José R. Lemos

Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3.

In many cells, receptor activation initiates sustained Ca2+ entry which is critical in signal transduction. Mammalian transient receptor potential (Trp) proteins, which are homologous to the Drosophila photoreceptor-cell Trp protein, have emerged as candidate subunits of the ion channels that mediate this influx. As a consequence of overexpression, these proteins produce cation currents that open either after depletion of internal Ca2+ stores or through receptor activation. However, determining the role of endogenous Trp proteins in signal transduction is complicated by the absence of selective antagonists. Here we examine Trp function during sperm–egg interaction. The sperm acrosome reaction is a Ca2+-dependent secretory event that must be completed before fertilization. In mammals, exocytosis is triggered during gamete contact by ZP3, a glycoprotein constituent of the eggs extracellular matrix, or zona pellucida (ZP). ZP3 activates trimeric G proteins and phospholipase C and causes a transient Ca2+ influx into sperm through T-type Ca2+ channels. These early responses promote a second Ca2+-entry pathway, thereby producing sustained increases in intracellular Ca2+ concentration ([Ca2+]i) that drive acrosome reactions. Our results show that Trp2 is essential for the activation of sustained Ca2+ influx into sperm by ZP3.

Interaction of SNX482 with Domains III and IV Inhibits Activation Gating of α1E (CaV2.3) Calcium Channels

We have investigated the action of SNX482, a toxin isolated from the venom of the tarantula Hysterocrates gigas, on voltage-dependent calcium channels expressed in tsa-201 cells. Upon application of 200 nM SNX482, R-type alpha(1E) calcium channels underwent rapid and complete inhibition, which was only poorly reversible upon washout. However, upon application of strong membrane depolarizations, rapid and complete recovery from inhibition was obtained. Tail current analysis revealed that SNX482 mediated an approximately 70 mV depolarizing shift in half-activation potential, suggesting that the toxin inhibits alpha(1E) calcium channels by preventing their activation. Experiments involving chimeric channels combining structural features of alpha(1E) and alpha(1C) subunits indicated that the presence of the domain III and IV of alpha(1E) is a prerequisite for a strong gating inhibition. In contrast, L-type alpha(1C) channels underwent incomplete inhibition at saturating concentrations of SNX482 that was paralleled by a small shift in half-activation potential and which could be rapidly reversed, suggesting a less pronounced effect of the toxin on L-type calcium channel gating. We conclude that SNX482 does not exhibit unequivocal specificity for R-type channels, but highly effectively antagonizes their activation.

Rat supraoptic magnocellular neurones show distinct large conductance, Ca2+-activated K+ channel subtypes in cell bodies versus nerve endings

1 Large conductance, Ca2+‐activated K+ (BK) channels were identified in freshly dissociated rat supraoptic neurones using patch clamp techniques. 2 The single channel conductance of cell body BK channels, recorded from inside‐out patches in symmetric 145 mM K+, was 246.1 pS, compared with 213 pS in nerve ending BK channels (P < 0.01). 3 At low open probability (Po), the reciprocal of the slope in the ln(NPo)‐voltage relationship (N, number of available channels in the patch) for cell body and nerve ending channels were similar: 11 vs. 14 mVper e‐fold change in NPo, respectively. 4 At 40 mV, the [Ca2+]i producing half‐maximal activation was 273 nM, as opposed to > 1.53 μM for the neurohypophysial channel, indicating the higher Ca2+ sensitivity of the cell body isochannel. 5 Cell body BK channels showed fast kinetics (open time constant, 8.5 ms; fast closed time constant, 1.6 and slow closed time constant, 12.7 ms), identifying them as ‘type I’ isochannels, as opposed to the slow gating (type II) of neurohypophysial BK channels. 6 Cell body BK activity was reduced by 10 nM charybdotoxin (NPo, 37 % of control), or 10 nM iberiotoxin (NPo, 5 % of control), whereas neurohypophysial BK channels are insensitive to charybdotoxin at concentrations as high as 360 nM. 7 Whilst blockade of nerve ending BK channels markedly slowed the repolarization of evoked single spikes, blockade of cell body channels was without effect on repolarization of evoked single spikes. 8 Ethanol reversibly increased neurohypophysial BK channel activity (EC50, 22 mM; maximal effect, 100 mM). In contrast, ethanol (up to 100 mM) failed to increase cell body BK channel activity. 9 In conclusion, we have characterized BK channels in supraoptic neuronal cell bodies, and demonstrated that they display different electrophysiological and pharmacological properties from their counterparts in the nerve endings.

Ca2+ Syntillas, Miniature Ca2+ Release Events in Terminals of Hypothalamic Neurons, Are Increased in Frequency by Depolarization in the Absence of Ca2+ Influx

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of ∼250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of –80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.

