Differential effects of temperature on E. coli and synthetic polyhydroxybutyrate/polyphosphate channels

Complexes of poly-(R)-3-hydroxybutyrate and inorganic polyphosphate (PHB/polyP), isolated from the plasma membranes of Escherichia coli or prepared synthetically (HB(128)/polyP(65)), form Ca(2+)-selective ion channels in planar lipid bilayers that exhibit indistinguishable gating and conductance characteristics at 22 degrees C. Here we examine the gating and conductance of E. coli and synthetic PHB/polyP complexes in planar lipid bilayers as a function of temperature from 15 to 45 degrees C. E. coli PHB/polyP channels remained effectively open throughout this range, with brief closures that became more rare at higher temperatures. Conversely, as temperatures were gradually increased, the open probability of HB(128)/polyP(65) channels progressively decreased. The effect was fully reversible. Channel conductance exhibited three distinct phases. Below 25 degrees C, as PHB approached its glass temperature (ca. 10 degrees C), the conductance of both E. coli and synthetic channels remained at about the same level (95-105 pS). Between 25 degrees C and ca. 40 degrees C, the conductance of E. coli and synthetic channels increased gradually with temperature coefficients (Q(10)) of 1.45 and 1.42, respectively. Above 40 degrees C, E. coli channel conductance increased sharply, whereas the conductance of HB(128)/polyP(65) channels leveled off. The discontinuities in the temperature curves for E. coli channels coincide with discontinuities in thermotropic fluorescence spectra and specific growth rates of E. coli cells. It is postulated that E. coli PHB/polyP complexes are associated with membrane components that inhibit their closure at elevated temperatures.     

Transmembrane ion transport by polyphosphate/poly-(R)-3-hydroxybutyrate complexes

Transmembrane ion transport, a critical process in providing energy for cell functions, is carried out by pore-forming macromolecules capable of discriminating among very similar ions and responding to changes in membrane potential. It is widely regarded that ion channels are exclusively proteins, relatively late arrivals in cell evolution. Here we discuss the formation of ion-selective, voltage-activated channels by complexes of two simple homopolymers, namely, inorganic polyphosphates (polyPs) and poly-(R)-3-hydroxybutyrates (PHBs), derived from phosphate and acetate, respectively. Each has unique molecular characteristics that facilitate ion selection, solvation, and transport. Complexes of the two polymers, isolated from bacterial plasma membranes or prepared from the synthetic polymers, form voltage-dependent, Ca2+-selective channels in planar lipid bilayers that are selective for divalent over monovalent cations, permeant to Ca2+, Sr2+, and Ba2+, and blocked by transition metal cations in a concentration-dependent manner. Recently, both polyP and PHB have been found to be components of ion-conducting proteins: namely, the human erythrocyte Ca2+-ATPase pump and the Streptomyces lividans potassium channel. The contribution of polyP and PHB to ion selection and/or transport in these proteins is yet unknown, but their presence gives rise to the hypothesis that these and other ion transporters are supramolecular structures in which proteins, polyP, and PHB cooperate in forming well-regulated and specific cation transfer systems.     

A high-conductance mode of a poly-3-hydroxybutyrate/calcium/polyphosphate channel isolated from competent Escherichia coli cells

Reconstitution into planar lipid bilayers of a poly-3-hydroxybutyrate/calcium/polyphosphate (PHB/Ca(2+)/polyP) complex from Escherichia coli membranes yields cationic-selective, 100 pS channels (Das, S., Lengweiler, U.D., Seebach, D. and Reusch, R.N. (1997) Proof for a non-proteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate. Proc. Natl. Acad. Sci. USA 94, 9075-9079). Here, we report that this complex can also form larger, weakly selective pores, with a maximal conductance ranging from 250pS to 1nS in different experiments (symmetric 150mM KCl). Single channels were inhibited by lanthanum (IC(50)=42+/-4microM, means+/-S.E.M.) with an unusually high Hill coefficient (8.4+/-1.2). Transition to low-conductance states (<250pS) was favored by increased membrane polarization (/V/ >or=50mV). High conductance states (>250pS) may reflect conformations important for genetic transformability, or "competence", of the bacterial cells, which requires the presence of the PHB/Ca(2+)/polyP complex in the membrane.     

Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations.

In earlier studies of genetic competence in Escherichia coli induced with calcium-containing buffers, a strong correlation was found between transformation efficiency and the formation of poly-beta-hydroxybutyrate/calcium polyphosphate (PHB/Ca2+/PPi) complexes in the plasma membranes. In this study, we replaced Ca2+ with one of a number of other cations--monovalent, divalent, and trivalent--and found significant numbers of transformants (transformation efficiency, > 10(5)/micrograms of pBR322 DNA) only when the cells had high levels of PHB/Ca2+/PPi and the medium contained at least one of the divalent cations Ca2+, Mn2+, Sr2+, or Mg2+. Cells with high levels of the complexes were not competent when the medium did not contain these cations, but the cations were also ineffectual when the cells had few complexes. Surprisingly, Mn, Sr, and Mg were not incorporated into the complexes in place of Ca. These results indicate that PHB/Ca2+/PPi complexes and the above-mentioned divalent cations each have essential but disparate roles in genetic competence. Moreover, the strong selectivity of PHB/PPi for Ca2+ suggests the binding sites in the complexes are ionophoretic.     

pH regulates cation selectivity of poly-(R)-3-hydroxybutyrate/polyphosphate channels from E. coli in planar lipid bilayers

Poly-(R)-3-hydroxybutyrate/polyphosphate (PHB/polyP) complexes, whether isolated from the plasma membranes of bacteria or prepared from the synthetic polymers, form ion channels in planar lipid bilayers that are highly selective for Ca(2+) over Na(+) at physiological pH. This preference for divalent over monovalent cations is attributed to a high density of negative charge along the polyP backbone and the higher binding energies of divalent cations. Here we modify the charge density of polyP by varying the pH, and observe the effect on cation selectivity. PHB/polyP complexes, isolated from E. coli, were incorporated into planar lipid bilayers, and unitary current-voltage relations were determined as a function of pH. When Ca(2+) was the sole permeant cation, conductance diminished steadily from 97 +/- 6 pS at pH 7.4 to 47 +/- 3 pS at pH 5.5. However, in asymmetric solutions of Ca(2+) and Na(+), there was a moderate increase in conductance from 98 +/- 4 at pH 7.4 to 129 +/- 4 pS at pH 6.5, and a substantially larger increase to 178 +/- 6 pS at pH 5.6, signifying an increase in Na(+) permeability or disorganization of channel structure. Reversal potentials point to a sharp decrease in preference for Ca(2+) over Na(+) over a relatively small decrease in pH. Ca(2+) was strongly favored over Na(+) at physiological pH, but the channels became nonselective near the pK(2) of phosphate (approximately 6.8), and displayed weak selectivity for Na(+) over Ca(2+) at acidic pH. Evidently, PHB/polyP complexes are versatile ion carriers whose selectivity may be modulated by small adjustments of the local pH. The results may be relevant to the physiological function of PHB/polyP channels in bacteria and the role of PHB and polyP in the Streptomyces lividans potassium channel.     

Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA

Here we present functional evidence for involvement of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP) in ion conduction and selection at the intracellular side of the Streptomyces lividans potassium channel, KcsA. At < or = 25 degrees C, KcsA forms channels in planar bilayers that display signal characteristics of PHB/polyP channels at the intracellular side; i.e., a preference for divalent Mg(2+) cations at pH 7.2, and a preference for monovalent K+ cations at pH 6.8. Between 25 and 26 degrees C, KcsA undergoes a transition to a new conformation in which the channel exhibits high selectivity for K+, regardless of solution pH. This suggests that basic residues of the C-terminal polypeptides have moved closer to the polyP end unit, reducing its negative charge. The data support a supramolecular structure for KcsA in which influx of ions is prevented by the selectivity pore, whereas efflux of K+ is governed by a conductive core of PHB/polyP in partnership with the C-terminal polypeptide strands.     

