The effects of prepulse-blink reflex trial repetition and prepulse change on blink reflex modification at short and long lead intervals

1. Thin slices of the posterior pituitary can be used as a preparation for the study of biophysical mechanisms underlying neuropeptide secretion. Patch-clamp techniques in this preparation have revealed the properties of ion channels that control the excitability of the nerve terminal membrane and have clarified the relation between Ca2+ and exocytosis. 2. Repetitive electrical activity at high frequencies broadens action potentials to allow more Ca2+ entry and thus enhance exocytosis. Action potential broadening results from the inactivation of a voltage-dependent K+ channel. 3. When repetitive electrical activity is sustained, secretion is depressed. This depression can be attributed in part to action potential failure caused by the opening of a Ca(2+)-activated K+ channel. This channel can be modulated by protein kinases, phosphatases, and G-proteins. 4. The inhibitory neurotransmitter GABA activates a GABAA receptor in the nerve terminal membrane. The gating of the associated Cl- channel depolarizes the membrane slightly to inactivate voltage-gated Na+ channels and block action potential propagation. 5. The response of the nerve terminal GABAA receptor is enhanced by neuroactive steroids and this can potentiate the inhibition of neurosecretion by GABA. The action of neurosteroids at this site could play a role in changes in neuropeptide secretion associated with reproductive transitions. 6. Ca2+ channels in the nerve terminal membrane are inactivated by sustained depolarization and by trains of brief pulses. Ca2+ entry promotes Ca2+ channel inactivation during trains by inhibiting the recovery of Ca2+ channels from inactivation. The inactivation of Ca2+ channels can play a role in defining the optimal frequency and train duration for evoking neuropeptide secretion. 7. Measurements of membrane capacitance in peptidergic nerve terminals have revealed rapid exocytosis and endocytosis evoked by Ca2+ entry through voltage-gated Ca2+ channels. Exocytosis is too rapid to account for the delays in neuropeptide secretion evoked by trains of action potentials. Endocytosis sets in rapidly after exocytosis with a time course comparable to that of the rapid endocytosis observed in nerve terminals at rapid synapses. Our results support the finding in rapid synaptic nerve terminals that endocytosis is inhibited by intracellular Ca2+. Multiple pools of vesicles were revealed, and these pools may reflect different stages in the mobilization and release of neuropeptide.     

Structure-activity relationships of astemizole derivatives for inhibition of store operated Ca2+ channels and exocytosis

The effects of a series of analogues of the antiallergic drug astemizole on the exocytosis of the enzyme beta-hexosaminidase were studied in a mast cell model, the rat basophilic leukemia (RBL-2H3) cell. Besides differences in the effects on Fc epsilonRI receptor-stimulated exocytosis, changes were also observed in Ca2+ influx and in the perturbation of the cell membrane. A strong correlation was found between the effects on antigen- and thapsigargin-stimulated 45Ca2+ influx. Furthermore, the inhibition of 45Ca2+ influx was correlated with the inhibition of beta-hexosaminidase release and membrane stabilization. It is concluded that the astemizole analogues are capable of inhibiting mast cell beta-hexosaminidase release through inhibition of Ca2+-store-operated Ca2+ channels (SOC). Compounds with high lipophilicity also released Ca2+ from intracellular stores. Lowering of the hydrophobicity by introduction of nitrogens or truncation at different sites in the astemizole structure decreased inhibitory activity on SOC channels. The inhibition of SOC channels cannot completely be ascribed to non-specific membrane effects. The piperidinyl-benzimidazole moiety was found to be important for inhibition of SOC channels. The observed differences in activity possibly depend on the way the compounds penetrate the membrane bilayer. Astemizole is an interesting new tool to study SOC channels and can be a lead for the design of mast cell-stabilizing antiallergic drugs.     

