpublished results), using antibodies raised against class E channels, has localized these channels to isolated neurohypophysial terminals.

The R-type Ca²⁺ channel controls OT, but not AVP, release

Our data suggest that in one group of terminals, there is a Ni²⁺- and SNX-482-sensitive Ca²⁺ channel able to regulate OT release preferentially, whereas in another group of terminals the P/Q-type Ca²⁺ channel plays a converse role in AVP release. We demonstrate here that the _1E class or R-type Ca²⁺ channel exists on these neurohypophysial terminals, where it participates in the control of neuropeptide secretion. These results lead to the hypothesis that the R-type channels are preferentially localized on OT peptide-containing nerve terminals and thus do not affect Ca²⁺ currents in vasopressinergic terminals. Interestingly, some (5%) terminals appear to have both types of channels (Fig. 4B), comparable with the observed percentage of terminals containing both OT and AVP (Wang et al., 1997b). In any case, the data clearly show that the R-type component plays an important role in OT, but not in AVP, secretion from these CNS terminals.

In conclusion, we have demonstrated that an R-type Ca²⁺ channel exists in at least one type of CNS terminal and is important in depolarization-secretion coupling. This lends support to the idea that R-type channels may play a specific role in synaptic transmission in other CNS synapses (Newcomb et al., 1998; Wu et al., 1998, 1999). The data presented here clarify the specific identities and functional importance of the Ca²⁺ channels actually located at nerve terminals and point out that the R- and P/Q-channels, at least, may be heterogeneously localized for different functions.

	FOOTNOTES

Received May 10, 1999; revised Aug. 16, 1999; accepted Aug. 18, 1999.

We would like to acknowledge support by the National Institutes of Health Grant NS29470 (J.R.L.). Portions of this work were supported by the Neurex Pharmaceutical Corporation, and we would like to thank members of their synthetic chemistry group for providing the synthetic peptides. We also thank G. Miljanich for support and advice.

Correspondence should be addressed to Dr. José R. Lemos, Department of Physiology and Neuroscience Program, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail: Jose.Lemos{at}umassmed.edu.

Dr. Wang's present address: Division of Neurobiology, Department of Neurology and Neuroscience, Cornell University Medical College, 411 East 69th Street, New York, NY 10021.

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Copyright © 1999 Society for Neuroscience  0270-6474/99/19219235-07$05.00/0

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