Action of Ca2+ agonists/antagonists in mammalian peripheral neurons

The action of several ligands on the low- (LVA,T) and high-threshold (HVA,L and N) Ca channels of adult rat sensory neurons and human neuroblastoma IMR32 cells has been investigated. In both cell types, 40 microM Cd2+ and 6.4 microM /omega-Conotoxin (omega-CgTx) selectively blocked the HVA channels, sparing the majority of LVA channels that were antagonized by amiloride and Ni2+. In 50% of the cells, however, /omega-CgTx spared also a 15% of HVA channels that proved to be sensitive to BAY K 8644. The agonistic action of BAY K 8644 on [omega-CgTx-resistant HVA channels caused a large Ba current increase, prolonged current deactivation and acceleration of HVA channels inactivation that was particularly evident in adult rat DRG.     

3,4-Dihydroquinazoline derivatives as novel selective T-type Ca2+ channel blockers

For LVA T-type Ca2+ channel blockers, 3,4-dihydroquinazoline derivatives as new scaffolds were prepared and evaluated for the inhibitory activity against two members of the recombinant T-type Ca2+ channel family. Among them, 8a (KYS05001, IC50=0.9 microM) was nearly equipotent with mibefradil (IC50=0.84 microM) and inhibited LVA T-type Ca2+ channel with greater efficacy than HVA Ca2+ channel.     

L, P-/Q- and T-type Ca2+ channels in smooth muscle cells from human umbilical artery.

The electrophysiological and pharmacological properties of Ca(2+) current (I(Ca)) were determined by the whole-cell configuration of the patch-clamp technique in smooth muscle cells from human umbilical artery. Using 5 mM extracellular Ca(2+), depolarizing step pulses from -60 to 50 mV from a holding membrane potential of -80 mV evoked an I(Ca) which activated at membrane potentials more positive than -50 mV and exhibited a maximum current density in a range of 10-20 mV. Steady-state inactivation protocols using a V(test) of 10 mV gave a voltage at one-half inactivation and a slope factor of -35.6 mV and 9.5 mV, respectively. Nifedipine (1 microM), an L-type Ca(2+) channels antagonist, completely inhibited I(Ca), while the L-type Ca(2+) channels agonist Bay-K 8644 (1 microM) significantly increased I(Ca) amplitude. Moreover, the selective blocker of P-/Q-type Ca(2+) channels omega-agatoxin IVA partially blocked I(Ca) (about 40 % inhibition at +20 mV by 20 nM). These pharmacological results suggest that L- and P-/Q-type Ca(2+) channels, both nifedipine-sensitive, underlie the I(Ca) registered using low extracellular Ca(2+). The presence of the P-/Q-type Ca(2+) channels was confirmed by immunoblot analysis. When I(Ca) was recorded in a high concentration (30 mM) of extracellular Ca(2+) or Ba(2+) as current carrier, it was evident the presence of a nifedipine-insensitive component which completely inactivated during the course of the voltage-step (75 ms) at all potentials tested, and was blocked by the T-type Ca(2+) channels blocker mibefradil (10 microM). Summarizing, this work shows for the first time the electrophysiological and pharmacological properties of voltage-activated Ca(2+) currents in human umbilical artery smooth muscle cells.     

Differential expression of voltage-gated Ca(2+)-currents in cultivated aortic myocytes.

The expression of different types of Ca(2+)-channels was studied using the whole-cell patch-clamp technique in cultured rat aortic smooth-muscle myocytes. Ca(2+)-currents were identified as either low- or high voltage-activated (ICa,LVA or ICa,HVA, respectively) based on their distinct voltage-dependences of activation and inactivation, decay kinetics using Ba2+ as the charge carrier and sensitivity to dihydropyridines. The heterogeneity in the functional expression of the two types of Ca(2+)-channels in the cultured myocytes delineated four distinct phenotypes; (i), cells exhibiting only LVA currents; (ii), cells exhibiting only HVA currents; (iii), cells exhibiting both LVA and HVA currents and (iv), cells exhibiting no current. The myocytes exclusively expressed HVA currents both during the first five days in primary culture and after the cells had reached confluence (> 15 days). In contrast, LVA currents were expressed transiently between 5 and 15 days, during which time the cells were proliferating and had transient loss of contractility. Thus, both LVA and HVA Ca(2+)-current types contribute to Ca(2+)-signalling in cultured rat aortic myocytes. However, the differential expression of the two Ca2+ current types associated with differences in contractile and proliferative phenotypes suggest that they serve distinct cellular functions. Our results are consistent with the idea that LVA current expression is important for cell proliferation.     