Cysteine string protein functions directly in regulated exocytosis

Cysteine string protein (Csp) is essential for neurotransmitter release in Drosophila. It has been suggested that Csp functions by regulating the activity of presynaptic Ca2+ channels, thus controlling exocytosis. We have examined the effect of overexpressing Csp1 in PC12 cells, a neuroendocrine cell line. PC12 cell clones overexpressing Csp1 did not show any changes in morphology, granule number or distribution, or in the levels of other key exocytotic proteins. This overexpression did not affect intracellular Ca2+ signals after depolarization, suggesting that Csp1 has no gross effect on Ca2+ channel activity in PC12 cells. In contrast, we show that Csp1 overexpression enhances the extent of exocytosis from permeabilized cells in response to Ca2+ or GTPgammaS in the absence of Ca2+. Because secretion from permeabilized cells is not influenced by Ca2+ channel activity, this represents the first demonstration that Csp has a direct role in regulated exocytosis.     

Control of Ca2+ channel current and exocytosis in rat lactotrophs by basally active protein kinase C and calcineurin

Modulation of voltage-activated Ca2+ channel activity by phosphorylation was studied in metabolically intact voltage-clamped rat lactotrophs. Experiments using Ba2+ as a charge carrier indicated that a phorbol ester protein kinase C activator stimulates high-voltage-activated Ca2+ channel currents, but has no effect on low-voltage-activated currents. Extracellular application of structurally and mechanistically distinct protein kinase C inhibitors (staurosporin, H7, calphostin C, chelerythrine and Ro 31-8220) preferentially inhibited the high-voltage-activated Ba2+ current. This suggests that protein kinase C is required for maintainance of Ca2+ channel activity even in the absence of modulators. Cyclosporin A, an inhibitor of the Ca2+/calmodulin-dependent protein phosphatase calcineurin, increased the high-voltage-activated Ca2+ channel current, and staurosporin reversed this effect. Thus, dephosphosphorylation by calcineurin may limit basal Ca2+ channel activity. Time-domain monitoring of cellular capacitance changes demonstrated that cyclosporin A and 12-O-tetradecanoyl-phorbol-13-acetate do not affect exocytosis at a hyperpolarized potential, but each enhances depolarization-induced exocytosis. Facilitation of exocytosis by cyclosporin A differed from 12-O-tetradecanoyl-phorbol-13-acetate in that it was biphasic. The delayed facilitation induced by cyclosporin A could be accounted for by stimulation of the voltage-gated Ca2+ current. These results suggest that the high-voltage activated Ca2+ channel current in rat lactotrophs is determined by the opposing basal activities of protein kinase C and calcineurin. Furthermore, it is concluded that the regulation of Ca2+ channels by protein kinase C and calcineurin affects depolarization-induced exocytosis.     

Modulation of neurotransmitter release by dihydropyridine-sensitive calcium channels involves tyrosine phosphorylation

Cultured rat cerebellar granule cells depolarized by high KCl, display a large component of Ca2+ influx through L-type voltage-dependent Ca2+ channels as defined by a sensitivity to 1 microM nifedipine. This Ca2+ influx is not coupled to neurotransmitter exocytosis but has implications for neuronal development. KCl stimulation in the absence of external Ca2+ followed by the readdition of Ca2+ allows the coupling of a class of L-type Ca2+ channels to neurotransmitter exocytosis as assessed by loading of glutamatergic pools with [3H]-D-aspartate. KCl stimulation in the absence of external Ca2+ ('predepolarization') enhances tyrosine phosphorylation of several cellular proteins, and inhibitors of tyrosine kinases block both phosphorylation and the neurotransmitter release coupled to the L-type Ca2+ channel. More specifically, an inhibitor of src family tyrosine kinases, PP1, blocks the effects of predepolarization suggesting a role for a src family kinase in the process. Furthermore, L-type Ca2+ channel recruitment and modulation of release could be activated with the tyrosine phosphatase inhibitor sodium orthovanadate. The phosphoproteins enhanced by predepolarization, which include the cytoskeletal proteins focal adhesion kinase (FAK) and vinculin, are also highly phosphorylated early on in culture when neurite outgrowth occurs. As the neurons develop a network of neurites, both tyrosine phosphorylation and L-type Ca2+ channel activity decrease. These results show a novel mechanism for the recruitment of L-type Ca2+ channels and their coupling to neurotransmitter release which involves tyrosine phosphorylation. This phenomenon has a role in cerebellar granule cell development.     

