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We investigated which subtypes of G-protein β 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 β subunits. Gβ1and Gβ2strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The Gβ5subunit produced much weaker effects than Gβ1and Gβ2, whereas Gβ3and Gβ4were 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, Gβ1or Gβ2coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a α1subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the Gβ5subunit interacted weakly, and Gβ3and Gβ4failed to interact. Together, these results suggest that Gβ1and/or Gβ2subunits 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 α1subunit is a principal receptor for this inhibition.

Muscarinic acetylcholine receptors are members of the G protein-coupled receptor superfamily expressed in neurons, cardiomyocytes, smooth muscle, and a variety of epithelia. Five subtypes of muscarinic acetylcholine receptors have been discovered by molecular cloning, but their pharmacological similarities and frequent colocalization make it difficult to assign functional roles for individual subtypes in specific neuronal responses. We have used gene targeting by homologous recombination in embryonic stem cells to produce mice lacking the m1 receptor. These mice show no obvious behavioral or histological defects, and the m2, m3, and m4 receptors continue to be expressed in brain with no evidence of compensatory induction. However, the robust suppression of the M-current potassium channel activity evoked by muscarinic agonists in sympathetic ganglion neurons is completely lost in m1 mutant mice. In addition, both homozygous and heterozygous mutant mice are highly resistant to the seizures produced by systemic administration of the muscarinic agonist pilocarpine. Thus, the m1 receptor subtype mediates M current modulation in sympathetic neurons and induction of seizure activity in the pilocarpine model of epilepsy.

Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca2+ uptake and extrusion modulate free cytosolic [Ca2+] (Cac) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca2+ entry, and to monitor Ca2+-activated currents that report near-surface [Ca2+]. We show that rapid recovery from elevations of Cac requires both the mitochondrial Ca2+ uniporter and the mitochondrial energization that drives Ca2+ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca2+] (Cam). The indicated resting Cam of 100–200 nM is similar to the resting Cac reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Cam and Cac at high temporal resolution shows that, although Cam increases less than Cac, mitochondrial sequestration of Ca2+ is fast and has high capacity. We find that mitochondrial Ca2+ uptake limits the rise and underlies the rapid decay of Cac excursions produced by Ca2+ entry or by mobilization of reticular stores. We also find that subsequent export of Ca2+ from mitochondria, seen as declining Cam, prolongs complete Cac recovery and that suppressing export of Ca2+, by inhibition of the mitochondrial Na+/ Ca2+ exchanger, reversibly hastens final recovery of Cac. We conclude that mitochondria are active participants in cellular Ca2+ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca2+.

Cl- secretion by pancreatic duct epithelial cells (PDEC) regulates cellular HCO3- secretion, an important component of the exocrine pancreas. In cystic fibrosis, for example, impaired function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel results in decreased pancreatic secretion and secondary pancreatic insufficiency. Studies of ion transport by PDEC have been hindered by the lack of a practical in vitro model. We have successfully cultured nontransformed dog PDEC on Vitrogen-coated permeable membranes overlying a feeder layer of myofibroblasts and report the characterization of Cl- channels in these cells. Cl- conductance, assessed through efflux of 125I from PDEC, was stimulated by agents acting via adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+. The Cl- conductances activated by cAMP and Ca2+ were distinct, since they were differentially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and, to a lesser extent, by 5-nitro-2-(3-phenylpropylamino)benzoic acid and diphenylamine-2 carboxylate. Patch-clamp studies confirmed the presence of Cl- channels activated by cAMP and Ca2+, with differential inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The presence of CFTR Cl- channels in PDEC was confirmed by immunoblotting. These cultured PDEC are an optimal model for studies of pancreatic duct secretion.

Local recycling of synaptic vesicle membrane at nerve terminals is necessary to maintain a readily releasable pool of transmitter. To what extent are the dynamics of vesicle recycling subject to modulation? We examined the influence of presynaptic GABAB receptors on vesicle dynamics at single synapses using optical imaging of FM1–43 in cultured rat hippocampal neurons. The kinetics of FM1–43 destaining indicate that synapses from a single neuron have a unimodal distribution of release probabilities, and GABAB-mediated inhibition occurs uniformly at all sites. Electrical and optical recordings from single cells show that the inhibition of excitatory transmission is entirely accounted for by a rapidly reversible reduction of exocytosis. In contrast, GABAB receptors do not alter the rate or extent of endocytosis.