Slow changes in cytosolic free Ca2+ in Escherichia coli highlight two putative influx mechanisms in response to changes in extracellular calcium.

Free intracellular Ca2+ ([Ca2+]i) in Escherichia coli was measured using the bioluminescent protein aequorin. Overall, the bacteria maintained a tight control on their free [Ca2+]i. The results indicated a slow Ca2+ influx, the magnitude of the initial rise in free [Ca2+]i being dependent upon the concentrations of external Ca2+. This was followed by the slow removal of free Ca2+ until normal levels were restored. Specifically, addition of external Ca2+ (0.25-10 mM) resulted in a gradual rise in intracellular free Ca2+ from a basal level of approximately 272 nM, maximally reaching a peak of 0.85-5.4 microM within 30-40 min. This was followed by a slow fall over the next 30 min, culminating in an oscillatory pattern of free [Ca2+]i (range 0.3-0.7 microM for 0.25 mM external Ca2+). In the presence of EGTA, free [Ca2+]i was dramatically reduced. Neither the influx of Ca2+ nor restoration of intracellular free Ca2+ required protein synthesis. Moreover, preincubation with Ca2+ increased the rising phase of intracellular Ca2+ in response to further exposure to external Ca2+. This was further evidence against a specific adaptation process such as the synthesis of calcium exporters. A putative Ca2+ influx channel was demonstrated in stationary phase cells in particular, which could be blocked by La3+. This channel was consistent with the voltage-activated poly-3-hydroxybutyrate/polyphosphate Ca2+ channels previously detailed by Reusch et al. [23] Even in the presence of La3+, however, the free [Ca2+]i of log phase and stationary phase bacteria still increased two-fold over resting values in response to external Ca2+. This suggested the presence of at least two Ca2+ influx processes, one inhibited by La3+ and the other not.     

Gating Kinetics of E. coli Poly-3-Hydroxybutyrate/Polyphosphate Channels in Planar Bilayer Membranes

Nonproteinaceous calcium channel complexes from Escherichia coli, composed of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP), exhibit two distinct gating modes (modes 1 and 2) in planar lipid bilayers. Here we report the kinetic characterization of the channel in mode 2, a mode characterized by two well-defined conductance levels, a fully open state (87 ± 3 pS), and a major subconductance state (56 ± 2 pS). Other subconductance states and full closures are rare (<0.5% of total time). Several kinetic properties of the channel showed asymmetric voltage-dependence indicating an asymmetry in the channel structure. Accordingly, single channels responded to potential change in one of two mirror-image patterns, postulated to arise from opposite orientations of the asymmetrical channel complex in the bilayer. The fraction of time spent in each conductance level was strongly voltage-sensitive. For channels reported in this study, presumably all oriented in the same direction, residence time in the fully open state increased as clamping potentials became more positive whereas residence time in the major subconductance state increased at more negative potentials. Analysis of open time distributions revealed existence of two kinetically distinct states for each level. The shorter time constants for both conductance states exhibited weak voltage-sensitivity; however, the longer time constants were strongly voltage-sensitive. A kinetic scheme, consistent with the complex voltage dependence of the channel, is proposed. Received: 1 February 1999/Revised: 2 April 1999     

Electrophysiological evidence that glycoprotein IIb-IIIa complex is involved in calcium channel activation on human platelet plasma membrane.