Voltage-dependent calcium channels

Voltage-activated calcium channels can be divided into two subgroups based on their activation threshold, low-voltage-activated (LVA) and high-voltage-activated (HVA). Auxiliary subunits of the HVA calcium channels contribute significantly to biophysical properties of the channels. We have cloned and characterized members of two families of auxiliary subunits: alpha2delta and gamma. Two new alpha2delta subunits, alpha2delta-2 and alpha2delta-3, regulate all classes of HVA calcium channels. While the ubiquitous alpha2delta-2 modulates both neuronal and non-neuronal channels with similar efficiency, the alpha2delta-3 subunit regulates Ca(v)2.3 channels more effectively. Furthermore, alpha2delta-2 may modulate the LVA Ca(v)3.1 channel. Four new gamma subunits, gamma-2, gamma-3, gamma-4 and gamma-5, were characterized. The gamma-2 subunit modulated both the non-neuronal Ca(v)1.2 channel and the neuronal Ca(v)2.1 channel. The gamma-4 subunit affected only the Ca(v)2.1 channel. The gamma-5 subunit may be a regulatory subunit of the LVA Ca(v)3.1 channel. The Ca(v)1.2 channel is a major target for treatment of cardiovascular diseases. We have mapped the interaction site for clinically important channel blockers - dihydropyridines (DHPs) - and analysed the underlying inhibition mechanism. High-affinity inhibition is characterized by interaction with inactivated state of the channel. Its structural determinants are amino acids of the IVS6 segment, with smaller contribution of the IS6 segment, which contributes to voltage-dependence of DHP inhibition. Removal of amino acids responsible for the high-affinity inhibition revealed a low-affinity open channel block, in which amino acids of the IIIS5 and IIIS6 segments take part. Experiments with a permanently charged DHP suggested that there is another low-affinity interaction site on the alpha(1) subunit. We have cloned and characterized murine neuronal LVA Ca(v)3.1 channel. The channel has high sensitivity to the organic blocker mibefradil, moderate sensitivity to phenytoin, and low sensitivity to ethosuximide, amiloride and valproat. The channel is insensitive to tetrodotoxin and DHPs. The inorganic blockers Ni2+ and Cd2+ are moderately effective compared to La3+. The current through the Ca(v)3.1 channel inactivates faster with Ba2+ compared to Ca2+. Molecular determinants of fast inactivation are located in amino side of the intracellular carboxy terminus. The voltage dependence of charge movement is very shallow compared to the voltage dependence of current activation. Transfer of 30 % of charge correlates with activation of 70 % of measurable macroscopic current. Prolonged depolarization does not immobilize charge movement of the Ca(v)3.1 channel.     

Calcium channel blockers inhibit galvanotaxis in human keratinocytes

Directed migration of keratinocytes is essential for wound healing. The migration of human keratinocytes in vitro is strongly influenced by the presence of a physiological electric field and these cells migrate towards the negative pole of such a field (galvanotaxis). We have previously shown that the depletion of extracellular calcium blocks the directional migration of cultured human keratinocytes in an electric field (Fang et al., 1998; J Invest Dermatol 111:751-756). Here we further investigate the role of calcium influx on the directionality and migration speed of keratinocytes during electric field exposure with the use of Ca(2+) channel blockers. A constant, physiological electric field strength of 100 mV/mm was imposed on the cultured cells for 1 h. To determine the role of calcium influx during galvanotaxis we tested the effects of the voltage-dependent cation channel blockers, verapamil and amiloride, as well as the inorganic Ca(2+) channel blockers, Ni(2+) and Gd(3+) and the Ca(2+) substitute, Sr(2+), on the speed and directionality of keratinocyte migration during galvanotaxis. Neither amiloride (10 microM) nor verapamil (10 microM) had any effect on the galvanotaxis response. Therefore, calcium influx through amiloride-sensitive channels is not required for galvanotaxis, and membrane depolarization via K(+) channel activity is also not required. In contrast, Sr(2+) (5 mM), Ni(2+) (1-5 mM), and Gd(3+) (100 microM) all significantly inhibit the directional migratory response to some degree. While Sr(2+) strongly inhibits directed migration, the cells exhibit nearly normal migration speeds. These findings suggest that calcium influx through Ca(2+) channels is required for directed migration of keratinocytes during galvanotaxis and that directional migration and migration speed are probably controlled by separate mechanisms.     

