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Competing Interests: Competing interests statement:The authors declare no competing interest.

Ca V -channel dependent activation of BK channels is critical for feedback control of both calcium influx and cell excitability. Here we addressed the functional and spatial interaction between BK and Ca V 1.3 channels, unique Ca V 1 channels that activate at low voltages. We found that when BK and Ca V 1.3 channels were co-expressed in the same cell, BK channels started activating near -50 mV, ~30 mV more negative than for activation of co-expressed BK and high-voltage activated Ca V 2.2 channels. In addition, single-molecule localization microscopy revealed striking clusters of Ca V 1.3 channels surrounding clusters of BK channels and forming a multi-channel complex both in a heterologous system and in rat hippocampal and sympathetic neurons. We propose that this spatial arrangement allows tight tracking between local BK channel activation and the gating of Ca V 1.3 channels at quite negative membrane potentials, facilitating the regulation of neuronal excitability at voltages close to the threshold to fire action potentials.

Phosphoinositides serve as signature motifs for different cellular membranes and often are required for the function of membrane proteins. Here, we summarize clear evidence supporting the concept that many ion channels are regulated by membrane phosphoinositides. We describe tools used to test their dependence on phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, and consider mechanisms and biological meanings of phosphoinositide regulation of ion channels. This lipid regulation can underlie changes of channel activity and electrical excitability in response to receptors. Since different intracellular membranes have different lipid compositions, the activity of ion channels still in transit towards their final destination membrane may be suppressed until they reach an optimal lipid environment. This article is part of a Special Issue entitled Phosphoinositides., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Store-operated Ca2+ channels (SOCs) are activated by depletion of intracellular Ca2+ stores following agonist-mediated Ca2+ release. Previously we demonstrated that Ca2+ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca2+ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca2+ influx as Ca2+ was released from intracellular stores. Direct measurement of [Ca2+] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP3 or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ∼8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La3+ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca2+ entry through SOCs fills internal Ca2+ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca2+ such as salt and mucin secretion from the exocrine pancreatic ducts., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Secretory granules (SGs) sequester significant calcium. Understanding roles for this calcium and potential mechanisms of release is hampered by the difficulty of measuring SG calcium directly in living cells. We adapted the Förster resonance energy transfer-based D1-endoplasmic reticulum (ER) probe to develop a unique probe (D1-SG) to measure calcium and pH in secretory granules. It significantly localizes to SGs and reports resting free Ca(2+) of 69 ± 15 μM and a pH of 5.8. Application of extracellular ATP to activate P2Y receptors resulted in a slow monotonic decrease in SG Ca(2+) temporally correlated with the occurrence of store-operated calcium entry (SOCE). Further investigation revealed a unique receptor-mediated mechanism of calcium release from SGs that involves SG store-operated Orai channels activated by their regulator stromal interaction molecule 1 (STIM1) on the ER. SG Ca(2+) release is completely antagonized by a SOCE antagonist, by switching to Ca(2+)-free medium, and by overexpression of a dominant-negative Orai1(E106A). Overexpression of the CRAC activation domain (CAD) of STIM1 resulted in a decrease of resting SG Ca(2+) by ∼75% and completely abolished the ATP-mediated release of Ca(2+) from SGs. Overexpression of a dominant-negative CAD construct(CAD-A376K) induced no significant changes in SG Ca(2+). Colocalization analysis suggests that, like the plasma membrane, SG membranes also possess Orai1 channels and that during SG Ca(2+) release, colocalization between SGs and STIM1 increases. We propose Orai channel opening on SG membranes as a potential mode of calcium release from SGs that may serve to raise local cytoplasmic calcium concentrations and aid in refilling intracellular calcium stores of the ER and exocytosis.