The involvement of platelet glycoprotein (GP) IIb-IIIa complex in calcium channel activity on the plasma membrane was investigated using an electrophysiological approach. Plasma membrane vesicles were prepared from thrombin-stimulated platelets and incorporated into planar lipid bilayers. Voltage-independent Ca2+ channel currents with a conductance of about 10 pS (in 53 mM Ba2+) were observed, in membranes derived from thrombin-stimulated, but not unstimulated platelet membranes. These channel activities were markedly reduced by exposure of membranes to EGTA at 37 degrees C. This reduction was specifically related to the dissociation of the GPIIb-IIIa complex since preincubation of the membranes with a monoclonal antibody to the GPIIb-IIIa complex (AP-2) could protect the channel activities from the effect of EGTA. Thrombasthenic platelets, which lack the GPIIb-IIIa complex, showed impaired channel activities characterized by decreased open probability and lowered conductance states. Furthermore, when platelets were stimulated by thrombin in the presence of EGTA, AP2, or the synthetic peptide RGDS, to prevent fibrinogen binding to the GPIIb-IIIa complex, open probabilities of the channel currents in these membrane vesicles were also decreased. These results suggest that the GPIIb-IIIa complex is involved in platelet Ca2+ channel activation and that ligand binding to the complex during platelet activation may modify the activation of Ca2+ channels.     

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.     

Single-channel calcium and barium currents of large and small conductance from sarcoplasmic reticulum.

Two types of divalent cation conducting channels from rabbit skeletal muscle sarcoplasmic reticulum (SR) were incorporated into planar lipid bilayers. A high conductance (100 pS in 53 mM trans Ca2+) Ca2+ channel was incorporated from heavy density SR fractions. The 100-pS channel was activated by adenine nucleotides and Ca2+ and inhibited by Mg2+ and ruthenium red. A 10-pS calcium and barium conducting channel could be incorporated into planar lipid bilayers from light, intermediate, and heavy density SR vesicles. 10-pS channel activity in bilayers was not dependent on cis Ca2+ and was only weakly dependent on adenine nucleotides. Ruthenium red at concentrations up to 1 mM had no effect and Mg2+ was only marginally effective in inhibiting macroscopic Ba2+ currents from this channel. Calcium releasing activity in intermediate and heavy density SR fractions was assayed according to a rapid quench protocol and compared with the results obtained in the bilayer. Results from this comparison indicate that the 10-pS channel is probably not involved in rapid Ca2+- and adenine nucleotide-induced Ca2+ release from isolated SR vesicles.     

Escherichia coli lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous,PHB-polyphosphate Ca2+ channels in the membrane

PHB(polyP) complexes bind calcium and form calcium channels in the cytoplasmic membrane in Escherichia coli and are likely to be important in Ca(2+) homeostasis in this organism. E. coli N43, which lacks the AcrA component of a major multidrug resistance pump, was shown to be defective in calcium handling, with an inability to maintain submicromolar levels of free Ca(2+) in the cytoplasm. Therefore, using an N-phenyl-1-napthylamine (NPN)-dependent fluorescence assay, we measured temperature-dependent phase transitions in the membranes of intact cells. These transitions specifically depend on the presence of PHB(Ca(2+)polyP) complexes. PHB(Ca(2+)polyP) channel complexes, particularly in stationary phase cultures, were detected in wild-type strains; however, in contrast, isogenic acrA(-) strains had greatly reduced amounts of the complexes. This indicates that the AcrAB transporter may have a novel, hitherto undetected physiological role, either directly in the membrane assembly of the PHB complexes or the transport of a component of the membrane, which is essential for assembly of the complexes into the membrane. In other experiments, we showed that the particular defective calcium handling detected in N43 was not due to the absence of AcrA but to other unknown factors in this strain.     

Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+ channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+ currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+ channels in rat arterial VSM and characterize endogenous LVA Ca2+ currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+ oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+ channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+ spiking can be abolished by mibefradil at low concentrations (<1 microM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+ currents but did not alter the AVP-stimulated Ca2+ spiking response. Together these data suggest that T-type Ca2+ channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+ entry through these channels is not limiting for repetitive Ca2+ spiking observed in A7r5 cells.     