Caffeine activates a mechanosensitive Ca(2+) channel in human red cells

Caffeine is known to activate influx of both mono- and divalent cations in various cell types, suggesting that this xanthine opens non-selective cation channels at the plasma membrane. This possibility was investigated in human erythrocytes, studying the caffeine action on net Ca(2+), Na(+) and K(+) movements in ATP-depleted cells. Whole populations and subpopulations of young and old erythrocytes were employed. Caffeine was tested in the presence of known mechanosensitive channel blockers (Gd(3+), neomycin and amiloride) and ruthenium red as a possible inhibitor. Caffeine enhanced net cation fluxes in a concentration-dependent way. In whole populations, the Ca(2+) entry elicited by 20 mM caffeine was fully suppressed by Gd(3+) (5 microM), amiloride (250 microM) and ruthenium red (100 microM) and partially blocked by neomycin (100 microM). The above blockers also inhibited caffeine-dependent Na(+) entry whilst showing antagonistic effects on the corresponding K(+) efflux. These compounds fully suppressed hypotonically-induced (-35 mOsm/kg) Ca(2+) influx at nearly the same concentrations completely blocking caffeine-stimulated Ca(2+) entry. The effect of inhibitors on Ca(2+) influx in young cells exceeded that in old cells at similar concentrations. The results clearly show that caffeine stimulates a stretch-activated Ca(2+) channel in human red cells and that aged cells are less susceptible to mechanosensitive channel blockers.     

Fast-deactivating calcium channels in chick sensory neurons

Whole-cell Ca and Ba currents were studied in chick dorsal root ganglion (DRG) cells kept 6-10 in culture. Voltage steps with a 15-microseconds rise time were imposed on the membrane using an improved patch-clamp circuit. Changes in membrane current could be measured 30 microseconds after the initiation of the test pulse. Currents through Ca channels were recorded under conditions that eliminate Na and K currents. Tail currents, associated with Ca channel closing, decayed in two distinct phases that were very well fitted by the sum of two exponentials. The time constants tau f and tau s were near 160 microseconds and 1.5 ms at -80 mV, 20 degrees C. The tail current components, called FD and SD (fast-deactivating and slowly deactivating), are Ca channel currents. They were greatly reduced when Mg2+ replaced all other divalent cations in the bath. The SD component inactivated almost completely as the test pulse duration was increased to 100 ms. It was suppressed when the cell was held at membrane potentials positive to -50 mV and was blocked by 100-200 microM Ni2+. This behavior indicates that the SD component was due to the closing of the low-voltage-activated (LVA) Ca channels previously described in this preparation. The FD component was fully activated with 10-ms test pulses to +20 mV at 20 degrees C, and inactivated to approximately 30% during 500-ms test pulses. It was reduced in amplitude by holding at -40 mV, but was only slightly reduced by micromolar concentrations of Ni2+. Replacement of Ca2+ with Ba2+ increased the FD tail current amplitudes by a factor of approximately 1.5. The deactivation kinetics did not change (a) as channels inactivated during progressively longer pulses or (b) when the degree of activation was varied. Further, tau f was affected neither by changing the holding potential nor by varying the test pulse amplitude. Lowering the temperature from 20 to 10 degrees C decreased tau f by a factor of 2.5. In all cases, the FD component was very well fitted by a single exponential. There was no indication of an additional tail component of significant size. Our findings indicate that the FD component is due to closing of a single class of Ca channels that coexist with the LVA Ca channel type in chick DRG neurons.     

Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G)

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(1G) Ca(2+) channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the alpha(1G) Ca(2+) channel subunit.     