The β-subunits of voltage-gated Ca(2+) (Ca(V)) channels regulate the functional expression and several biophysical properties of high-voltage-activated Ca(V) channels. We find that Ca(V) β-subunits also determine channel regulation by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)). When Ca(V)1.3, -2.1, or -2.2 channels are cotransfected with the β3-subunit, a cytosolic protein, they can be inhibited by activating a voltage-sensitive lipid phosphatase to deplete PIP(2). When these channels are coexpressed with a β2a-subunit, a palmitoylated peripheral membrane protein, the inhibition is much smaller. PIP(2) sensitivity could be increased by disabling the two palmitoylation sites in the β2a-subunit. To further test effects of membrane targeting of Ca(V) β-subunits on PIP(2) regulation, the N terminus of Lyn was ligated onto the cytosolic β3-subunit to confer lipidation. This chimera, like the Ca(V) β2a-subunit, displayed plasma membrane localization, slowed the inactivation of Ca(V)2.2 channels, and increased the current density. In addition, the Lyn-β3 subunit significantly decreased Ca(V) channel inhibition by PIP(2) depletion. Evidently lipidation and membrane anchoring of Ca(V) β-subunits compete with the PIP(2) regulation of high-voltage-activated Ca(V) channels. Compared with expression with Ca(V) β3-subunits alone, inhibition of Ca(V)2.2 channels by PIP(2) depletion could be significantly attenuated when β2a was coexpressed with β3. Our data suggest that the Ca(V) currents in neurons would be regulated by membrane PIP(2) to a degree that depends on their endogenous β-subunit combinations.

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca(2+) entry evoked by alkaline depolarization. In the absence of external Ca(2+), Na(+) carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na(+)](i)-reporting probe SBFI in populations of intact sperm. Removal of external Ca(2+) increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na(+)](i) is greater for sperm alkalinized with NH(4)Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na(+)](i) rise is slowed by candidate CatSper blocker HC-056456 (IC(50) approximately 3 microM). HC-056456 similarly slows the rise of [Ca(2+)](i) that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO(3) (-) but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca(2+) entry through the CatSper channel is terminated by removal of external Ca(2+). Thus, open CatSper channels and entry of external Ca(2+) through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.

Two proteins, STIM1 in the endoplasmic reticulum and Orai1 in the plasma membrane, are required for the activation of Ca(2+) release-activated Ca(2+) (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orai1 and STIM1 molecules are required to form a functional CRAC channel. We engineered several genetically expressed fluorescent Orai1 tandem multimers and a fluorescent, constitutively active STIM1 mutant. The tandem multimers assembled into CRAC channels, as seen by rectifying inward currents and by cytoplasmic calcium elevations. CRAC channels were visualized as fluorescent puncta in total internal reflection microscopy. With single-molecule imaging techniques, it was possible to observe photo-bleaching of individual fluorophores and to count the steps of bleaching as a measure of the stoichiometry of each CRAC channel complex. We conclude that the subunit stoichiometry in an active CRAC channel is four Orai1 molecules and two STIM1 molecules. Fluorescence resonance energy transfer experiments also showed that four Orai1 subunits form the assembled channel. From the fluorescence intensity of single fluorophores, we could estimate that our transfected HEK293 cells had almost 400,000 CRAC channels and that, when intracellular Ca(2+) stores were depleted, the channels clustered in aggregates containing approximately 1,300 channels, amplifying the local Ca(2+) entry.

Among several candidate Ca(2+) entry channels in sperm, only CatSper1 and CatSper2 are known to have required roles in male fertility. Past work with CatSper1 null sperm indicates that a critical lesion in hyperactivated motility underlies the infertility phenotype and is associated with an absence of depolarization-evoked Ca(2+)entry. Here we show that failure of hyperactivation of CatSper2 null sperm similarly correlates with an absence of depolarization evoked Ca(2+) entry. Additional shared aspects of the phenotypes of CatSper1 and -2 null sperm include unperturbed regional distributions of conventional voltage-gated Ca(2+) channel proteins and robust acceleration of the flagellar beat by bicarbonate. Further study reveals that treatment of both wild-type and CatSper2 null sperm with procaine increases beat asymmetry, a characteristic of the flagellar waveform of hyperactivation. This partial rescue of the loss-of-hyperactivation phenotype suggests that an absence of CatSper2 precludes hyperactivation by preventing delivery of needed Ca(2+) messenger rather than by preventing flagellar responses to Ca(2+). CatSper2 null sperm also have an increased basal cAMP content and beat frequency. Protein kinase A inhibitor H89 lowers beat frequency to that of wild-type sperm, suggesting that CatSper2 is required for protein kinase A-mediated, tonic control of resting cAMP content. Relative to wild-type testis, CatSper1 and -2 null testes contain normal amounts of CatSper2 and -1 transcripts, respectively. However, CatSper1 null sperm lack CatSper2 protein and CatSper2 null sperm lack CatSper1 protein. Hence, stable expression of CatSper1 protein requires CatSper2 and vice versa. This co-dependent expression dictates identical loss-of-function sperm phenotypes for CatSper1 and -2 null mutants.

