Blockade of current through single calcium channels by trivalent lanthanide cations. Effect of ionic radius on the rates of ion entry and exit

Currents flowing through single dihydropyridine-sensitive Ca2+ channels were recorded from cell-attached patches on C2 myotubes. In the presence of dihydropyridine agonist to prolong the duration of single-channel openings, adding micromolar concentrations of lanthanum (La), cerium (Ce), neodymium (Nd), gadolinium (Gd), dysprosium (Dy), or ytterbium (Yb) to patch electrodes containing 110 mM BaCl2 caused the unitary Ba2+ currents to fluctuate between fully open and shut states. The kinetics of channel blockade followed the predictions of a simple open channel block model in which the fluctuations of the single-channel current arose from the entry and exit of blocking ions from the pore. Entry rates for all the lanthanides tested were relatively insensitive to membrane potential, however, exit rates depended strongly on membrane potential increasing approximately e-fold per 23 mV with hyperpolarization. Individual lanthanide ions differed in both the absolute rates of ion entry and exit: entry rates decreased as cationic radius decreased; exit rates also decreased with cationic radius during the first part of the lanthanide series but then showed little change during the latter part of the series. Overall, the results support the idea that smaller ions enter the channel more slowly, presumably because they dehydrate more slowly; smaller ions also bind more tightly to a site within the channel pore, but lanthanide residence time within the channel approaches a maximum for the smaller cations with radii less than or equal to that of Ca2+.     

Raman spectroscopy of DNA-metal complexes. II. The thermal denaturation of DNA in the presence of Sr2+, Ba2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+.

Differential scanning calorimetry, laser Raman spectroscopy, optical densitometry, and pH potentiometry have been used to investigate DNA melting profiles in the presence of the chloride salts of Ba2+, Sr2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+. Metal-DNA interactions have been observed for the molar ratio [M2+]/[PO2-] = 0.6 in aqueous solutions containing 5% by weight of 160 bp mononucleosomal calf thymus DNA. All of the alkaline earth metals, plus Mn2+, elevate the melting temperature of DNA (Tm > 75.5 degrees C), whereas the transition metals Co2+, Ni2+, and Cd2+ lower Tm. Calorimetric (delta Hcal) and van't Hoff (delta HVH) enthalpies of melting range from 6.2-8.7 kcal/mol bp and 75.6-188.6 kcal/mol cooperative unit, respectively, and entropies from 17.5 to 24.7 cal/K mol bp. The average number of base pairs in a cooperative melting unit (<nmelt>) varied from 11.3 to 28.1. No dichotomy was observed between alkaline earth and transition DNA-metal complexes for any of the thermodynamic parameters other than their effects on Tm. These results complement Raman difference spectra, which reveal decreases in backbone order, base unstacking, distortion of glycosyl torsion angles, and rupture of hydrogen bonds, which occur after thermal denaturation. Raman difference spectroscopy shows that transition metals interact with the N7 atom of guanine in duplex DNA. A broader range of interaction sites with single-stranded DNA includes ionic phosphates, the N1 and N7 atoms of purines, and the N3 atom of pyrimidines. For alkaline earth metals, very little interaction was observed with duplex DNA, whereas spectra of single-stranded complexes are very similar to those of melted DNA without metal. However, difference spectra reveal some metal-specific perturbations at 1092 cm-1 (nPO2-), 1258 cm-1 (dC, dA), and 1668 cm-1 (nC==O, dNH2 dT, dG, dC). Increased spectral intensity could also be observed near 1335 cm-1 (dA, dG) for CaDNA. Optical densitometry, employed to detect DNA aggregation, reveals increased turbidity during the melting transition for all divalent DNA-metal complexes, except SrDNA and BaDNA. Turbidity was not observed for DNA in the absence of metal. A correlation was made between DNA melting, aggregation, and the ratio of Raman intensities I1335/I1374. At room temperature, DNA-metal interactions result in a pH drop of 1.2-2.2 units for alkaline earths and more than 2.5 units for transition metals. Sr2+, Ba2+, and Mg2+ cause protonated sites on the DNA to become thermally labile. These results lead to a model that describes DNA aggregation and denaturation during heating in the presence of divalent metal cations; 1) The cations initially interact with the DNA at phosphate and/or base sites, resulting in proton displacement. 2) A combination of metal-base interactions and heating disrupts the base pairing within the DNA duplex. This allows divalent metals and protons to bind to additional sites on the DNA bases during the aggregation/melting process. 3) Strands whose bases have swung open upon disruption are linked to neighboring strands by metal ion bridges. 4) Near the midpoint of the melting transition, thermal energy breaks up the aggregate. We have no evidence to indicate whether metal ion cross-bridges or direct base-base interactions rupture first. 5) Finally, all cross-links break, resulting in single-stranded DNA complexed with metal ions.     