In rat pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) stimulates rhythmic release of Ca2+ from stores sensitive to inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which in turn induces an oscillatory activation of apamin-sensitive Ca2+-activated K+ current, IK(Ca). Since GnRH also activates protein kinase C (PKC), we investigate the action of PKC while simultaneously measuring intracellular Ca2+ concentration ([Ca2+]i) and IK(Ca). Stimulation of PKC by application of phorbol 12-myristate 13-acetate (PMA) did not affect basal [Ca2+]i. However, PMA or phorbol 12,13-dibutyrate (PdBu), but not the inactive 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), reduced the frequency of GnRH-induced [Ca2+]i oscillation and augmented the IK(Ca) induced by any given level of [Ca2+]i. The slowing of oscillations and the enhancement of IK(Ca) were mimicked by synthetic diacylglycerol (1,2-dioctanoyl-sn-glycerol) and could be induced during ongoing oscillations that had been initiated irreversibly in cells loaded with guanosine 5'-O-(3-thiotriphosphate) (GTP-[gammaS]). In contrast, when oscillations were initiated by loading cells with Ins(1,4,5)P3, phorbol esters enhanced IK(Ca) without affecting the frequency of oscillation. The protein kinase inhibitor, staurosporine, reduced IK(Ca) without affecting [Ca2+]i and partially reversed the phorbol-ester-induced slowing of oscillation. Therefore, activation of PKC has two rapid effects on gonadotropes. It slows [Ca2+]i oscillations probably by actions on phospholipase C, and it enhances IK(Ca) probably by a direct action on the channels.

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.

Publisher Summary The pituitary gland is a versatile hormonal interface between the nervous system and the rest of the body, a warehouse of peptides waiting for nervous requisitions. In mammals, the anterior pituitary can secrete at least six major peptide hormones from at least five cell types. Each cell is under the control of specific releasing hormones and neurotransmitters secreted into the pituitary portal circulation by hypothalamic neurons. This chapter describes a study of G protein-mediated modulation of ion channels, which involved working with muscarinic activation of a K+ channel in cardiac trial cells and GnRH and muscarinic inhibition of a K+ channel in frog sympathetic ganglia, where GnRH was a neurotransmitter. The work with GnRH has explained the activities of gondaotrope, where a chain of events is triggered by GnRH. The primary secretory response to GnRH is unambiguously attributable to a steady rise of IP3that leads to an oscillatory release of Ca2+ from intracellular stores. Each elevation brings [Ca2+]i well above the 300 nM level that suffices to initiate exocytosis of numerous secretory granules. The secretory stimulus for gonadotropes does not initiate a major entry of extracellular Ca2+ as a primary signal for exocytosis. The electrophysiological responses of stimulated gonadotropes include a hyperpolarization during periods of [Ca2+]i rise instead of a depolarization.

Phosphatidylinositol −4,5-bisphosphate (PI(4,5)P2) is an important regulator of activity of a variety of ion channels including inwardly rectifying potassium channels, KCNQ, TRP, and voltage-gated calcium channels. Several groups have provided evidence that also voltage-gated potassium (Kv) channels might be regulated by PI(4,5)P2. Taking the wide expression of Kv channels in a variety of different tissues into account, such regulation of activity by PI(4,5)P2 could potentially be of great physiological importance. To determine whether several Kv channels show a regulation of their activity by PI(4,5)P2 we have coexpressed them in tsA201-cells with either a voltage-gated lipid 5-phosphatase (VSP), a G-protein coupled receptor (M1R), or an engineered fusion protein carrying both a 4- and 5-lipid phosphatase activity (Pseudojanin, PJ). These tools deplete PI(4,5)P2 with application of depolarization, muscarinic agonists, or rapamycin, respectively. We monitored PI(4,5)P2 amounts at the plasma membrane by FRET with PH-probes from PLCδ1 simultaneously with whole-cell patch-clamp recordings. Activating VSP or PJ inhibited KCNQ2/3 channel current leaving only about 5-10 % remaining current. Activation of M1R inhibited KCNQ2/3 current a comparable amount. Thus the tools used for our assays are working. We tested with them for potential regulation of activity by PI(4,5)P2 of Kv1.1/Kvβ1.1, Kv1.3, Kv1.4 and Kv1.5/Kvβ1.3, Kv3.4, Kv4.2, Kv4.3 (both with different KChIPs and DPP6-s) and HERG/KCNE2. Interestingly, we found a substantial upregulation of current density and a removal of inactivation for Kv1.1/Kvβ1.1 and Kv3.4 upon activation of M1R, but no changes in activity upon only activating VSP or PJ. All other channels tested showed no alteration in activity in any of the assays we used. In conclusion, “physiological” depletion of PI(4,5)P2 at the plasma membrane does not seem to influence activity of most tested Kv channels. Supported by NIH grant NS08174 and the AvH Foundation.