MgATP activates the beta cell KATP channel by interaction with its SUR1 subunit

ATP-sensitive potassium (KATP) channels in the pancreatic beta cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca2+ influx and exocytosis. Metabolic regulation of KATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The beta cell KATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate KATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg2+ caused an apparent reduction in the inhibitory action of ATP on wild-type KATP channels, and MgATP actually activated KATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates KATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg2+.     

Memory performance among women with parental abuse histories: enhanced directed forgetting or directed remembering?

Progesterone interaction with human spermatozoa promotes a rise in intracellular Ca2+ and can trigger acrosomal exocytosis in capacitated cells. We have used nifedipine, a 1,4-dihydropyridine Ca2+ channel antagonist, to investigate the possibility that Ca2+ channels play a role in the progesterone-stimulated exocytotic response. Cells were assessed biochemically for the generation of diacylglycerol (DAG) and microscopically for acrosome loss using chlortetracycline fluorescence. When motile cells were preincubated for 5 hr using culture conditions similar to those used for successful human in vitro fertilization, a short exposure to progesterone significantly stimulated DAG formation and acrosomal exocytosis. The addition of nifedipine (10 and 100 nM), either at time 0 or just prior to progesterone introduction, significantly inhibited both DAG formation and exocytosis, suggesting that Ca2+ channels are involved in the responses observed. Treatment of capacitated cells with a synthetic permeant DAG stimulated exocytosis irrespective of whether nifedipine was present, indicating that Ca2+ channels function prior to DAG generation. The possibility that an influx of Na+, as well as Ca2+, might be involved in the exocytotic pathway was investigated using the monovalent cation ionophores monensin and nigericin. Both significantly stimulated DAG generation and acrosome loss, but the prior inclusion of nifedipine significantly inhibited all responses. These results strongly suggest that the entry of Ca2+ through Ca2+ channels, with characteristics similar to those of L-type, voltage-sensitive Ca2+ channels found in cardiac and skeletal muscle, is a crucial step in the sequence of events leading to exocytosis in progesterone-stimulated human spermatozoa. An influx of Na+ also may play a role, but at a point prior to the opening of Ca2+ channels.     

Signal transduction and ion channels in guard cells

Our understanding of the signalling mechanisms involved in the process of stomatal closure is reviewed. Work has concentrated on the mechanisms by which abscisic acid (ABA) induces changes in specific ion channels at both the plasmalemma and the tonoplast, leading to efflux of both K+ and anions at both membranes, requiring four essential changes. For each we need to identify the specific channels concerned, and the detailed signalling chains by which each is linked through signalling intermediates to ABA. There are two global changes that are identified following ABA treatment: an increase in cytoplasmic pH and an increase in cytoplasmic Ca2+, although stomata can close without any measurable global increase in cytoplasmic Ca2+. There is also evidence for the importance of several protein phosphatases and protein kinases in the regulation of channel activity. At the plasmalemma, loss of K+ requires depolarization of the membrane potential into the range at which the outward K+ channel is open. ABA-induced activation of a non-specific cation channel, permeable to Ca2+, may contribute to the necessary depolarization, together with ABA-induced activation of S-type anion channels in the plasmalemma, which are then responsible for the necessary anion efflux. The anion channels are activated by Ca2+ and by phosphorylation, but the precise mechanism of their activation by ABA is not yet clear. ABA also up-regulates the outward K+ current at any given membrane potential; this activation is Ca(2+)-independent and is attributed to the increase in cytoplasmic pH, perhaps through the marked pH-sensitivity of protein phosphatase type 2C. Our understanding of mechanisms at the tonoplast is much less complete. A total of two channels, both Ca(2+)-activated, have been identified which are capable of K+ efflux; these are the voltage-independent VK channel specific to K+, and the slow vacuolar (SV) channel which opens only at non-physiological tonoplast potentials (cytoplasm positive). The SV channel is permeable to K+ and Ca2+, and although it has been argued that it could be responsible for Ca(2+)-induced Ca2+ release, it now seems likely that it opens only under conditions where Ca2+ will flow from cytoplasm to vacuole. Although tracer measurements show unequivocally that ABA does activate efflux of Cl- from vacuole to cytoplasm, no vacuolar anion channel has yet been identified. There is clear evidence that ABA activates release of Ca2+ from internal stores, but the source and trigger for ABA-induced increase in cytoplasmic Ca2+ are uncertain. The tonoplast and another membrane, probably ER, have IP3-sensitive Ca2+ release channels, and the tonoplast has also cADPR-activated Ca2+ channels. Their relative contributions to ABA-induced release of Ca2+ from internal stores remain to be established. There is some evidence for activation of phospholipase C by ABA, by an unknown mechanism; plant phospholipase C may be activated by Ca2+ rather than by the G-proteins used in many animal cell signalling systems. A further ABA-induced channel modulation is the inhibition of the inward K+ channel, which is not essential for closing but will prevent opening. It is suggested that this is mediated through the Ca(2+)-activated protein phosphatase, calcineurin. The question of Ca(2+)-independent stomatal closure remains controversial. At the plasmalemma the stimulation of K+ efflux is Ca(2+)-independent and, at least in Arabidopsis, activation of anion efflux by ABA may also be Ca(2+)-independent. But there are no indications of Ca(2+)-independent mechanisms for K+ efflux at the tonoplast, and the appropriate anion channel at the tonoplast is still to be found. There is also evidence that ABA interferes with a control system in the guard cell, resetting its set-point to lower contents, suggesting that stretch-activated channels also feature in the regulation of guard cell ion channels, perhaps through interactions with cytoskeletal proteins. (ABSTRACT TRUN     