Characterization and localization of two ion-binding sites within the pore of cardiac L-type calcium channels

L-type Ca channels from porcine cardiac sarcolemma were incorporated into planar lipid bilayers. We characterized interactions of permeant and blocking ions with the channel's pore by (a) studying the current-voltage relationships for Ca2+ and Na+ when equal concentrations of the ions were present in both internal and external solutions, (b) testing the dose-dependent block of Ba2+ currents through the channels by internally applied cadmium, and (c) examining the dose and voltage dependence of the block of Na+ currents through the channels by internally and externally applied Ca2+. We found that the I-V relationship for Na+ appears symmetrical through the origin when equal concentrations of Na+ are present on both sides of the channel (gamma = 90 pS in 200 mM NaCl). The conductance for outward Ca2+ currents with 100 mM Ca2+ on both sides of the channel is approximately 8 pS, a value identical to that observed for inward currents when 100 mM Ca2+ was present outside only. This provides evidence that ions pass through the channel equally well regardless of the direction of net flux. In addition, we find that internal Cd2+ is as effective as external Cd2+ in blocking Ba2+ currents through the channels, again suggesting identical interactions of ions with each end of the pore. Finally, we find that micromolar Ca2+, either in the internal or in the external solution, blocks Na+ currents through the channels. The affinity for internally applied Ca2+ appears the same as that for externally applied Ca2+. The voltage dependence of the Ca(2+)-block suggests that the sites to which Ca2+ binds are located approximately 15% and approximately 85% of the electric field into the pore. Taken together, these data provide direct experimental evidence for the existence of at least two ion binding sites with high affinity for Ca2+, and support the idea that the sites are symmetrically located within the electric field across L-type Ca channels.     

Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells

Ion channels, activated upon IgE-Fc epsilon receptor aggregation by specific antigen, were studied in micropipet-supported lipid bilayers. These bilayers were reconstituted with purified IgE-Fc epsilon receptor complex and the intact 110-kDa channel-forming protein, both isolated from plasma membranes of rat basophilic leukemia cells (line RBL-2H3). In order to identify the current carrier through these ion channels and to determine their ion selectivity, we investigated the currents flowing through the IgE-Fc epsilon receptor gated channels in the presence of a gradient of Ca2+ ions. Thus, the solution in which the micropipet-supported bilayer was immersed contained 1.8 mM CaCl2, while the interior of the micropipet contained 0.1 microM Ca2+ (buffered with EGTA). Both solutions also contained 150 mM of a monovalent cation chloride salt (either K+ or Na+). The currents induced upon specific aggregation of the IgE (by either antigen or anti-IgE antibodies) were examined over a range of potentials imposed on the bilayer. The type of conductance event most frequently observed under the employed experimental conditions was a channel that has a slope conductance of 3 pS and a reversal potential practically identical with the calculated value for the reversal potential of calcium (134 +/- 11 mV in the presence of sodium, 125 +/- 13 mV in the presence of potassium). These results indicate that this channel is highly selective for calcium against the monovalent cations sodium and potassium. This same channel has a conductance of 4-5 pS in the presence of symmetrical solutions containing only 100 mM CaCl2 and 8 pS in the presence of 0.5 M NaCl with no calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abnormal ryanodine receptor channels in malignant hyperthermia.