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 GH-releasing hexapeptide (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 or GHRP-6) on single rat somatotropes were studied using whole cell patch clamp electrophysiology and indo-1 Ca2+ photometry. GHRP-6 elevated intracellular free Ca2+ ([Ca2+]i) in two phases: a rapid transient phase, followed by a persistent phase. Based on its insensitivity to treatments that block Ca2+ entry [removal of external Ca2+, addition of the dihydropyridine Ca2+ channel blocker nitrendipine (1 microM), and the hyperpolarizing action of zero external Na+ or 100 nM somatostatin], the transient elevation is the result of release of Ca2+ from intracellular stores. The half-maximal concentration for the peak [Ca2+]i rise during Ca2+ release was 49 nM GHRP-6. Prior treatment of cells with caffeine (10 mM) or ryanodine (50 microM) abolished or partially occluded GHRP-6-induced Ca2+ release. Simultaneous measurement of [Ca2+]i and membrane current or potential revealed that the transient release of Ca2+ by GHRP-6 activates a voltage-independent Ca(2+)-activated K+ conductance, which transiently hyperpolarizes the somatotrope. The GHRP-6-induced persistent [Ca2+]i elevation is abolished by removal of external Ca2+ or external Na+ or the addition of 1 microM nitrendipine or 100 nM somatostatin, consistent with Ca2+ entry through voltage-dependent Ca2+ channels. In nondialyzed cells (perforated patch recording), we have identified a long-lasting GHRP-6-induced depolarization which may be responsible for the persistent [Ca2+]i elevation.

1. Whole-cell voltage clamp was used in conjunction with the fluorescent Ca2+ indicator indo-1 to measure extracellular Ca2+ entry and intracellular Ca2+ concentrations ([Ca2+]i) in rat gonadotrophs identified with the reverse haemolytic plaque assay. 2. Depolarizations to potentials more positive than -40 mV elicited inward Ca2+ current (ICa) and transient elevations of [Ca2+]i. 3. The relationship between [Ca2+]i elevations and Ca2+ entry with different Ca2+ buffer concentrations in the pipette showed that endogenous Ca2+ buffers normally bind approximately 99% of the Ca2+ entering the cell. 4. With [Ca2+]i elevations less than 500 nM, decay of [Ca2+]i could be approximated by an exponential whose time constant increased with the concentration of exogenous Ca2+ buffers. 5. Inhibitors of intracellular Ca(2+)-ATPases, thapsigargin, cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ), caused [Ca2+]i to rise. Application of BHQ during [Ca2+]i oscillations induced by gonadotrophin-releasing hormone (GnRH) terminated the oscillation in a slowly decaying elevation. BHQ slowed the decay of depolarization-induced [Ca2+]i elevations about 3-fold. 6. Taking into account the Ca2+ buffering properties of the cytoplasm permitted estimation of the fluxes and rate constants for Ca2+ movements in gonadotrophs. The intracellular store is a major determinant of Ca2+ homeostasis in gonadotrophs.

Publisher Summary The secretion of hormones from endocrine cells in the anterior pituitary regulates a variety of physiological functions including reproduction, lactation, growth, and metabolism. However, until recently, cellular mechanisms underlying the stimulus-secretion coupling in these cells have been hard to study for two reasons: (1) it was difficult to isolate specific cell types from the heterogenous population of anterior pituitary cells and (2) the intracellular environment could not be manipulated or monitored accurately. The intracellular environment can be altered by diffusing various activators or inhibitors of second messenger pathways, caged molecules, and ion-selective fluorescent indicator dyes into the cell. A thin glass-bottomed Cunningham chamber can be constructed as follows. A circular hole of approximately 20 mm diameter is drilled in the bottom of a 35-mm plastic culture dish (Falcon). A 25-mm circular glass coverslip is cemented onto the bottom of the culture dish with Sylgard 184 elastomer (Dow Corning). The chamber is rinsed with 100% methanol and then several times with distilled water to remove any grease. To facilitate cell attachment, the bottom of the chamber is coated by adding 1 ml of 1% gelatin solution (Sigma). After 5 min, the solution is removed and the chamber is allowed to dry under UV light.

Gq-coupled plasma membrane receptors modulate cellular functions by activating phospholipase C (PLC), which hydrolyses the membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) into the second messengers inositoltrisphosphate (IP3) and diacylgycerol (DAG). To better understand the mechanisms that govern these partially independent signals we monitored in single, living tsA-201 cells levels of PIP2, IP3, calcium, DAG, and PKC by optical probes and current. We compared (i) activation of (low-abundance) endogenous purinergic receptors and overexpressed M1 muscarinic receptors, and (ii) different concentrations of the muscarinic agonist oxotremorine-M (oxo-M). Whereas the peak responses from reporters of IP3 (LIBRAvIII) and DAG (C1 domains of PKCγ) scale with abundance of receptor or agonist, downstream production of calcium (Fura4F) and PKC activation (CKAR) do not. Amplitude and duration of calcium signals elicited by 100 µM UTP, 10 nM oxo-M, or 10 µM oxo-M are almost identical. The only difference is a shorter latency with 10 µM oxo-M. These data suggest that a relatively low amount of IP3 is required for calcium release. This interpretation is supported by the finding that a full-size calcium response can still be elicited after PIP2 is depleted by recruiting a PI 5-phosphatase to the plasma membrane (by rapamycin-induced dimerization). Duration and late recovery time courses are different between IP3 (duration=68 s; τoff =55 s) and calcium (duration=110 s; τoff =34 s), suggesting that once a threshold of IP3 is reached, the calcium signal unfolds. Therefore we conclude that the IP3 requirement for calcium release must be low. The time point and IP3 level (from LIBRAvIII) at which the calcium response starts can provide an estimate of this IP3 threshold. Supported by NIH grants NS08174 & GM83913 and the HFSP.