T-type Ca(2+) channels mediate neurotransmitter release in retinal bipolar cells

Transmitter release in neurons is thought to be mediated exclusively by high-voltage-activated (HVA) Ca(2+) channels. However, we now report that, in retinal bipolar cells, low-voltage-activated (LVA) Ca(2+) channels also mediate neurotransmitter release. Bipolar cells are specialized neurons that release neurotransmitter in response to graded depolarizations. Here we show that these cells express T-type Ca(2+) channel subunits and functional LVA Ca(2+) currents sensitive to mibefradil. Activation of these currents results in Ca(2+) influx into presynaptic terminals and exocytosis, which we detected as a capacitance increase in isolated terminals and the appearance of reciprocal currents in retinal slices. The involvement of T-type Ca(2+) channels in bipolar cell transmitter release may contribute to retinal information processing.     

Forced-exercise delays neuropathic pain in experimental diabetes: effects on voltage-activated calcium channels

Physical exercise produces a variety of psychophysical effects, including altered pain perception. Elevated levels of centrally produced endorphins or endocannabinoids are implicated as mediators of exercise-induced analgesia. The effect of exercise on the development and persistence of disease-associated acute/chronic pain remains unclear. In this study, we quantified the physiological consequence of forced-exercise on the development of diabetes-associated neuropathic pain. Euglycemic control or streptozotocin (STZ)-induced diabetic adult male rats were subdivided into sedentary or forced-exercised (2-10 weeks, treadmill) subgroups and assessed for changes in tactile responsiveness. Two weeks following STZ-treatment, sedentary rats developed a marked and sustained hypersensitivity to von Frey tactile stimulation. By comparison, STZ-treated diabetic rats undergoing forced-exercise exhibited a 4-week delay in the onset of tactile hypersensitivity that was independent of glucose control. Exercise-facilitated analgesia in diabetic rats was reversed, in a dose-dependent manner, by naloxone. Small-diameter (< 30 μm) DRG neurons harvested from STZ-treated tactile hypersensitive diabetic rats exhibited an enhanced (2.5-fold) rightward (depolarizing) shift in peak high-voltage activated (HVA) Ca(2+) current density with a concomitant appearance of a low-voltage activated (LVA) Ca(2+) current component. LVA Ca(2+) currents present in DRG neurons from hypersensitive diabetic rats exhibited a marked depolarizing shift in steady-state inactivation. Forced-exercise attenuated diabetes-associated changes in HVA Ca(2+) current density while preventing the depolarizing shift in steady-state inactivation of LVA Ca(2+) currents. Forced-exercise markedly delays the onset of diabetes-associated neuropathic pain, in part, by attenuating associated changes in HVA and LVA Ca(2+) channel function within small-diameter DRG neurons possibly by altering opioidergic tone.     

Differential interactions of Na+ channel toxins with T-type Ca2+ channels

Two types of voltage-dependent Ca(2+) channels have been identified in heart: high (I(CaL)) and low (I(CaT)) voltage-activated Ca(2+) channels. In guinea pig ventricular myocytes, low voltage-activated inward current consists of I(CaT) and a tetrodotoxin (TTX)-sensitive I(Ca) component (I(Ca(TTX))). In this study, we reexamined the nature of low-threshold I(Ca) in dog atrium, as well as whether it is affected by Na(+) channel toxins. Ca(2+) currents were recorded using the whole-cell patch clamp technique. In the absence of external Na(+), a transient inward current activated near -50 mV, peaked at -30 mV, and reversed around +40 mV (HP = -90 mV). It was unaffected by 30 microM TTX or micromolar concentrations of external Na(+), but was inhibited by 50 microM Ni(2+) (by approximately 90%) or 5 microM mibefradil (by approximately 50%), consistent with the reported properties of I(CaT). Addition of 30 microM TTX in the presence of Ni(2+) increased the current approximately fourfold (41% of control), and shifted the dose-response curve of Ni(2+) block to the right (IC(50) from 7.6 to 30 microM). Saxitoxin (STX) at 1 microM abolished the current left in 50 microM Ni(2+). In the absence of Ni(2+), STX potently blocked I(CaT) (EC(50) = 185 nM) and modestly reduced I(CaL) (EC(50) = 1.6 microM). While TTX produced no direct effect on I(CaT) elicited by expression of hCa(V)3.1 and hCa(V)3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni(2+) (IC(50) increased to 550 microM Ni(2+) for Ca(V)3.1 and 15 microM Ni(2+) for Ca(V)3.2); in contrast, 30 microM TTX directly inhibited hCa(V)3.3-induced I(CaT) and the addition of 750 microM Ni(2+) to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni(2+) alone. 1 microM STX directly inhibited Ca(V)3.1-, Ca(V)3.2-, and Ca(V)3.3-mediated I(CaT) but did not enhance the ability of Ni(2+) to block these currents. These findings provide important new implications for our understanding of structure-function relationships of I(CaT) in heart, and further extend the hypothesis of a parallel evolution of Na(+) and Ca(2+) channels from an ancestor with common structural motifs.     