Permeation and gating in CaV3.1 (alpha1G) T-type calcium channels effects of Ca2+, Ba2+, Mg2+, and Na+

We examined the concentration dependence of currents through Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current-voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by P(Ca)/P(Na) = 87 and P(Ca)/P(Ba) = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent K(d) values were similar for Ca(2+) and Ba(2+), both for block of currents carried by Na(+) (3 muM for Ca(2+) vs. 4 muM for Ba(2+), at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca(2+) vs. 2.5 mM for Ba(2+); nearly voltage independent). Block by 3-10 muM Ca(2+) was time dependent, described by bimolecular kinetics with binding at approximately 3 x 10(8) M(-1)s(-1) and voltage-dependent exit. Ca(2+)(o), Ba(2+)(o), and Mg(2+)(o) also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e(-) per 98 A(2) from Gouy-Chapman theory. Additionally, inward currents inactivated approximately 35% faster in Ba(2+)(o) (vs. Ca(2+)(o) or Na(+)(o)). The accelerated inactivation in Ba(2+)(o) correlated with the transition from Na(+) to Ba(2+) permeation, suggesting that Ba(2+)(o) speeds inactivation by occupying the pore. We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca(2+) or Ba(2+).     

Voltage control of Ca2+ permeation through N-type calcium (CaV2.2) channels

Voltage-gated calcium (Ca_V) channels deliver Ca²⁺ to trigger cellular functions ranging from cardiac muscle contraction to neurotransmitter release. The mechanism by which these channels select for Ca²⁺ over other cations is thought to involve multiple Ca²⁺-binding sites within the pore. Although the Ca²⁺ affinity and cation preference of these sites have been extensively investigated, the effect of voltage on these sites has not received the same attention. We used a neuronal preparation enriched for N-type calcium (Ca_V2.2) channels to investigate the effect of voltage on Ca²⁺ flux. We found that the EC₅₀ for Ca²⁺ permeation increases from 13 mM at 0 mV to 240 mM at 60 mV, indicating that, during permeation, Ca²⁺ ions sense the electric field. These data were nicely reproduced using a three-binding-site step model. Using roscovitine to slow Ca_V2.2 channel deactivation, we extended these measurements to voltages <0 mV. Permeation was minimally affected at these hyperpolarized voltages, as was predicted by the model. As an independent test of voltage effects on permeation, we examined the Ca²⁺-Ba²⁺ anomalous mole fraction (MF) effect, which was both concentration and voltage dependent. However, the Ca²⁺-Ba²⁺ anomalous MF data could not be reproduced unless we added a fourth site to our model. Thus, Ca²⁺ permeation through Ca_V2.2 channels may require at least four Ca²⁺-binding sites. Finally, our results suggest that the high affinity of Ca²⁺ for the channel helps to enhance Ca²⁺ influx at depolarized voltages relative to other ions (e.g., Ba²⁺ or Na⁺), whereas the absence of voltage effects at negative potentials prevents Ca²⁺ from becoming a channel blocker. Both effects are needed to maximize Ca²⁺ influx over the voltages spanned by action potentials.     

Folding pathway of apo-metallothionein induced by Zn2+, Cd2+ and Co2+.

Metal ion binding to the sulfhydryl groups of apometallothionein (apo-MT) causes both the formation of native metal-thiolate clusters and the folding of the polypeptide chain of each domain. Cd2+ and Zn2+ react with apo-MT to form metal-thiolate bonds in reactions that are complete within milliseconds and which are pH-dependent. Dual mixing experiments were conducted that involve the initial reaction of metal ion and apo-MT followed by mixing with 5,5'-N-dithio-bis(2-nitrobenzoate) or EDTA after 26 ms. They showed that structures had formed within the brief reaction period which were resistant to rapid reaction with reagents that interact with sulfhydryl groups or metal ions, respectively. It was concluded that native metallothionein domains had been constituted within this brief period. Apo-MT was also titrated with Co2+ to yield Co(n)-MT (n=1-7). Initially, Co2+ bound to independent, tetrahedral thiolate sites. Spectrophotometric analysis of the titration suggested that the independent Co(II) sites began to coalesce into clusters at n=4 (pH 7.2) or n=5 (pH 8.4). Back titration of free sulfhydryl groups (S) in Co(n)-MT (n=1-7) with iodoacetamide at pH 7.2 confirmed that clustering began at n=4. Upon conversion of these alkylated structures to the corresponding 113Cd2+ species 113Cd NMR spectroscopy established that the location of Co(II) in Co(n)-MT (n=1-3) was non-specific and that at n=4, the only observable structure was Co(II)4S11. The results suggest possible kinetic pathways of folding that are conceptually similar to those hypothesized for other small proteins.     