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.

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.

1. Whole-cell and single-channel voltage-clamp techniques were used to record the 5-HT3 receptor-mediated currents in neurons freshly dissociated from rat superior cervical ganglia. 2. Whole-cell currents elicited by brief pressure ejection of 5-HT (10 microM) reversed at -4.5 mV when extracellular and intracellular solutions mainly contained NaCl and CsCl. The peak current-voltage relation showed modest inward rectification that was fully developed within less than 2 ms of the applied voltage step. 3. With prolonged application of 5-HT (10 microM) using a fast perfusion system, the response desensitized in two phases with fast and slow time constants of 0.57 and 6.0 s at -74 mV. The time constants showed little voltage dependence; however, the relative amplitude of the two components was significantly dependent on voltage. The time course of desensitization was not affected by agents that increase the levels of intracellular cyclic AMP. 4. The relative permeability of the channel was determined from reversal potential changes. The channel passed small cations non-selectively, with permeability ratios (PX/PNa) of 0.93 and 1.24 for Cs+ and K+. The organic cations Tris and glucosamine were measurably permeant with permeability ratios of 0.19 and 0.06. Ca2+ was fairly permeant with a relative permeability of 0.55 in 20 mM solution and of 0.16 when the concentration of CaCl2 was increased to 115 mM. No permeability was detected for Cl-. 5. Fluctuation analysis of the whole-cell current revealed an apparent single-channel current of approximately 0.18 pA at -74 mV. 6. 5-HT-activated single-channel currents were recorded in excised outside-out patches. When 5-HT (10 microM) was delivered by pressure ejection, channel openings appeared rapidly with a delay of 28 ms. The unitary current was about approximately 0.80 pA at -74 mV. The channel activity induced by bath perfusion of 5-HT (0.8 microM) was significantly reduced by 100 nM of the 5-HT3 receptor-specific antagonists 3-tropanyl-3,5-dichlorobenzoate (MDL 72222) or 3-tropanyl-indole-3-carboxylate (ICS 205-930). 7. The single-channel current-voltage relation was non-linear, with moderate inward rectification similar to that of the whole-cell current. The chord conductance of the channel decreased with membrane depolarization from 14.6 pS at -104 mV to only 9.9 pS at -54 mV. Open-time distributions consisted of two components with mean time constants of 0.45 and 2.8 ms at -104 mV. Burst-length distributions were also made up of two components with time constants of 0.45 and 4.6 ms.(ABSTRACT TRUNCATED AT 400 WORDS)

Secretion of gonadotropic hormones from pituitary gonadotropes in response to gonadotropin-releasing hormone (GnRH) is essential for regulation of reproductive potential. Gonadotropes from male rats exhibited an unusual form of cellular excitation that resulted from periodic membrane hyperpolarization. GnRH induced an oscillatory release of intracellular Ca2+ via a guanosine triphosphate (GTP) binding protein-coupled phosphoinositide pathway and hyperpolarized the gonadotrope periodically by opening apamin-sensitive Ca(2+)-activated K+ (SK) channels. Each hyperpolarization was terminated by firing of a few action potentials that may result from removal of inactivation from voltage-gated Na+ and Ca2+ channels.

Agonist-induced suppression of current in voltage-gated Ca 2+ channels was studied in rat sympathetic neurons. We have previously distinguished two intracellular signaling pathways used by muscarinic agonists to suppress neuronal Ca 2+ 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 Ca 2+ 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. α-Adrenergic agonists use the fast pertussis toxin-sensitive and the fast insensitive pathways, but not the slow one.