Calcium current subtypes in GnRH neurons

Calcium plays roles in excitability, rhythm generation, and neurosecretion. Identifying channel subtypes that regulate calcium influx is thus important to understanding rhythmic GnRH secretion, which is a prerequisite for reproduction. Whole-cell voltage-clamp recordings were made from short-term dissociated GnRH adult ovariectomized (OVX) mice (n = 21) to identify channel subtypes that carry calcium current using selective channel blockers and voltage characteristics. Low-voltage activated (LVA) currents were not observed in 42 GnRH neurons tested, although most non-GnRH neurons (4/6) displayed LVA current. The L-type component of the high-voltage activated (HVA) calcium current was 25% +/- 2%. The remaining HVA calcium current passed through N-type (27% +/- 3%), P-type (15% +/- 1%), Q-type (18% +/- 3%), and R-type (15% +/- 1%) channels. Because these data differ substantially from reports on cultured GnRH neurons, which may represent reproductively immature models, we also examined GnRH neurons from gonadal-intact young (Postnatal Days 4-10, n = 8 mice) mice. LVA currents were still rare (2/28) in young mice. Although the same HVA components were observed, the proportions were shifted toward significantly more L-type and less N-type current, suggesting a possible developmental shift in calcium currents in GnRH neurons. These data suggest that calcium channel subtypes in GnRH neurons prepared in the short term from brain slices differ substantially from those in long-term cultured GnRH models. These findings provide a vital foundation to examine the role of calcium channels in the secretory and rhythmic machinery of GnRH neurons.     

Molecular determinant for specific Ca/Ba selectivity profiles of low and high threshold Ca2+ channels

Voltage-gated Ca(2+) channels (VGCC) play a key role in many physiological functions by their high selectivity for Ca(2+) over other divalent and monovalent cations in physiological situations. Divalent/monovalent selection is shared by all VGCC and is satisfactorily explained by the existence, within the pore, of a set of four conserved glutamate/aspartate residues (EEEE locus) coordinating Ca(2+) ions. This locus however does not explain either the choice of Ca(2+) among other divalent cations or the specific conductances encountered in the different VGCC. Our systematic analysis of high- and low-threshold VGCC currents in the presence of Ca(2+) and Ba(2+) reveals highly specific selectivity profiles. Sequence analysis, molecular modeling, and mutational studies identify a set of nonconserved charged residues responsible for these profiles. In HVA (high voltage activated) channels, mutations of this set modify divalent cation selectivity and channel conductance without change in divalent/monovalent selection, activation, inactivation, and kinetics properties. The Ca(V)2.1 selectivity profile is transferred to Ca(V)2.3 when exchanging their residues at this location. Numerical simulations suggest modification in an external Ca(2+) binding site in the channel pore directly involved in the choice of Ca(2+), among other divalent physiological cations, as the main permeant cation for VGCC. In LVA (low voltage activated) channels, this locus (called DCS for divalent cation selectivity) also influences divalent cation selection, but our results suggest the existence of additional determinants to fully recapitulate all the differences encountered among LVA channels. These data therefore attribute to the DCS a unique role in the specific shaping of the Ca(2+) influx between the different HVA channels.     