CatSper family proteins are putative ion channels expressed exclusively in membranes of the sperm flagellum and required for male fertility. Here, we show that mouse CatSper1 is essential for depolarization-evoked Ca2+ entry and for hyperactivated movement, a key flagellar function. CatSper1 is not needed for other developmental landmarks, including regional distributions of CaV1.2, CaV2.2, and CaV2.3 ion channel proteins, the cAMP-mediated activation of motility by HCO3-, and the protein phosphorylation cascade of sperm capacitation. We propose that CatSper1 functions as a voltage-gated Ca2+ channel that controls Ca2+ entry to mediate the hyperactivated motility needed late in the preparation of sperm for fertilization.

Many different G protein-coupled receptors modulate the activity of Ca2+ and K+ channels in a variety of neuronal types. There are five known subtypes (M1-M5) of muscarinic acetylcholine receptors. Knockout mice lacking the M1, M2, or M4 subtypes are studied to determine which receptors mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons. In these cells, muscarinic agonists modulate N- and L-type Ca2+ channels and the M-type K+ channel through two distinct, G-protein mediated pathways. The fast and voltage-dependent pathway is lacking in the M2 receptor knockout mice. The slow and voltage-independent pathway is absent in the M1 receptor knockout mice. Neither pathway is affected in the M4 receptor knockout mice. Muscarinic modulation of the M current is absent in the M1 receptor knockout mice, and can be reconstituted in a heterologous expression system using cloned channels and M1 receptors. Our results using knockout mice are compared with pharmacological data in the rat.

There are five known subtypes of muscarinic receptors (M(1)-M(5)). We have used knockout mice lacking the M(1), M(2), or M(4) receptors to determine which subtypes mediate modulation of voltage-gated Ca(2+) channels in mouse sympathetic neurons. Muscarinic agonists modulate N- and L-type Ca(2+) channels in these neurons through two distinct G-protein-mediated mechanisms. One pathway is fast and membrane-delimited and inhibits N- and P/Q-type channels by shifting their activation to more depolarized potentials. The other is slow and voltage-independent and uses a diffusible cytoplasmic messenger to inhibit both Ca(2+) channel types. Using patch-clamp methods on acutely dissociated sympathetic neurons, we isolated each pathway by pharmacological and kinetic means and found that each one is nearly absent in a particular knockout mouse. The fast and voltage-dependent pathway is lacking in the M(2) receptor knockout mice; the slow and voltage-independent pathway is absent from the M(1) receptor knockout mice; and neither pathway is affected in the M(4) receptor knockout mice. The knockout effects are clean and are apparently not accompanied by compensatory changes in other muscarinic receptors.

We investigated which subtypes of G-protein beta subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein beta subunits. Gbeta1 and Gbeta2 strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The Gbeta5 subunit produced much weaker effects than Gbeta1 and Gbeta2, whereas Gbeta3 and Gbeta4 were nearly inactive in these electrophysiological studies. The specificity implied by these results was confirmed and extended using the yeast two-hybrid system to test for protein-protein interactions. Here, Gbeta1 or Gbeta2 coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a alpha1 subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the Gbeta5 subunit interacted weakly, and Gbeta3 and Gbeta4 failed to interact. Together, these results suggest that Gbeta1 and/or Gbeta2 subunits account for most of the voltage-dependent inhibition of N-type calcium channels and that the linker between domains I and II of the calcium channel alpha1 subunit is a principal receptor for this inhibition.

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B2 bradykinin and M1 muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP3 receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP3 to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca2+]i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca2+]i were blocked by depletion of Ca2+ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca2+ from IP3-sensitive Ca2+ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.

Mitochondria, the metabolic powerhouses of the cell, can sequester and release large amounts of Ca2+. This import and export of Ca2+ helps to adjust energy production to cellular needs. Recent advances show that mitochondrial Ca2+ fluxes play a major role in normal Ca2+ signaling.