Alcohol Tolerance in Large-Conductance, Calcium-Activated Potassium Channels of CNS Terminals Is Intrinsic and Includes Two Components: Decreased Ethanol Potentiation and Decreased Channel Density

Tolerance is an important element of drug addiction and provides a model for understanding neuronal plasticity. The hypothalamic–neurohypophysial system (HNS) is an established preparation in which to study the actions of alcohol. Acute application of alcohol to the rat neurohypophysis potentiates large-conductance calcium-sensitive potassium channels (BK), contributing to inhibition of hormone secretion. A cultured HNS explant from adult rat was used to explore the molecular mechanisms of BK tolerance after prolonged alcohol exposure. Ethanol tolerance was intrinsic to the HNS and consisted of: (1) decreased BK potentiation by ethanol, complete within 12 min of exposure, and (2) decreased current density, which was not complete until 24 hr after exposure, indicating that the two components of tolerance represent distinct processes. Single-channel properties were not affected by chronic exposure, suggesting that decreased current density resulted from downregulation of functional channels in the membrane. Indeed, we observed decreased immunolabeling against the BK α-subunit on the surface of tolerant terminals. Analysis using confocal microscopy revealed a reduction of BK channel clustering, likely associated with the internalization of the channel.

Dihydropyridine Receptors and Type 1 Ryanodine Receptors Constitute the Molecular Machinery for Voltage-Induced Ca2+ Release in Nerve Terminals

Ca2+ stores were studied in a preparation of freshly dissociated terminals from hypothalamic magnocellular neurons. Depolarization from a holding level of −80 mV in the absence of extracellular Ca2+ elicited Ca2+ release from intraterminal stores, a ryanodine-sensitive process designated as voltage-induced Ca2+ release (VICaR). The release took one of two forms: an increase in the frequency but not the quantal size of Ca2+ syntillas, which are brief, focal Ca2+ transients, or an increase in global [Ca2+]. The present study provides evidence that the sensors of membrane potential for VICaR are dihydropyridine receptors (DHPRs). First, over the range of −80 to −60 mV, in which there was no detectable voltage-gated inward Ca2+ current, syntilla frequency was increased e-fold per 8.4 mV of depolarization, a value consistent with the voltage sensitivity of DHPR-mediated VICaR in skeletal muscle. Second, VICaR was blocked by the dihydropyridine antagonist nifedipine, which immobilizes the gating charge of DHPRs but not by Cd2+ or FPL 64176 (methyl 2,5 dimethyl-4[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate), a non-dihydropyridine agonist specific for L-type Ca2+ channels, having no effect on gating charge movement. At 0 mV, the IC50 for nifedipine blockade of VICaR in the form of syntillas was 214 nm in the absence of extracellular Ca2+. Third, type 1 ryanodine receptors, the type to which DHPRs are coupled in skeletal muscle, were detected immunohistochemically at the plasma membrane of the terminals. VICaR may constitute a new link between neuronal activity, as signaled by depolarization, and a rise in intraterminal Ca2+.

Modulation/physiology of calcium channel sub-types in neurosecretory terminals.

The hypothalamic-neurohypophysial system (HNS) controls diuresis and parturition through the release of arginine-vasopressin (AVP) and oxytocin (OT). These neuropeptides are chiefly synthesized in hypothalamic magnocellular somata in the supraoptic and paraventricular nuclei and are released into the blood stream from terminals in the neurohypophysis. These HNS neurons develop specific electrical activity (bursts) in response to various physiological stimuli. The release of AVP and OT at the level of neurohypophysis is directly linked not only to their different burst patterns, but is also regulated by the activity of a number of voltage-dependent channels present in the HNS nerve terminals and by feedback modulators. We found that there is a different complement of voltage-gated Ca(2+) channels (VGCC) in the two types of HNS terminals: L, N, and Q in vasopressinergic terminals vs. L, N, and R in oxytocinergic terminals. These channels, however, do not have sufficiently distinct properties to explain the differences in release efficacy of the specific burst patterns. However, feedback by both opioids and ATP specifically modulate different types of VGCC and hence the amount of AVP and/or OT being released. Opioid receptors have been identified in both AVP and OT terminals. In OT terminals, μ-receptor agonists inhibit all VGCC (particularly R-type), whereas, they induce a limited block of L-, and P/Q-type channels, coupled to an unusual potentiation of the N-type Ca(2+) current in the AVP terminals. In contrast, the N-type Ca(2+) current can be inhibited by adenosine via A(1) receptors leading to the decreased release of both AVP and OT. Furthermore, ATP evokes an inactivating Ca(2+)/Na(+)-current in HNS terminals able to potentiate AVP release through the activation of P2X2, P2X3, P2X4 and P2X7 receptors. In OT terminals, however, only the latter receptor type is probably present. We conclude by proposing a model that can explain how purinergic and/or opioid feedback modulation during bursts can mediate differences in the control of neurohypophysial AVP vs. OT release.