Voltage-sensitive phosphatases (VSPs) have a voltage sensor linked to a phosphoinositide (PI) 5-phosphatase, which hydrolyzes plasma membrane PI-(4,5)-bisphosphate (PIP2) to PI(4)P (Iwasaki, PNAS 105, 7970). We used PIP2 hydrolysis by VSP from Ciona intestinalis (ci-VSP) and zebrafish (dr-VSP) to better understand PIP2 binding and resynthesis. PIP2 was monitored using the PIP2-sensitive M-current (KCNQ2/3) and FRET between a pair of PIP2-binding probes (PH-PLCδ1-CFP & PH-PLCδ1-YFP). Depolarizations to +100 mV lasting >50 ms reduced M-current and PH-probe FRET. PIP2 depletion was saturated by depolarizations lasting 500-1000 ms. Evidently PH-probe FRET and M-current respond quickly to changes in plasma membrane PIP2.After repolarization, PH-probe FRET and M-current relaxed to baseline values with time constants of ~10 s in a wortmannin-insensitive manner. This reflects endogenous PIP 5-kinase converting PI(4)P back to PI(4,5)P2. Overexpression of PIP 5-kinase increased the length of depolarization required to deplete PIP2, and speeded PIP2 recovery after repolarization.Recovery of PIP2 after VSP activation is ~10x faster than after PLC activation. However, it only requires PIP 5-kinase, whereas recovery after PLC activation requires PI 4-kinase and PIP 5-kinase in series. Thus PI 4-kinase must be the slower enzyme. To estimate the rate of PI 4-kinase, we compared translocation of a fluorescent probe that reports plasma membrane PI(4)P (PH-OSH2, T.Balla) to translocation of the PIP2-binding probe in confocal time-lapse imaging. Upon PLC activation through M1 receptors, the plasma membrane PI(4)P signal decreased 20 s later than the PIP2 signal. Both probes recovered with similar time courses. FRET photometry between either PIP2-probes or PI(4)P-probes showed comparable results. The simultaneous recovery of both probes is consistent with the hypothesis that recovery of PIP2 is governed by rate-limiting synthesis of PI(4)P by the PI 4-kinase, followed by rapid conversion of PI(4)P into PI(4,5)P2 by the PIP 5-kinase.NIH-NS008174&HFSP

Background: Intracellular organelles move along microtubules and filamentous actin (F-actin) using molecular motors. Previously we reported that movement of secretory granules was hindered by Ca2+-dependent formation of a dense cytoplasmic F-actin meshwork in pancreatic duct epithelial cells (PDEC). Aim: Here we addressed whether other organelles such as mitochondria and lysosomes also are controlled by the same mechanism. Methods: Mitochondria and lysosomes were labeled with fluorescent MitoTracker Red and LysoTracker Red, respectively. Their movements were monitored every 1.2 s with high resolution fluorescence microscopy and measured by comparing subsequent images. Cells were stimulated with UTP to activate an endogenous P2Y2 G-protein coupled receptor to increase intracellular Ca2+. Results: Mitochondria and lysosomes moving actively at rest stopped rapidly (τ ∼ 2.6 s) after Ca2+ rise. This organelle ‘freezing’ was accompanied by the formation of F-actin in the whole cytoplasm as stained with phalloidin-Alexa 488. EGFP-actin binding domain 2 (ABD2) expressed in PDEC, indicated a rapid formation of cytoplasmic F-actin (τ ∼ 7.8 s). In addition, the freezing of the organelles was blocked by latrunculin B, an inhibitor of F-actin formation. Conclusion: Ca2+-dependent formation of a fine F-actin meshwork reduces the movement of intracellular organelles physically in PDEC. The freezing mechanism controls exocytosis of secretory granules and may affect local ATP supply by controlling mitochondrial distribution in PDEC.Supported by UW Royalty Research Fund and an NIH grant (GM083913).

Two proteins, STIM1 in the endoplasmic reticulum and Orai1 in the plasma membrane, are required for the activation of Ca2+ releaseactivated Ca2+ (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 Ca2+ stores were depleted, the channels clustered in aggregates containing ∼1,300 channels, amplifying the local Ca2+ entry.

We recently derived a GnRH-responsive pituitary cell line of the gonadotrope lineage (alpha T3-1) by targeted oncogenesis in transgenic mice. Here, we report studies characterizing the GnRH receptors present in these cells and the intracellular responses to GnRH treatment. The receptors in alpha T3-1 cells show specificity for different GnRH analogs, with dissociation constants very similar to those found in normal rat and mouse pituitary. The concentration of receptors is within the range found in normal pituitary. The addition of GnRH or GnRH agonists increases phosphoinositide turnover and protein kinase-C translocation to membranes, and enhances activation of voltage-sensitive calcium channels. However, GnRH does not affect cAMP levels. Analysis of alpha-subunit mRNA levels demonstrated induction by GnRH and phorbol esters. Our results indicate that GnRH initiates a cascade of intracellular events that generate a set of second messengers, one or more of which is involved in the regulation of gene expression. The responses of alpha T3-1 cells to GnRH appear to have characteristics equivalent to those of primary pituitary gonadotropes, indicating the utility of this cell line as a model system for the study of GnRH responses.