Previous studies have demonstrated a defect associated with the calcium release mechanism of sarcoplasmic reticulum (SR) from individuals susceptible to malignant hyperthermia (MH). To examine whether SR calcium release channels were indeed altered in MH, SR vesicles were purified from normal and MH susceptible (MHS) porcine muscle. The Ca2+ dependence of calcium efflux rates from 45Ca2(+)-filled SR vesicles was then compared with the Ca2+ dependence of single-channel recordings of SR vesicles incorporated into planar lipid bilayers. The rate constants of 45Ca2+ efflux from MHS SR were two to threefold larger than from normal SR over a wide range of myoplasmic Ca2+. Normal and MHS single channels were progressively activated in a similar fashion by cis Ca2+ from pCa 7 to 4. However, below pCa 4, normal channels were inactivated by cis Ca2+, whereas MHS channels remained open for significantly longer times. The altered Ca2+ dependence of channel inactivation in MHS SR was also evident when Ca2+ was increased on the trans side while cis Ca2+ was held constant. We propose that a defect in a low-affinity Ca2+ binding site is responsible for the altered gating of MHS SR channels. Such a defect could logically result from a mutation in the gene encoding the calcium release channel, providing a testable hypothesis for the molecular basis of this inherited disorder.     

Inorganic Polyphosphate Modulates TRPM8 Channels

Polyphosphate (polyP) is an inorganic polymer built of tens to hundreds of phosphates, linked by high-energy phosphoanhydride bonds. PolyP forms complexes and modulates activities of many proteins including ion channels. Here we investigated the role of polyP in the function of the transient receptor potential melastatin 8 (TRPM8) channel. Using whole-cell patch-clamp and fluorescent calcium measurements we demonstrate that enzymatic breakdown of polyP by exopolyphosphatase (scPPX1) inhibits channel activity in human embryonic kidney and F-11 neuronal cells expressing TRPM8. We demonstrate that the TRPM8 channel protein is associated with polyP. Furthermore, addition of scPPX1 altered the voltage-dependence and blocked the activity of the purified TRPM8 channels reconstituted into planar lipid bilayers, where the activity of the channel was initiated by cold and menthol in the presence of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P₂). The biochemical analysis of the TRPM8 protein also uncovered the presence of poly-(R)-3-hydroxybutyrate (PHB), which is frequently associated with polyP. We conclude that the TRPM8 protein forms a stable complex with polyP and its presence is essential for normal channel activity.     

Effects of mebudipine and dibudipine, two new calcium channel blockers on voltage-activated calcium currents of PC12 cells

Mebudipine and dibudipine are two newly synthesized dihydropyridine (DHP) calcium channel blockers that have been shown to have considerable relaxant effects on vascular and atrial smooth muscle. The in vitro half-lives of mebudipine and dibudipine are reported to be significantly longer than that of nifedipine. In this study, we investigated the effects of mebudipine and dibudipine on voltage-activated Ca2+ channels on differentiated PC12 cells and compared their potencies to amlodipine. Our results point to absence of voltage-activated Ca2+ currents in undifferentiated PC12 cells. It is also concluded that mebudipine and dibudipine, like amlodipine are L-type calcium channel blockers. When tested in a range of 10-100 microM, mebudipine is at least as potent as amlodipine in inhibition of peak Ba2+ currents in differentiated PC12 cells while dibudipine is significantly less potent compared to amlodipine and mebudipine.     

Differential expression of voltage-gated calcium channels in identified visual cortical neurons.

Using the whole-cell patch-clamp technique, Ca2+ channel currents were examined in three distinct types of neurons derived from rat primary visual cortex. Callosal-projecting and superior colliculus-projecting neurons were identified following in vivo retrograde labeling with fluorescent "beads." A subset of intrinsic GABAergic visual cortical neurons was identified with the monoclonal antibody VC1.1. Although high voltage-activated Ca2+ channel currents were measured in all three cell types, clear differences in the densities of these channels were observed. There were also marked variations in the relative amplitudes of the inactivating and noninactivating components of the high voltage-activated currents, suggesting that N- and L-type Ca2+ channels are differentially distributed. Although low voltage-activated or T-type currents were measured in subsets of both types of projection neurons, they were not observed in VC1.1-positive cells. These results provide a direct demonstration that voltage-gated Ca2+ channels are expressed in neurons of the mammalian visual cortex and reveal that the distribution and densities of different Ca2+ channel types in diverse classes of visual cortical neurons are distinct.     

Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.