We have measured the onset and recovery speed of inhibition of N-type Ca2+ channels in adult rat superior cervical ganglion neurons by somatostatin (SS), norepinephrine (NE), and oxotremorine-M (oxo-M, a muscarinic agonist), using the whole cell configuration of the patch-clamp method with 5 mM external Ca2+. With a local perfusion pipette system that changed the solution surrounding the cell within 50 ms, we applied agonists at various times before a brief depolarization from -80 mV that elicited I(Ca). At concentrations that produced maximal inhibition, the onset time constants for membrane-delimited inhibition by SS (0.5 microM), NE (10 microM), and oxo-M (20 microM) were 2.1, 0.7, and 1.0 s, respectively. The time constants for NE inhibition depended only weakly on the concentration, ranging from 1.2 to 0.4 s in the concentration range from 0.5 to 100 microM. Inhibition by oxo-M (20 microM) through a different G-protein pathway that uses a diffusible cytoplasmic messenger had a time constant near 9 s. The recovery rate constant from membrane-delimited inhibition was between 0.09 and 0.18 s(-1), significantly higher than the intrinsic GTPase rate of purified G protein Go, suggesting that Ca2+ channels or other proteins in the plasma membrane act as GTPase activating proteins. We also measured the rate of channel reinhibition after relief by strong depolarizing prepulses, which should reflect the kinetics of final steps in the inhibition process. In the presence of different concentrations of NE, reinhibition was four to seven times faster than the onset of inhibition, indicating that the slowest step of inhibition must precede the binding of G protein to the channel. We propose a kinetic model for the membrane-delimited NE inhibition of Ca2+ channels. It postulates two populations of receptors with different affinities for NE, a single population of G proteins, and a single population of Ca2+ channels. This model closely simulated the time courses of onset and recovery of inhibition and reinhibition, as well as the dose-response curve for inhibition of Ca2+ channels by NE.

Many neuromodulators inhibit N-type Ca2+ currents via G protein-coupled pathways in acutely isolated superior cervical ganglion (SCG) neurons. Less is known about which neuromodulators affect release of norepinephrine (NE) at varicosities and terminals of these neurons. To address this question, we used carbon fiber amperometry to measure catecholamine secretion evoked by electrical stimulation at presumed sites of high terminal density in cultures of SCG neurons. The pharmacological properties of action potential-evoked NE release paralleled those of N-type Ca2+ channels: Release was completely blocked by Cd2+ or omega-conotoxin GVIA, reduced 50% by 10 microM NE or 62% by 2 microM UK-14,304, an alpha2-adrenergic agonist, and reduced 63% by 10 microM oxotremorine M (Oxo-M), a muscarinic agonist. Consistent with action at M2 or M4 receptor subtypes, Oxo-M could be antagonized by 10 microM muscarinic antagonists methoctramine and tropicamide but not by pirenzepine. After overnight incubation with pertussis toxin, inhibition by UK-14,304 and Oxo-M was much reduced. Other neuromodulators known to inhibit Ca2+ channels in these cells, including adenosine, prostaglandin E2, somatostatin, and secretin, also depressed secretion by 34-44%. In cultures treated with omega-conotoxin GVIA, secretion dependent on L-type Ca2+ channels was evoked with long exposure to high K+ Ringer's solution. This secretion was not sensitive to UK-14,304 or Oxo-M. Evidently, many neuromodulators act on the secretory terminals of SCG neurons, and the depression of NE release at terminals closely parallels the membrane-delimited inhibition of N-type Ca2+ currents in the soma.

We investigated actions of somatostatin (Som) on voltagegated calcium channels in acutely isolated rat amygdaloid neurons. Somatostatin caused a dose-dependent inhibition of the high voltage-activated (HVA) Ca2+ current, with little or no effect on the low voltage-activated (LVA) current. Nifedipine (2-10 microM) reduced the peak current by approximately 15% without reducing inhibition of current by Som significantly, ruling out L-type channels as the target of modulation. The modulation appears to involve N- and P/Q-type calcium channels. After pretreatment with omega-conotoxin-GVIA (omega-CgTx) or omega-agatoxin-IVA, the inhibition was reduced but not abolished, whereas the combined application of both toxins nearly abolished the modulation. The Som analog BIM-23060 mimicked the effects of Som, whereas BIM-23058 had no effect, implicating Som type-2 receptors (SSTR-2). The inhibition was voltage-dependent, being minimal for small depolarizations, and was often accompanied by a slowing of the activation time course. Strong depolarizing prepulses partially relieved the inhibition and restored the time course of activation. Intracellular dialysis with GTP gamma S led to spontaneous inhibition and a slowing of the current like that with Som and occluded the effects of the peptide. Dialysis with GDP beta S also diminished the inhibition. A short preincubation with 50 microM of the alkylating agent N-ethylmaleimide (NEM) prevented the action of somatostatin. These results suggest a role for NEM-sensitive G-proteins in the Som inhibition. Application of 8-CPT-cAMP and IBMX did not mimic or prevent the effects of Som.