Carbon-fiber amperometry detects oxidizable molecules released by exocytosis. We extended this electrochemical technique to cells that do not normally secrete oxidizable transmitters. We incubated AtT-20 cells, pituitary gonadotropes, cultured cerebellar granule cells, and yeast with high concentrations of dopamine (DA) and observed spontaneous and evoked quantal release of DA by amperometry. The rate of detectable spontaneous amperometric events was used as a measure of loading in AtT-20 cells. With 70 mm DA in the bath, loading was complete within 40 min. Cytoplasmic accumulation preceded vesicular loading. Loading decreased proportionally as the bath DA concentration was lowered. Loading rates were similar at 37 and 25 degrees C and much slower at 15 degrees C. Loading was blocked by bafilomycin A(1), a proton pump inhibitor, but not by bupropion, an inhibitor of the plasma membrane DA transporter. Other cells were tested. Spontaneous quantal events became more frequent and evoked events became larger and more frequent when PC12 cells were loaded with DA. Fluid-phase loading of neurons by short stimulation in DA solutions seemed selective for the synaptic vesicles. Thus, many cell types can be loaded with DA to study spontaneous and evoked exocytosis. The amine molecules enter these cells passively and may become concentrated in acidic vesicles by protonation.

A new family of K + channels has been defined in a paper in this week9s issue [see Kohler et al . (p. 1709)], reporting the cloning of three members. The Perspective by Hille explains why this class of channels may underlie the control of attention in the brain by the neurotransmitter norepinephrine.

CALCIUM ions entering cells through voltage-gated Ca 2+ channels initiate rapid release of neurotransmitters and secretion of hormones. Ca 2+ currents can be inhibited in many cell types by neurotransmitters acting through G proteins via a membrane-delimited pathway independently of soluble intracellular messengers 1-4 . 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 Ca 2+ currents 1,4-6 . This pathway regulates the activity of N-type and P/Q-type Ca 2+ channels 1,2,7 , which are localized in presynaptic terminals 8,9 and participate in neurotransmitter release 10-13 . Synaptic transmission is inhibited by neurotransmitters through this mechanism 1,4 . G-protein α subunits confer specificity in receptor coupling 1-4,14-17 , but it is not known whether the Gα or Gβγ subunits are responsible for modulation of Ca 2+ channels. Here we report that Gβγ subunits can modulate Ca 2+ channels. Transfection of Gβγ into cells expressing P/Q-type Ca 2+ channels induces modulation like that caused by activation of G protein-coupled receptors, but Gα subunits do not. Similarly, injection or expression of Gβγ subunits in sympathetic ganglion neurons induces facilitation and occludes modulation of N-type channels by noradrenaline, but Gα subunits do not. In both cases, the Gγ subunit is ineffective by itself, but overexpression of exogenous Gβ subunits is sufficient to cause channel modulation.

Potassium currents were recorded from voltage-clamped nodes of isolated, myelinated axons of Rana pipiens. Nodes were maintained in a modified Ringer solution containing tetrodotoxin to block sodium current and 47.5 mM-potassium to minimize effects of extracellular potassium accumulation. Voltage protocols included depolarizing pulses lasting a few milliseconds to several seconds. Fluctuations about the ensemble average of the current were characterized in terms of non-stationary variance and autocovariance. The fluctuations had a Gaussian amplitude distribution and were virtually free of contaminations from systematic variations of the membrane current. Corrections for background noise were based on measurements done while potassium current was blocked with tetraethylammonium, and on simulations of extrinsic current fluctuations expected to arise from noise in the actual membrane voltage. The fluctuations were attributed to variations of nodal potassium conductance, since they were absent at the reversal potential of the potassium current and at membrane voltages that do not activate potassium current. Covariances indicated that voltage steps that reversed a macroscopic potassium current also reversed the sign of the fluctuation. Plots of the conductance variance versus the mean potassium conductance were generated from both the activation and deactivation (tail) phases of the potassium currents at various voltages between -80 and +70 mV. When the current was activated by a small depolarization (-50 mV) the trajectories from both phases were indistinguishable and were fitted by the parabola expected for a single population of channels with only one open-channel conductance. Apparent single-channel conductance from the early activation phase averaged 24 pS and was not significantly voltage dependent. In contrast, experiments with large depolarizations (+10 to +70 mV) gave significantly different variance--mean trajectories during activation and deactivation and these trajectories were poorly fitted by parabola. This result indicates that the fluctuations reflect several populations of channels and/or a population of channels that can have several levels of non-zero conductance. Projections of the fluctuation covariance showed long correlations, as well as the rapidly decaying component expected from the activation gating of channels. A slow fluctuation arose at a time slightly later than the rise of potassium current, spanned the entire length of brief depolarizations, and extended up to 880 ms during long depolarizations.(ABSTRACT TRUNCATED AT 400 WORDS)