Differential regulation of N- and Q-type Ca2+ channels by cyclic nucleotides and G-proteins

Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway delimited to the membrane. In addition, modulation of Ca2+ channel activity by protein kinases also contributes to the dynamic regulation of neuronal physiology. Recently, differences in these modulations between Ca2+ channel subtypes have been shown in several neuronal preparations. Here we show that two types of presynaptic Ca2+ channel (N-type and Q-type) are differentially regulated by cAMP and G-proteins using a Xenopus oocyte expression system. Treatment to increase cytosolic cAMP concentration with forskolin and 3-isobutyl-1-methylxanthine (IBMX) markedly potentiated Q-type channel current, and the enhancement was reversed by protein kinase A inhibitors. Much smaller enhancement was observed in N-type channel current after the cAMP elevation. When large depolarizing prepulse was applied to the oocytes for evaluation of the tonic inhibition of Ca2+ channels by intrinsic G-protein activity, N-type channel current elicited a large prepulse facilitation but Q-type channels did not. The tonic inhibition of N-type channels was abolished by an intracellular perfusion with a 'cut-open' recording configuration, or by co-expression with G(alpha o). When kappa opioid receptors were co-expressed and stimulated with agonists, depolarization-resistant inhibition was more apparent in Q-type channels than in N-type channels. These results suggest that Q-type channels are more susceptible to the protein kinase A-mediated facilitation than N-type channels, and that activity of N-type channels can be more highly regulated in a voltage-dependent manner by G(betagamma) than that of Q-type channels. These differences may account for the selective regulation of neurotransmitter release by these Ca2+ channels.     

The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery

Although N- and P-type Ca2+ channels predominant in fast-secreting systems, Lc-type Ca2+ channels (C-class) can play a similar role in certain secretory cells and synapses. For example, in retinal bipolar cells, Ca2+ entry through the Lc channels triggers ultrafast exocytosis, and in pancreatic beta-cells, evoked secretion is highly sensitive to Ca2+. These findings suggest that a rapidly release pool of vesicles colocalizes with the Ca2+ channels to allow high Ca2+ concentration and a tight coupling of the Lc channels at the release site. In binding studies, we show that the Lc channel is physically associated with synaptotagmin (p65) and the soluble N-ethylmaleimide-sensitive attachment proteins receptors: syntaxin and synaptosomal-associated protein of 25 kDa. Soluble N-ethylmaleimide-sensitive attachent proteins receptors coexpressed in Xenopus oocytes along with the Lc channel modify the kinetic properties of the channel. The modulatory action of syntaxin can be overcome by coexpressing p65, where at a certain ratio of p65/syntaxin, the channel regains its unaltered kinetic parameters. The cytosolic region of the channel, Lc753-893, separating repeats II-III of its alpha1C subunit, interacts with p65 and "pulls" down native p65 from rat brain membranes. Lc753-893 injected into single insulin-secreting beta-cell, inhibits secretion in response to channel opening, but not in response to photolysis of caged Ca2+, nor does it affect Ca2+ current. These results suggest that Lc753-893 competes with the endogenous channel for the synaptic proteins and disrupts the spatial coupling with the secretory apparatus. The molecular organization of the Lc channel and the secretory machinery into a multiprotein complex (named excitosome) appears to be essential for an effective depolarization evoked exocytosis.     