1. We studied the effects of phorbol-12-myristate, 13-acetate (PMA) on G-protein-mediated inhibition of Ca2+ channels by several neurotransmitters in rat superior cervical ganglion (SCG) sympathetic neurons, with the use of the whole cell patch clamp. PMA attenuated membrane-delimited inhibition of calcium currents (ICa) by norepinephrine (NE) and somatostatin by more than half, but did not attenuate inhibition by M1 muscarinic receptors, which use a diffusible cytoplasmic messenger. Inhibition of ICa by NE through pertussis-toxin-sensitive and -insensitive G proteins was equally attenuated by PMA. PMA enhanced ICa in about half the neurons (enhancement of 10 +/- 1%, mean +/- SE) and strongly reduced the holding current in 44 of 61 cells. 2. The M-type K+ current (IM) was not suppressed by PMA, and PMA did not attenuate inhibition of IM by muscarinic agonists, which is also via a diffusible cytoplasmic messenger. 3. Attenuation of NE and somatostatin inhibition by PMA was blocked by 1 microM staurosporine, a broad-spectrum protein kinase inhibitor. Tests with three inhibitors selective for distinct isoforms of protein kinase C (PKC) gave mixed results. PMA's actions were unaffected by 1 microM calphostin C, blocked by 500 nM bisindolylmaleimide, and unaffected by the pseudosubstrate inhibitor PKC19-36. 4. Thus we find that two membrane-delimited signaling pathways that inhibit ion channels in rat SCG neurons are strongly attenuated by PMA, but signaling pathway(s) that use a diffusible cytoplasmic messenger are not. We speculate that a nonstandard PKC isoform, perhaps PKC mu, mediates PMA actions.

Calcium ions entering cells through voltage-gated Ca2+ channels initiate rapid release of neurotransmitters and secretion of hormones. Ca2+ currents can be inhibited in many cell types by neurotransmitters acting through G proteins via a membrane-delimited pathway independently of soluble intracellular messengers. Inhibition is typically caused by a positive shift in the voltage dependence and a slowing of channel activation and is relieved by strong depolarization resulting in facilitation of Ca2+ currents. This pathway regulates the activity of N-type and P/Q-type Ca2+ channels, which are localized in presynaptic terminals and participate in neurotransmitter release. Synaptic transmission is inhibited by neurotransmitters through this mechanism. G-protein alpha subunits confer specificity in receptor coupling, but it is not known whether the G alpha or G beta gamma subunits are responsible for modulation of Ca2+ channels. Here we report that G beta gamma subunits can modulate Ca2+ channels. Transfection of G beta gamma into cells expressing P/Q-type Ca2+ channels induces modulation like that caused by activation of G protein-coupled receptors, but G alpha subunits do not. Similarly, injection or expression of G beta gamma subunits in sympathetic ganglion neurons induces facilitation and occludes modulation of N-type channels by noradrenaline, but G alpha subunits do not. In both cases, the G gamma subunit is ineffective by itself, but overexpression of exogenous G beta subunits is sufficient to cause channel modulation.

1. We studied modulation of N-type Ca2+ channels in adult rat superior cervical ganglion (SCG) neurons by pancreatic polypeptide (PP) using whole cell clamp. In large (> 20 pF) SCG neurons, PP inhibited ICa (35 +/- 2%, mean +/- SE) in a concentration-dependent fashion, with one-half maximal inhibition at 19 nM. 2. One-third of the inhibition was blocked by pertussis toxin, about one-half was blocked by N-ethylmaleimide (NEM) treatments, and about one-half was voltage dependent. The NEM-insensitive component of the PP inhibition was voltage independent and not significantly blocked by intracellular Ca2+ chelators. 3. The NEM-insensitive component was only weakly attenuated by GDP-beta-S, and moderately reversible with guanosine 5'-triphosphate (GTP)-gamma-S, in the whole cell pipette, leaving open the possibility that it is not mediated by a G protein. 4. Hence, PP inhibits ICa via two mechanisms: one G-protein-mediated and the other possibly G-protein independent. The former pathway is sensitive to pertussis toxin (PTX) and NEM, voltage dependent, and shared by several other transmitters in these cells. The latter pathway is PTX-and NEM-insensitive, not voltage dependent, and not affected by the presence of intracellular Ca2+ chelators.

Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling pathways. One is sensitive to pertussis toxin and acts rapidly by a membrane-delimited pathway on the channels. The other is not sensitive to pertussis toxin and acts more slowly through an unknown second messenger. These pathways are shared with several other agonists.

Neurotransmitters acting through G-protein-coupled receptors change the electrical excitability of neurons. Activation of receptors can affect the voltage dependence, the speed of gating, and the probability of opening of various ion channels, thus changing the computational state and outputs of a neuron. Each cell expresses many kinds of receptors, and uses several intracellular signaling pathways to modulate channel function in different ways. It has become possible to dissect these pathways by combining pharmacological and biophysical experiments. Recent patch-clamp work in sympathetic neurons will be summarized to illustrate the mechanisms underlying modulation and its significance.

The actions of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, on G-protein-mediated inhibition of N-type Ca2+ channels in adult rat superior cervical ganglion (SCG) neurons were studied using whole-cell voltage clamp. In SCG neurons, inhibition of ICa occurs by at least three separable pathways: one pertussis toxin (PTX) sensitive and voltage dependent, and two PTX insensitive and voltage independent. NEM blocked PTX-sensitive inhibition nearly completely, with only small effects on PTX-insensitive inhibition. Somatostatin inhibition is completely PTX sensitive and was wholly blocked by a 120 sec exposure to 50 microM NEM, with shorter exposure times producing a less complete block. Inhibition of ICa by norepinephrine (NE) is approximately half PTX sensitive and was also approximately half NEM sensitive. One component of muscarinic inhibition is PTX insensitive, voltage independent, and mediated by a diffusible cytoplasmic messenger; this pathway was largely spared by NEM treatment. Another pathway is also PTX insensitive and voltage independent, used by substance P, and was also largely NEM insensitive. Hence, in SCG neurons, NEM selectively inactivates PTX-sensitive G-proteins. We also find evidence that the PTX-insensitive action of NE is distinct from the other PTX-insensitive pathways, and therefore assign it to a fourth signaling pathway.

Actions of growth-hormone-releasing hormone (GHRH) on single rat anterior pituitary cells were studied using indo-1 fluorescence to monitor changes in intracellular calcium, [Ca2+]i, and perforated-patch recording to measure changes in membrane potential and ionic currents. GHRH elevated [Ca2+]i in non-voltage-clamped cells by a mechanism that was dependent upon extracellular Na+ and Ca2+ and was blocked by the dihydropyridine Ca(2+)-channel blocker, nitrendipine. Resting cells had a fluctuating membrane potential whose a mean value depolarized by 9 mV in response to GHRH. The membrane-permeant cAMP analogue, 8-(4-chlorophenylthio)cAMP, mimicked the action of GHRH on membrane potential. Under voltage clamping, GHRH activated a small inward current (1-5 pA). Two types of response could be distinguished. The type I response had an inward current that was largest at more negative potentials (-90 mV), and the type II response had inward current that was larger at more positive potentials (-40 to -70 mV). Both types of response were reversible and blocked by removal of extracellular Na+. These results suggest that the rise in [Ca2+]i produced by GHRH in non-voltage-clamped cells results from the activation via cAMP of a Na(+)-dependent conductance, which depolarizes the cell and increases the Ca2+ influx through voltage-gated Ca2+ channels.

We characterized inhibition of N-type Ca2+ and M current K+ channels in rat superior cervical ganglion neurons by angiotensin II (angioII) using the patch clamp. Of 120 neurons, 97 showed inhibition of ICa (mean 32%), which was slow in onset and very slow to reverse under whole-cell recording conditions. This inhibition was blocked by the AT1 receptor antagonist losartan, attenuated by inclusion of 2 mM GDP-beta-S in the pipette, mostly pertussis toxin insensitive, half-sensitive to N-ethylmaleimide, and wholly voltage independent. With 20 mM instead of 0.1 mM BAPTA in the pipette, the inhibition was strongly attenuated; however, we detected no angioII-induced [Ca2+]i signal using the fluorescent indicator indo-1. IBa from cell-attached patches was reduced by bath-applied angioII (mean 33%), suggesting use of a diffusible cytoplasmic messenger. M currents were inhibited by angioII in 8 of 11 neurons (mean 50%) cultured overnight. Hence, a second agonist, angioII, may share the slow, second messenger-utilizing, pertussis toxin-insensitive signaling pathway used by muscarinic agonists.