Spatial and temporal Ca2+, Mg2+, and ATP2- dynamics in cardiac dyads during calcium release

We have constructed a three-dimensional reaction-diffusion model of the mammalian cardiac calcium release unit. We analyzed effects of diffusion coefficients, single channel current amplitude, density of RyR channels, and reaction kinetics of ATP(2-) with Ca(2+) and Mg(2+) ions on spatiotemporal concentration profiles of Ca(2+), Mg(2+), and ATP(2-) in the dyadic cleft during Ca(2+) release. The model revealed that Ca(2+) concentration gradients persist near RyRs in the steady state. Even with low number of open RyRs, peak [Ca(2+)] in the dyadic space reached values similar to estimates of luminal [Ca(2+)] in approximately 1 ms, suggesting that during calcium release the Ca(2+) gradient moves from the cisternal membrane towards the boundary of the dyadic space with the cytosol. The released Ca(2+) bound to ATP(2-), and thus substantially decreased ATP(2-) concentration in the dyadic space. The released Ca(2+) could also replace Mg(2+) in its complex with ATP(2-) during first milliseconds of release if dissociation of MgATP was fast. The results suggest that concentration changes of Ca(2+), Mg(2+), and ATP(2-) might be large and fast enough to reduce dyadic RyR activity. Thus, under physiological conditions, termination of calcium release may be facilitated by the synergic effect of the construction and chemistry of mammalian cardiac dyads.     

Interconversion of androgen receptor forms by divalent cations and 8 S androgen receptor-promoting factor. Effects of Zn2+, Cd2+, Ca2+, and Mg2+

The effects of divalent cations (Zn2+, Cd2+, Ca2+, Mg2+) on the cytosol androgen receptor were determined by sedimentation into sucrose gradients. At low ionic strength (25 mM KCl, 50 mM Tris, pH 7.4), Zn2+ (200 microM total, which calculates to 130 nM free Zn2+ in 10 mM mercaptoethanol) causes a shift in the sedimentation coefficient of the rat Dunning prostate tumor (R3327H) cytosol receptor and rat ventral prostate cytosol receptor from 7.5 +/- 0.3 S to 8.6 +/- 0.3 S. Zn2+ stabilizes the 8.6 S receptor form in salt concentrations up to 0.15 M KCl in 50 mM Tris, pH 7.2. In low ionic strength gradients containing Ca2+ (greater than or equal to 200 microM) or Mg2+ (greater than or equal to 1 mM), the receptor sediments as 4.7 +/- 0.3 S. The dissociating effects of Ca2+ and Mg2+ can be fully reversed by sedimentation into gradients containing Zn2+ (200 microM total) or Cd2+ (10 microM total). In the presence of Zn2+ (200 microM total), Ca2+ (10 microM to 3 mM) converts the receptor to an intermediate form with sedimentation coefficient 6.2 +/- 0.2 S, Stokes radius 73 A, and apparent Mr approximately 203,000. The potentiating effect of Zn2+ on formation of the 8.6 S receptor (in the absence of Ca2+) and the 6.2 S receptor (in the presence of Ca2+) requires both the 4.5 S receptor and the 8 S androgen receptor-promoting factor. Sodium molybdate stabilizes the untransformed cytosol receptor but, unlike Zn2+, does not promote reconstitution of the 8.6 S receptor from its partially purified components. These results indicate that divalent cations alter the molecular size of the androgen receptor in vitro and thus may have a role in altering the state of transformation of the receptor.     