Modulation of native T-type calcium channels by omega-3 fatty acids

Low voltage-activated, rapidly inactivating T-type Ca(2+) channels are found in a variety of cells where they regulate electrical activity and Ca(2+) entry. In whole-cell patch clamp recordings from bovine adrenal zona fasciculata cells, cis-polyunsaturated omega-3 fatty acids including docosahexaenoic acid (DHA), eicosapentaenoic acid, and alpha-linolenic acid inhibited T-type Ca(2+) current (I(T-Ca)) with IC(50)s of 2.4, 6.1, and 14.4microM, respectively. Inhibition of I(T-Ca) by DHA was partially use-dependent. In the absence of stimulation, DHA (5microM) inhibited I(T-Ca) by 59.7+/-8.1% (n=5). When voltage steps to -10mV were applied at 12s intervals, block increased to 80.5+/-7.2%. Inhibition of I(T-Ca) by DHA was accompanied by a shift of -11.7mV in the voltage dependence of steady-state inactivation, and a smaller -3.3mV shift in the voltage dependence of activation. omega-3 fatty acids also selectively altered the gating kinetics of T-type Ca(2+) channels. DHA accelerated T channel recovery from inactivation by approximately 3-fold, but did not affect the kinetics of T channel activation or deactivation. Arachidonic acid, an omega-6 polyunsaturated fatty acid, also inhibited T-type Ca(2+) current at micromolar concentrations, while the trans polyunsaturated fatty acid linolelaidic acid was ineffective. These results identify cis polyunsaturated fatty acids as relatively potent, new T-type Ca(2+) channel antagonists. omega-3 fatty acids are essential dietary components that have been shown to possess remarkable neuroprotective and cardioprotective properties that are likely mediated through suppression of electrical activity and associated Ca(2+) entry. Inhibition of T-type Ca(2+) channels in neurons and cardiac myocytes could contribute significantly to their protective actions.     

The dihydropyridine-sensitive calcium channel subtype in cone photoreceptors

High-voltage activated Ca channels in tiger salamander cone photoreceptors were studied with nystatin-permeabilized patch recordings in 3 mM Ca2+ and 10 mM Ba2+. The majority of Ca channel current was dihydropyridine sensitive, suggesting a preponderance of L- type Ca channels. However, voltage-dependent, incomplete block (maximum 60%) by nifedipine (0.1-100 microM) was evident in recordings of cones in tissue slice. In isolated cones, where the block was more potent, nifedipine (0.1-10 microM) or nisoldipine (0.5-5 microM) still failed to eliminate completely the Ca channel current. Nisoldipine was equally effective in blocking Ca channel current elicited in the presence of 10 mM Ba2+ (76% block) or 3 mM Ca2+ (88% block). 15% of the Ba2+ current was reversibly blocked by omega-conotoxin GVIA (1 microM). After enhancement with 1 microM Bay K 8644, omega-conotoxin GVIA blocked a greater proportion (22%) of Ba2+ current than in control. After achieving partial block of the Ba2+ current with nifedipine, concomitant application of omega-conotoxin GVIA produced no further block. The P-type Ca channel blocker, omega-agatoxin IVA (200 nM), had variable and insignificant effects. The current persisting in the presence of these blockers could be eliminated with Cd2+ (100 microM). These results indicate that photoreceptors express an L-type Ca channel having a distinguishing pharmacological profile similar to the alpha 1D Ca channel subtype. The presence of additional Ca channel subtypes, resistant to the widely used L-, N-, and P-type Ca channel blockers, cannot, however, be ruled out.     

Site-specific regulation of ion transport by prolactin in rat colon epithelium

The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 μg/ml) induced a sustained decrease in short-circuit current (I(sc)) in the distal colon with an EC(50) value of 100 ng/ml and increased I(sc) in the proximal colon with an EC(50) value of 49 ng/ml. In the distal colon, the PRL-induced decrease in I(sc) was not affected by Na(+) channel blocker amiloride or Cl(-) channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K(+) channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K(+) channel blockers, Ba(2+), tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRL-induced decrease in I(sc) was also inhibited by Na(+)-K(+)-2Cl(-) transporter inhibitor bumetanide, Ba(2+), and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in I(sc) was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in I(sc) in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca(2+) chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K(+) secretion in the distal colon and activation of Cl(-) secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca(2+) mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon.     