We have previously reported that GnRH induces rhythmic hyperpolarizations in male rat (35- to 45-day-old) gonadotropes by periodically opening apamin-sensitive Ca(2+)-activated K+ channels. Using the whole cell recording technique, we now show that these gonadotropes, identified with the reverse hemolytic plaque assay, express tetrodotoxin-sensitive Na+ channels and omega-conotoxin-insensitive, high voltage-activated Ca2+ channels that are partially sensitive to dihydropyridines. We found no low voltage-activated Ca2+ channels in these cells. At the normal resting potential, about 93% of the Na+ channels and 50% of the Ca2+ channels are inactivated. The GnRH-induced hyperpolarizations transiently remove the resting inactivation of Na+ and Ca2+ channels, enabling them to initiate action potentials at the termination of each hyperpolarization. Opening of Na+ channels accounts for the high rate of rise and the positive peak of the action potential. In addition, a significant fraction of Ca2+ channels should be activated during the action potentials, allowing a voltage-gated entry of extracellular Ca2+ that can enhance the frequency and amplitude of GnRH-induced intracellular Ca2+ oscillations. Therefore, we envision the following role for action potentials in GnRH-stimulated Ca2+ responses: action potentials will open voltage-gated Ca2+ channels that allow entry of extracellular Ca2+, which can help to replenish the intracellular Ca2+ store and act as a coactivator in the stimulation of intracellular Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive store.

We studied inhibition of N-type Ca2+ channels in rat superior cervical ganglion neurons by substance P (SP) and somatostatin-14 (Som). In whole-cell clamp, 70 of 82 acutely dissociated neurons showed inhibition (mean 37%) by 500 nM SP, and 54 of 61 showed inhibition by 240 nM Som (mean 57%). Pertussis toxin (PTX) blocked Som but not SP inhibition; intracellular dialysis with 2 mM GDP-beta-S attenuated inhibition with either peptide. Inhibition was voltage dependent with Som but not with SP. Neurokinin A (1 microM) or B was without effect, implicating NK1 tachykinin receptors. In cell-attached patches with bath-applied drugs, to test for a diffusible messenger, inhibition by SP or Som was only 8%. Thus, SP signaling is voltage independent and PTX insensitive; Som inhibition is voltage dependent and PTX sensitive; and both are membrane delimited.

Muscarinic receptors mediating suppression of Ca2+ current and of M-type K+ current in rat superior cervical ganglion neurons were subclassified pharmacologically by using the muscarinic receptor antagonists pirenzepine and himbacine. Our voltage clamp experiments previously distinguished fast and slow intracellular signaling pathways coupling muscarinic receptors to calcium channels. We now establish that the fast, pertussis toxin-sensitive suppression of Ca2+ current is mediated primarily by muscarinic receptors of the M4 subtype, whereas the slow, bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetate (BAPTA)-sensitive suppression of Ca2+ current is mediated primarily by muscarinic receptors of the M1 subtype. Both actions on Ca2+ current are blocked by guanosine 5'-[beta-thio]diphosphate. Muscarinic suppression of M current is slow, BAPTA-sensitive, and mediated by receptors of the M1 subtype. Hence the two muscarinic pathways use different receptors and different guanine nucleotide binding proteins to produce different actions on channels.

The role of ion channels in the secretion of gonadotropins from anterior pituitary gonadotropes has been difficult to study at the single cell level because the cells are difficult to distinguish from other pituitary cell types. Recently, a cell line, alpha T3-1, has been generated that makes and secretes the alpha-subunit of gonadotropins. These cells have GnRH receptors, but not TRH receptors, and are, thus, specific to the gonadotrope lineage. We have used the patch clamp technique to investigate the types of ion channels expressed in alpha T3-1 cells and to test for electrophysiological responses to GnRH and a phorbol ester. These cells express TTX-sensitive sodium channels with rapid kinetics, several types of potassium channels, including Ca2(+)-sensitive ones, and two types of calcium channels. The currents through calcium channels are augmented by application of 100 nM GnRH or 10 nM phorbol 12-myristate 13-acetate, a phorbol ester. The augmentation by GnRH and phorbol 12-myristate 13-acetate is consistent with other reports that a portion of stimulated gonadotropin release is dependent on external calcium and sensitive to block by dihydropyridine antagonists. Thus, this cell line may be useful for studies of mechanisms underlying responses to GnRH.