Cette etude considere la structure et l'evolution de deux grandes familles de canaux ioniques: les canaux sensibles au voltage et les canaux couples aux recepteurs. De plus elle decrit quelques roles physiologiques recents de ces canaux: le couplage excitation-contraction et le couplage excitation-secretion et discute d'un eventuel controle de ces canaux par des systemes de seconds messagers

Local anesthetic solutions were applied suddenly to the outside of single myelinated nerve fibers to measure the time course of development of block of sodium channels. Sodium currents were measured under voltage clamp with test pulses applied several times per second during the solution change. The rate of block was studied by using drugs of different lipid solubility and of different charge type, and the external pH was varied from pH 8.3 to pH 6 to change the degree of ionization of the amine compounds. At pH 8.3 the half-time of action of amine anesthetics such as lidocaine, procaine, tetracaine, and others was always less than 2 s and usually less than 1 s. Lowering the pH to 6.0 decreased the apparent potency and slowed the rate of action of these drugs. The rate of action of neutral benzocaine was fast (1 s) and pH independent. The rate of action of cationic quaternary QX-572 was slow (greater than 200 s) and also pH independent. Other quaternary anesthetic derivatives showed no action when applied outside. The result is that neutral drug forms act much more rapidly than charged ones, suggesting that externally applied local anesthetics must cross a hydrophobic barrier to reach their receptor. A model representing diffusion of drug into the nerve fiber gives reasonable time courses of action and reasonable membrane permeability coefficients on the assumption that the hydrophobic barrier is the nodal membrane. Arguments are given that there may be a need for reinterpretation of many published experiments on the location of the anesthetic receptor and on which charge form of the drug is active to take into account the effects of unstirred layers, high membrane permeability, and high lipid solubility.

1. Na current fluctuations in nodes of Ranvier were measured under voltage clamp conditions as described in the preceding paper (Conti, Hille, Neumcke, Nonner & Stämpfli, 1976) and analysed in terms of power spectral density calculated for frequencies between 30 Hz and 5 kHz. 2. External (10(-5) g/ml.) Leiurus scorpion venom or Anemonia Toxin II (3 X 10(-5) g/ml.) or internal 20 mM iodate were applied in order to remove or slow down inactivation in part of the Na channels. The treatment increased the steady-state Na current during the noise measurement one-to eight fold over that in normal fibres. 3. Noise spectra were interpreted as the sum of 1/f noise and noise SNa(f) due to all-or-none, open-close transitions of single Na channels. The drug effects on the inactivation could be accounted for either by assuming two populations of channels, one with and one without inactivation, or by postulating a single population with modified inactivation characteristics. 4. Except for an increase in amplitude, the fluctuation spectra SNa(f) were similar to the ones in normal nodes. Again, the time constants taum obtained from the fit of the spectra agreed within a factor of 2 with the values of taum found in the macroscopic Na currents. 5. From the fluctuation spectra, single Na channel conductances gamma of 5-4 +/- 0-4 pS (iodate), 6-7 +/- 0-5 pS (Leiurus) and 7-0 +/- 0-6 pS (Anemonia) were calculated. The value of gamma was not significantly voltage dependent. 6. Our observations indicate that inactivation of Na channels can be modified with at most small effects on the microscopic properties of the activation process and on the conductance of the open channel. They suggest that the h mechanism normally produces all-or-none, open-close changes of conductance.

Macroscopic currents in Na channels were recorded from adult frog skeletal muscle under voltage clamp as various toxins were added to the bathing medium. Veratridine, cevadine, and 3-(4-ethoxybenzoyl)-veracevine modified the Na channels in a use-dependent manner during depolarizations and held them open for 3, 2.4, and 1.2 s, respectively, at -90 mV. The three alkaloids modified channels in the same way. Activation gating was shifted about -100 mV by the modification, and reversible closing of the channels by strong hyperpolarizations slowed reversal of the modification. The synthetic insecticides deltamethrin, EDO, GH739, and GH414 also modified channels during depolarizations that opened channels. The modification lasted 3 s with deltamethrin, but only 3-5 ms with the others. Hyperpolarization speeded the shutting off of current in insecticide-modified channels, but no reversible activation gating could be demonstrated. The ionic selectivity, PNa/PNH4, of channels was decreased by all of the toxins. This ratio was 0.11 in normal channels, 0.26 in insecticide-modified channels, and 0.7-1.6 in veratrum-alkaloid-modified channels. During use-dependent modification, the veratrum alkaloids reduced the total Na current markedly, while deltamethrin did not. Thus, alkaloid and insecticide modifications share many features but differ in how much the conducting properties of the pore are changed and whether the channel can close reversibly while the toxin remains bound.