Cardiovascular T-type calcium channels: physiological and pharmacological significance

CELLULAR CALCIUM REGULATION: A variety of Ca2+ control processes are responsible for Ca2+ homeostasis and signaling. Voltage-gated Ca2+ channels are dominant in the cardiovascular system. VOLTAGE-GATED Ca2+ CHANNELS: There are several distinct subclasses of Ca2+ channels, distinguished by location, biophysical, structural and pharmacological characteristics. They include both high- and low-voltage-activated channels. The long-lasting (L) type of high-voltage-activated channel is well characterized and is the site of action for the existing clinically available Ca2+ channel antagonists: nifedipine, verapamil and diltiazem. T-TYPE Ca2+ CHANNELS: The low-voltage-activated transient (T-type) channel is widespread in the cardiovascular system and in neurons. It serves pacemaking functions and supports Ca2+ signaling in secretory cells and vascular smooth muscle. The T-type channel also functions in cell growth processes under physiological and pathological conditions. MIBEFRADIL AS A T-TYPE Ca2+ CHANNEL ANTAGONIST: Mibefradil (Ro 40-5967) is a structurally novel Ca2+ antagonist with selectivity for T-type over L-type channels. This selectivity may underlie its vasodilating activity and heart rate depressive effect, its lack of negative inotropy and its cardioprotective properties.     

Ca2+ permeation in cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels conduct Na+, K+ and Ca2+ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca2+ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non-excitable cells, co-assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca2+ signaling depends on its specific Ca2+ conductance, it is necessary to analyze Ca2+ permeation for each individual channel type. We have analyzed Ca2+ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca2+ current over the physiological range of Ca2+ concentrations and found that Ca2+ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca2+ permeation is controlled by the Ca2+-binding affinity of the intrapore cation-binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca2+ signals.     

Alterations in basal and glucose-stimulated voltage-dependent Ca2+ channel activities in pancreatic beta cells of non-insulin-dependent diabetes mellitus GK rats

In genetically occurring non-insulin-dependent diabetes mellitus (NIDDM) model rats (GK rats), the activities of L- and T-type Ca2+ channels in pancreatic beta cells are found to be augmented, by measuring the Ba2+ currents via these channels using whole-cell patch-clamp technique, while the patterns of the current-voltage curves are indistinguishable. The hyper-responsiveness of insulin secretion to nonglucose depolarizing stimuli observed in NIDDM beta cells could be the result, therefore, of increased voltage-dependent Ca2+ channel activity. Perforated patch-clamp recordings reveal that the augmentation of L-type Ca2+ channel activity by glucose is markedly less pronounced in GK beta cells than in control beta cells, while glucose-induced augmentation of T-type Ca2+ channel activity is observed neither in the control nor in the GK beta cells. This lack of glucose-induced augmentation of L-type Ca2+ channel activity in GK beta cells might be causatively related to the selective impairment of glucose-induced insulin secretion in NIDDM beta cells, in conjunction with an insufficient plasma membrane depolarization due to impaired closure of the ATP-sensitive K+ channels caused by the disturbed intracellular glucose metabolism in NIDDM beta cells.     

Localization of Ca2+ channel subtypes on rat spinal motor neurons, interneurons, and nerve terminals