µ‐Opioid Receptor Preferentially Inhibits Oxytocin Release from Neurohypophysial Terminals by Blocking R‐type Ca2+ Channels

Oxytocin release from neurophypophysial terminals is particularly sensitive to inhibition by the µ‐opioid receptor agonist, DAMGO. Because the R‐type component of the neurophypophysial terminal Ca2+ current (ICa) mediates exclusively oxytocin release, we hypothesised that µ‐opioids could preferentially inhibit oxytocin release by blocking this channel subtype. Whole‐terminal recordings showed that DAMGO and the R‐type selective blocker SNX‐482 inhibit a similar ICa component. Measurements of [Ca2+]i levels and oxytocin release confirmed that the effects of DAMGO and SNX‐482 are not additive. Finally, isolation of the R‐type component and its associated rise in [Ca2+]i and oxytocin release allowed us to demonstrate the selective inhibition by DAMGO of this channel subtype. Thus, µ‐opioid agonists modulate specifically oxytocin release in neurophypophysial terminals by selectively targeting R‐type Ca2+ channels. Modulation of Ca2+ channel subtypes could be a general mechanism for drugs of abuse to regulate the release of specific neurotransmitters at central nervous system synapses.

Adenosine inhibition via A1 receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat

Effects of adenosine on voltage‐gated Ca2+ channel currents and on arginine vasopressin (AVP) and oxytocin (OT) release from isolated neurohypophysial (NH) terminals of the rat were investigated using perforated‐patch clamp recordings and hormone‐specific radioimmunoassays. Adenosine, but not adenosine 5′‐triphosphate (ATP), dose‐dependently and reversibly inhibited the transient component of the whole‐terminal Ba2+ currents, with an IC50 of 0.875 μm. Adenosine strongly inhibited, in a dose‐dependent manner (IC50= 2.67 μm), depolarization‐triggered AVP and OT release from isolated NH terminals. Adenosine and the N‐type Ca2+ channel blocker ω‐conotoxin GVIA, but not other Ca2+ channel‐type antagonists, inhibited the same transient component of the Ba2+ current. Other components such as the L‐, Q‐ and R‐type channels, however, were insensitive to adenosine. Similarly, only adenosine and ω‐conotoxin GVIA were able to inhibit the same component of AVP release. A1 receptor agonists, but not other purinoceptor‐type agonists, inhibited the same transient component of the Ba2+ current as adenosine. Furthermore, the A1 receptor antagonist 8‐cyclopentyltheophylline (CPT), but not the A2 receptor antagonist 3, 7‐dimethyl‐1‐propargylxanthine (DMPGX), reversed inhibition of this current component by adenosine. The inhibition of AVP and OT release also appeared to be via the A1 receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A1 receptors, specifically blocks the terminal N‐type Ca2+ channel thus leading to inhibition of the release of both AVP and OT.

µ‐Opioid Receptor Modulates Peptide Release From Rat Neurohypophysial Terminals By Inhibiting Ca2+ Influx

The activation of opioid receptors in neurones of the central nervous system leads to a variety of effects including the modulation of diuresis and parturition, processes that are directly controlled by the hypothalamic–neurohypophysial system (HNS). The effects of µ‐opioid receptor activation on peptide release, voltage‐gated Ca2+ currents and intracellular calcium levels ([Ca2+]i) were studied in isolated nerve terminals of the HNS. The µ‐receptor agonist, DAMGO ([d‐Ala2,N‐Me‐Phe4,Gly5‐ol]‐enkephalin) inhibited high K+‐induced peptide release in a dose‐dependent manner, with oxytocin release being more sensitive to block than vasopressin release at all concentrations tested. The addition of the µ‐receptor antagonist CTOP (d‐Phe‐Cys‐Tyr‐d‐Trp‐Orn‐Thr‐Pen‐Thr amide) was able to overcome the inhibitory effects of DAMGO. By contrast to previous results, voltage‐gated Ca2+ currents were sensitive to blockage by DAMGO and this inhibition was also prevented by CTOP. Furthermore, [Ca2+]i measurements with Fura‐2 corroborated the inhibition by DAMGO of calcium entry and its reversal by the µ‐receptor antagonist in these nerve terminals. Thus, the decrease in neuropeptide release, particularly for oxytocin, induced by the activation of µ‐opioid receptors in neurohypophysial terminals is mediated, at least in part, by a corresponding decrease in Ca2+ entry due to the inhibition of voltage‐gated Ca2+ channels.