The effects of external anions on gating of Na channels of frog skeletal muscle were studied under voltage clamp. Anions reversibly shift the voltage dependence of peak sodium permeability and of steady state sodium inactivation towards more negative potentials in the sequence: methanesulfonate less than or equal to Cl- less than or equal to acetate less than Br- less than or equal to NO-3 less than or equal to SO2-4 less than benzenesulfonate less than SCN- less than ClO-4; approximately the lyotropic sequence. Voltage shifts are graded with mole fraction in mixtures and are roughly additive to calcium shifts. The peak PNa is not greatly affected. Except for SO2-4, these anions did not change the Ca++ activity of the solutions as measured with the dye murexide. Shifts of gating can be explained as the electrostatic effect of anion adsorption to the Na channel or to nearby lipid. Such adsorption is expected to follow the lyotropic series. Anions also interfere significantly with the response of a Ca-sensitive membrane electrode following the same sequence of effectiveness as the shifts of gating. The lyotropic anions decrease the Ca++ sensitivity and cause anomalously negative responses of the Ca electrode because these anions are somewhat permeant in the hydrophobic detector membrane.

Ionic fluxes in Na channels of myelinated axons show ionic competition, block, and deviations from simple flux independence. These phenomena are particularly evident when external Na+ ions are replaced by other permeant or impermeant ions. The observed currents require new flux equations not based on the concepts of free diffusion. A specific permeability model for the Na channel is developed from Eyring rate theory applied to a chain of saturable binding sites. There are four energy barriers in the pore and only one ion is allowed inside at a time. Deviations from independence arise from saturation. The model shows that ionic permeability ratios measured from zero-current potentials can differ from those measured from relative current amplitudes or conductances. The model can be fitted to experiments with various external sodium substitutes by varying only two parameters: For each ion the height of the major energy barrier (the selectivity filter) determines the biionic zero-current potential and the depth of the energy well (binding site) just external to that barrier then determines the current amplitudes. Voltage clamp measurements with myelinated nerve fibers are given showing numerous examples of deviations from independence in ionic fluxes. Strong blocks of ionic currents by guanidinium compounds and Tl+ ions are fitted by binding within the channel with apparent dissociation constants in the range 50-122 mM. A small block with high Na+ concentrations can be fitted by Na+ ion binding with a dissociation constant of 368 mM. The barrier model is given a molecular interpretation that includes stepwise dehydration of the permeating ion as it interacts with an ionized carboxylic acid.

Evidence is given for a high density of negative surface charge near the sodium channel of myelinated nerve fibres. The voltage dependence of peak sodium permeability is measured in a voltage clamp. The object is to measure voltage shifts in sodium activation as the following external variables are varied: divalent cation concentration and type, monovalent concentration, and pH. With equimolar substitution of divalent ions the order of effectiveness for giving a positive shift is: Ba = Sr < Mg < Ca < Co ≈ Mn < Ni < Zn. A tenfold increase of concentration of any of these ions gives a shift of + 20 to + 25 mV. At low pH, the shift with a tenfold increase in Ca 2+ is much less than at normal pH, and conversely for high pH. Solutions with no added divalent ions give a shift of — 18 mV relative to 2 mM Ca 2+ . Removal of 7/8 of the cations from the calcium-free solution gives a further shift of — 35 mV. All shifts are explained quantitatively by assuming that changes in an external surface potential set up by fixed charges near the sodium channel produce the shifts. The model involves a diffuse double layer of counterions at the nerve surface and some binding of H+ ions and divalent ions to the fixed charges. Three types of surface groups are postulated: (1) an acid p K a = 2.88, charge density —0.9 nm -2 ; (2) an acid p K a = 4.58, charge density —0.58 nm -2 ; (3) a base p K a = 6.28, charge density + 0.33 nm -2 . The two acid groups also bind Ca 2+ ions with a dissociation constant K = 28 M. Reasonable agreement can also be obtained with a lower net surface charge density and stronger binding of divalent ions and H + ions.

Frog sympathetic ganglion cells were studied under whole-cell voltage clamp to determine whether protein kinase C (PKC) mediates peptide-induced suppression of M current (IM) or desensitization of peptide receptors. Low concentrations (10 mM) of chicken II luteinizing hormone-releasing hormone (LHRH) or substance P (SP) suppressed IM; in addition, higher concentrations (1 microM) desensitized receptors. Desensitization is homologous (specific to the peptide) and lasts at least 25 min. Two stimulators of PKC, phorbol 12-myristate 13-acetate and dioctanoylglycerol, partially depressed IM and occluded the response to SP but not to LHRH. The two actions of PKC stimulators were blocked by PKC inhibitors (staurosporine, a pseudosubstrate peptide, and H-7), but SP- and LHRH-mediated suppression of IM and receptor desensitization were not affected. Thus, we conclude that PKC is not necessary for normal IM suppression or receptor desensitization.