Ca2+ channels in distinct subcellular compartments of neurons mediate voltage-dependent Ca2+ influx, which integrates synaptic responses, regulates gene expression, and initiates synaptic transmission. Antibodies that specifically recognize the alpha1 subunits of class A, B, C, D, and E Ca2+ channels have been used to investigate the localization of these voltage-gated ion channels on spinal motor neurons, interneurons, and nerve terminals of the adult rat. Class A P/Q-type Ca2+ channels were present mainly in a punctate pattern in nerve terminals located along the cell bodies and dendrites of motor neurons. Both smooth and punctate staining patterns were observed over the surface of the cell bodies and dendrites with antibodies to class B N-type Ca2+ channels, indicating the presence of these channels in the cell surface membrane and in nerve terminals. Class C and D L-type and class E R-type Ca2+ channels were distributed mainly over the cell soma and proximal dendrites. Class A P/Q-type Ca2+ channels were present predominantly in the presynaptic terminals of motor neurons at the neuromuscular junction. Occasional nerve terminals innervating skeletal muscles from the hindlimb were labeled with antibodies against class B N-type Ca2+ channels. Staining of the dorsal laminae of the rat spinal cord revealed a complementary distribution of class A and class B Ca2+ channels in nerve terminals in the deeper versus the superficial laminae. Many of the nerve terminals immunoreactive for class B N-type Ca2+ channels also contained substance P, an important neuropeptide in pain pathways, suggesting that N-type Ca2+ channels are predominant at synapses that carry nociceptive information into the spinal cord.     

Potassium channels in epithelial cells

The broad array of K- channels in epithelial cells includes voltage-dependent (mainly outward) and Ca(2+)-activated channels, and K+ channels modulated by adenosine triphosphate (ATP). Voltage-dependent K+ channels mediate Na+/K+ absorption and secretion; typically, they are found in the basolateral membrane and exhibit burst activity. Ca(2+)-activated K+ (Ca2+/K+) channels regulate activity by decreasing Ca2+ influx via voltage-gated Ca2+ channels. Ca2+/K+ channels exhibit conductances of 4-300 pS, and have a low open probability (< 10(-7)) at the level of the resting membrane potential. ATP-sensitive channels have been observed mainly in insulin-secreting pancreatic beta-cells and in the urinary tract, where the open state is rapidly closed by ATP. The channels are voltage-dependent, exhibit burst activity, and, in the case of the urinary-tract cells, are Ca2+ dependent. Chemical compounds that selectively open or block K+ channels have been exploited to characterize channels found in different cells, but no opener or blocker has been found that specifically affects only one type of K+ channel. Specialized model systems and recombinant techniques have led to a general understanding of the structure of K+ channels, but many important details remain to be determined.     

Clinical study on five myeloperoxidase specific anti-neutrophil cytoplasmic antibody (MPO-ANCA) positive Churg-Strauss syndrome cases

Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, Vh=-80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell IBa could be fractionated into various subcomponents. Thus, IBa had a 25% fraction sensitive to 1 microM nifedipine (L-type channels), 21% sensitive to 1 microM omega-conotoxin GVIA (N-type channels), and 60% sensitive to 2 microM omega-agatoxin IVA (P/Q-type channels). The activation of IBa was considerably slowed down, and the peak current was inhibited upon superfusion with 10 microM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of IBa was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5-50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates.     

Voltage-dependent Ca2+ channels in arterial smooth muscle cells

The past years have seen some significant advances in our understanding of the functional and molecular properties of voltage-dependent Ca2+ channels in arterial smooth muscle. Molecular cloning and expression studies together with experiments on native voltage-dependent Ca2+ channels revealed that these channels are built upon a molecular structure with properties appropriate to function as the main source for Ca2+ entry into arterial smooth muscle cells. This Ca2+ entry regulates intracellular free Ca2+, and thereby arterial tone. We summarize several avenues of recent research that should provide significant insights into the functioning of voltage-dependent Ca2+ channels under conditions that occur in arterial smooth muscle. These experiments have identified important features of voltage-dependent Ca2+ channels, including the steep steady-state voltage-dependence of the channel open probability at steady physiological membrane potentials between -60 and -30 mV, and a relatively high permeation rate at physiological Ca2+ concentrations, being about one million Ca2+ ions/s at -50 mV. This calcium permeation rate seems to be a feature of the pore-forming Ca2+ channel alpha1 subunit, since it was identical for native channels and the expressed alpha1 subunit alone. The channel activity is regulated by dihydropyridines, vasoactive hormones and intracellular signaling pathways. While the membrane potential of smooth muscle cells primarily regulates arterial muscle tone through alterations in Ca2+ influx through dihydropyridine-sensitive voltage-dependent ('L-type') Ca2+ channels, the role of these channels in the differentiation and proliferation of vascular smooth muscle cells is less clear. We discuss recent findings suggesting that other Ca2+ permeable ion channels might be important for the control of Ca2+ influx in dedifferentiated vascular smooth muscle cells.     