Modulation of N- and L-type Ca2+ channels by oxotremorine-M (oxo-M) acting on muscarinic receptors and norepinephrine (NE) acting on alpha-adrenergic receptors was studied in superior cervical ganglion neurons. Oxo-M depresses dihydropyridine-augmented tail currents in whole-cell recordings, whereas NE does not. This modulation of L-type Ca2+ channels by oxo-M is abolished by adding 20 mM BAPTA to the pipette solution. Oxo-M, acting via a diffusible messenger, reduces the probability of opening of single N- and L-type channels recorded in cell-attached patches. We conclude that a diffusible messenger signaling pathway activated by oxo-M inhibits both N- and L-type Ca2+ channels, whereas a membrane-delimited pathway activated by oxo-M and NE inhibits only N-type Ca2+ channels.

The psychoactive properties of Cannabis sativa and its major biologically active constituent, delta 9-tetrahydrocannabinol, have been known for years. The recent identification and cloning of a specific cannabinoid receptor suggest that cannabinoids mimic endogenous compounds affecting neural signals for mood, memory, movement, and pain. Using whole-cell voltage clamp and the cannabinomimetic aminoalkylindole WIN 55,212-2, we have found that cannabinoid receptor activation reduces the amplitude of voltage-gated calcium currents in the neuroblastoma-glioma cell line NG108-15. The inhibition is potent, being half-maximal at less than 10 nM, and reversible. The inactive enantiomer, WIN 55,212-3, does not reduce calcium currents even at 1 microM. Of the several types of calcium currents in NG108-15 cells, cannabinoids predominantly inhibit an omega-conotoxin-sensitive, high-voltage-activated calcium current. Inhibition was blocked by incubation with pertussis toxin but was not altered by prior treatment with hydrolysis-resistant cAMP analogues together with a phosphodiesterase inhibitor, suggesting that the transduction pathway between the cannabinoid receptor and calcium channel involves a pertussis toxin-sensitive GTP-binding protein and is independent of cAMP metabolism. However, the development of inhibition is considerably slower than a pharmacologically similar pathway used by an alpha 2-adrenergic receptor in these cells. Our results suggest that inhibition of N-type calcium channels, which could decrease excitability and neurotransmitter release, may underlie some of the psychoactive effects of cannabinoids.

Agonist-induced suppression of current in voltage-gated Ca2+ channels was studied in rat sympathetic neurons. We have previously distinguished two intracellular signaling pathways used by muscarinic agonists to suppress neuronal Ca2+ current-one fast and membrane delimited, the other slow and acting via a diffusible second messenger. We now show that the fast pathway is sensitive mainly to pertussis toxin and shifts the gating of Ca2+ channels to more positive voltages (voltage dependent). The slow pathway is pertussis toxin insensitive and depresses currents at all test potentials (voltage independent). Muscarinic agonists may also activate a pertussis toxin-insensitive fast pathway. alpha-Adrenergic agonists use the fast pertussis toxin-sensitive and the fast insensitive pathways, but not the slow one.

Muscarinic and alpha-adrenergic suppression of current through Ca2+ channels was studied in adult rat superior cervical ganglion neurons using whole-cell and cell-attached configurations of the patch-clamp technique. Oxotremorine methiodide suppressed ICa by both a rapid (much less than 1 s) and a slow (greater than 4 s) process, whereas norepinephrine suppressed ICa only by a rapid process. The slow muscarinic suppression could be prevented by adding 20 mM BAPTA, a Ca2+ chelator, to the recording pipette, whereas the adrenergic suppression was not affected. Muscarinic, but not alpha-adrenergic, receptors can couple to Ca2+ channels by a second messenger capable of diffusing into an on-cell patch. This signal seems not to be carried by intracellular Ca2+, cGMP, cAMP, or protein kinase C.

The role of intracellular Ca2+ concentration ([Ca2+]i) in the muscarinic suppression of Ca2+ current and M-type K+ current has been investigated in isolated rat sympathetic neurons using the whole-cell patch-clamp technique and fura-2 fluorescence measurements. Muscarinic stimulation suppressed currents without raising [Ca2+]i. Nonetheless, intracellular bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) (11-12 mM), a Ca2+ chelator, reduced Ca2(+)-current suppression from 82 to 15%. For the latter, we explain the BAPTA action by a requirement for a certain minimum [Ca2+]i for continued operation of the pathway coupling muscarinic receptors to M-type K+ channels. The pathway coupling muscarinic receptors to Ca channels also showed some dependence on [Ca2+]i, but there may also be a blocking action of BAPTA that is independent of Ca2+ chelation.