L-type calcium channels are preferentially coupled to endocytosis in bovine chromaffin cells

Exocytosis and endocytosis are Ca(2+)-dependent processes. The contribution of high-voltage activated Ca(2+) channels subtypes to exocytosis has been thoroughly studied in chromaffin cells. However, similar reports concerning endocytosis are unavailable. Thus, we studied here the effects of blockers of L (nifedipine), N (omega-conotoxin GVIA) and P/Q (omega-agatoxin IVA) Ca(2+) channel on Ca(2+) currents (I(Ca)), Ca(2+) entry (Q(Ca)), as well as on the changes in membrane capacitance (C(m)) in perforated-patch voltage-clamped bovine adrenal chromaffin cells. Using 500-ms pulses to 0 or +10 mV, given from a holding potential of -80 mV and 2 mM Ca(2+) we found that omega-conotoxin GVIA affected little the exo-endocytotic responses while omega-agatoxin IVA markedly blocked those responses. However, nifedipine blocked little exocytosis but almost completely inhibited endocytosis. We conclude that L-type Ca(2+) channels seem to be selectively coupled to endocytosis.     

Voltage- and calcium-activated currents in cultured olfactory receptor neurons of male Mamestra brassicae (Lepidoptera)

Insect olfactory receptor neurons (ORNs) grown in primary cultures were studied using the patch-clamp technique in both conventional and amphotericin B perforated whole-cell configurations under voltage-clamp conditions. After 10-24 days in vitro, ORNs had a mean resting potential of -62 mV and an average input resistance of 3.2 GOmega. Five different voltage-dependent ionic currents were isolated: one Na(+), one Ca(2+) and three K(+) currents. The Na(+) current (35-300 pA) activated between -50 and -30 mV and was sensitive to 1 microM tetrodotoxin (TTX). The sustained Ca(2+) current activated between -30 and -20 mV, reached a maximum amplitude at 0 mV (-4.5 +/- 6.0 pA) that increased when Ba(2+) was added to the bath and was blocked by 1 mM Co(2+). Total outward currents were composed of three K(+) currents: a Ca(2+)-activated K(+) current activated between -40 and -30 mV and reached a maximum amplitude at +40 mV (605 +/- 351 pA); a delayed-rectifier K(+) current activated between -30 and -10 mV, had a mean amplitude of 111 +/- 67 pA at +60 mV and was inhibited by 20 mM tetraethylammonium (TEA); and, finally, more than half of ORNs exhibited an A-like current strongly dependent on the holding potential and inhibited by 5 mM 4-aminopyridine (4-AP). Pheromone stimulation evoked inward current as measured by single channel recordings.     

Multi-ion occupancy alters gating in high-conductance, Ca(2+)-activated K+ channels

In this study, single-channel recordings of high-conductance Ca(2+)-activated K+ channels from rat skeletal muscle inserted into planar lipid bilayer were used to analyze the effects of two ionic blockers, Ba2+ and Na+, on the channel's gating reactions. The gating equilibrium of the Ba(2+)-blocked channel was investigated through the kinetics of the discrete blockade induced by Ba2+ ions. Gating properties of Na(+)-blocked channels could be directly characterized due to the very high rates of Na+ blocking/unblocking reactions. While in the presence of K+ (5 mM) in the external solution Ba2+ is known to stabilize the open state of the blocked channel (Miller, C., R. Latorre, and I. Reisin. 1987. J. Gen. Physiol. 90:427-449), we show that the divalent blocker stabilizes the closed-blocked state if permeant ions are removed from the external solution (K+ less than 10 microM). Ionic substitutions in the outer solution induce changes in the gating equilibrium of the Ba(2+)-blocked channel that are tightly correlated to the inhibition of Ba2+ dissociation by external monovalent cations. In permeant ion-free external solutions, blockade of the channel by internal Na+ induces a shift (around 15 mV) in the open probability--voltage curve toward more depolarized potentials, indicating that Na+ induces a stabilization of the closed-blocked state, as does Ba2+ under the same conditions. A kinetic analysis of the Na(+)-blocked channel indicates that the closed-blocked state is favored mainly by a decrease in opening rate. Addition of 1 mM external K+ completely inhibits the shift in the activation curve without affecting the Na(+)-induced reduction in the apparent single-channel amplitude. The results suggest that in the absence of external permeant ions internal blockers regulate the permeant ion occupancy of a site near the outer end of the channel. Occupancy of this site appears to modulate gating primarily by speeding the rate of channel opening.