Effects of excitatory neurotransmitters on Ca2+ channel current in smooth muscle cells isolated from guinea-pig urinary bladder

1. A whole-cell voltage clamp technique was used to examine the effects of purinoceptor and muscarinic receptor agonists on voltage-sensitive Ca2+ channels in guinea-pig isolated urinary bladder cells. 2. When the cell membrane was clamped at the holding potential, rapid application of ATP elicited a large inward current in normal solution containing 2.5 mM Ca2+, and reduced the subsequent Ca2+ channel current evoked by a depolarizing pulse (0 mV). Carbachol (CCh) elicited little membrane current, but similarly reduced the Ca2+ current. 3. When purinoceptor agonists were rapidly applied during conditioning depolarizations at +80 mV, an outward current was elicited, and the Ca2+ channel current evoked by the subsequent test potential of 0 mV was not affected. Application of CCh at +80 mV also elicited an outward current, but it reduced the subsequently evoked Ca2+ current. 4. The inhibitory effect of muscarinic agonists on the Ca2+ channel current was attenuated by caffeine (10 mM). 5. In Ca(2+)-free, low-Mg2+ solution, a Na+ current flowing through voltage-dependent Ca2+ channels was evoked by depolarization. This current was not reduced by bath application of purinoceptor agonists (ATP and alpha,beta-methylene ATP). 6. These results suggest that the main effect of purinoceptor stimulation is opening of non-selective cation channels, and that muscarinic stimulation triggers Ca2+ release from intracellular stores. Voltage-sensitive Ca2+ channels are inactivated through an increase in intracellular Ca2+ induced by either activation of purinoceptor or muscarinic receptors.     

Separation of gating properties from permeation and block in mslo large conductance Ca-activated K+ channels

In this and the following paper we have examined the kinetic and steady-state properties of macroscopic mslo Ca-activated K+ currents in order to interpret these currents in terms of the gating behavior of the mslo channel. To do so, however, it was necessary to first find conditions by which we could separate the effects that changes in Ca2+ concentration or membrane voltage have on channel permeation from the effects these stimuli have on channel gating. In this study we investigate three phenomena which are unrelated to gating but are manifest in macroscopic current records: a saturation of single channel current at high voltage, a rapid voltage-dependent Ca2+ block, and a slow voltage-dependent Ba2+ block. Where possible methods are described by which these phenomena can be separated from the effects that changes in Ca2+ concentration and membrane voltage have on channel gating. Where this is not possible, some assessment of the impact these effects have on gating parameters determined from macroscopic current measurements is provided. We have also found that without considering the effects of Ca2+ and voltage on channel permeation and block, macroscopic current measurements suggest that mslo channels do not reach the same maximum open probability at all Ca2+ concentrations. Taking into account permeation and blocking effects, however, we find that this is not the case. The maximum open probability of the mslo channel is the same or very similar over a Ca2+ concentration range spanning three orders of magnitude indicating that over this range the internal Ca2+ concentration does not limit the ability of the channel to be activated by voltage.     

Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation

A T-type Ca2+ channel is expressed during differentiation of the male germ lineage in the mouse and is retained in sperm, where is it activated by contact with the the egg's extracellular matrix and controls sperm acrosomal exocytosis. Here, we examine the regulation of this Ca2+ channel in dissociated spermatogenic cells from the mouse using the whole-cell patch-clamp technique. T currents were enhanced, or facilitated, after strong depolarizations or high frequency stimulation. Voltage-dependent facilitation increased the Ca2+ current by an average of 50%. The same facilitation is produced by antagonists of protein tyrosine kinase activity. Conversely, antagonists of tyrosine phosphatase activity block voltage-dependent facilitation of the current. These data are consistent with the presence of a two-state model, in which T channels are maintained in a low (or zero) conductance state by tonic tyrosine phosphorylation and can be activated to a high conductance state by a tyrosine phosphatase activity. The positive and negative modulation of this channel by the tyrosine phosphorylation state provides a plausible mechanism for the control of sperm activity during the early stages of mammalian fertilization.