Block of current through single calcium channels by Fe, Co, and Ni. Location of the transition metal binding site in the pore

The blocking actions of Fe2+, Co2+, and Ni2+ on unitary currents carried by Ba2+ through single dihydropyridine-sensitive Ca2+ channels were recorded from cell-attached patches on myotubes from the mouse C2 cell line. Adding millimolar concentrations of blocker to patch electrodes containing 110 mM BaCl2 produced discrete excursions to the closed channel level. The kinetics of blocking and unblocking were well described with a simple model of open channel block. Hyperpolarization speeded the exit of all of the blockers from the channel, as expected if the blocking site resides within the pore. The block by Ni2+ differs from that produced by Fe2+ and Co2+ because Ni2+ enters the channel approximately 20 times more slowly and exits approximately 50 times more slowly. Ni2+ also differs from the other transition metals because at millimolar concentrations it reduces the amplitude of the unitary current in a concentration-dependent manner. The results are consistent with the idea that the rate-limiting step for ion entry into the channel is water loss at its inner coordination sphere; unblocking, on the other hand, cannot be explained in terms of simple coulombic interactions arising from differences in ion size.     

Interactions of protons with single open L-type calcium channels. pH dependence of proton-induced current fluctuations with Cs+, K+, and Na+ as permeant ions

We studied the pH dependence of the proton-induced current fluctuations that appear in single open L-type Ca channels when monovalent ions are the charge carriers. We used different methods of analysis to obtain kinetic measurements even under conditions where the individual transitions were too fast to be resolved directly as discrete current steps between two conductance levels. The reciprocal of the dwell times at the high conductance level increased linearly with the pipette proton activity, with a slope that was similar for Cs, K, and Na as permeant ions. Contrary to the expectation for a simple model in which the high and low conductances represent the unprotonated and protonated states of the channel, respectively, the dwell times at the low conductance level were also pH dependent and lengthened with increasing proton activity. At all pH values the dwell times at the low conductance level were longest with Cs as permeant ion and shortened in the order Cs greater than K greater than Na. We introduce a more general model of the protonation cycle in which the channel is represented by four states and can be protonated and deprotonated both at the high and low conductance levels. The conductance change is represented by a conformational change of the channel protein. We discuss the validity of this model and its implications for the mechanism by which protons interact with ion permeation through L-type Ca channels.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle: effects of Sr2+ and Cd2+ on Ca2+ uptake and formation of the phosphorylated intermediate of the Ca2+,Mg2+-ATPase

The effects of various divalent cations on the Ca2+ uptake by microsomes from bovine aortic smooth muscle were studied. High concentrations (1 mM) of Co2+, Zn2+, Mn2+, Fe2+, and Ni2+ inhibited neither the Ca2+ uptake by the microsomes nor the formation of the phosphorylated intermediate (E approximately P) of the Ca2+,Mg2+-ATPase of the microsomes. The cadmium ion, however, inhibited both the Ca2+ uptake and the E approximately P formation by the microsomes. Dixon plot analysis indicated Cd2+ inhibited (Ki = 135 microM) the Ca2+ dependent E approximately P formation in a non-competitive manner. The inhibitory effect of Cd2+ was lessened by cysteine or dithiothreitol. The strontium ion inhibited the Ca2+ uptake competitively, while the E approximately P formation increased on the addition of Sr2+ at low Ca2+ concentrations. At a low Ca2+ concentration (1 microM), Sr2+ was taken up by the aortic microsomes in the presence of 1 mM ATP. It is thus suggested that Sr2+ replaces Ca2+ at the Ca2+ binding site on the ATPase.     

Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides

A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of heavy sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a Ca2+ release channel which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.     

Monovalent ion current through single calcium channels of skeletal muscle transverse tubules.

Akali monovalents, Li, Na, K, Cs, and organic monovalents of molecular cross section less than 20 A2, ammonium, methylammonium, hydrazinium, guanidinium, are shown to have a measurable conductance through Ca channels of muscle transverse tubules reconstituted into planar bilayers. For the alkali series, single channel conductances follow the sequence Cs approximately equal to K greater than Na greater than Li with a conductance ratio [g(Cs)/g(Li)] = 1.7. For permeability ratios, the sequence is Li greater than Na greater than K approximately equal to Cs with [P(Li)/P(Cs)] = 1.5. Monovalent current is only unmasked when Ba ions are not present. In mixtures of Cs and Ba, single channel current reverses close to the Ba equilibrium potential and more than 100 mV away from the Cs equilibrium potential. A cutoff in conduction is found for organic cations larger than trimethylammonium; this suggests an apparent pore aperture of about 5 X 5 A. Even in such a large pore, the fact that the alkali cation permeability sequence and conductance sequence are inverted rules out molecular sieving as the mechanism of selection among monovalents.     

The activation of adrenal cortex mitochondrial malic enzyme by Ca2+ and Mg2+.

Adrenal cortex mitochondria prepared by a standard method do not exhibit malic enzyme activity. Addition of physiological concentrations of Ca2+ and Mg2+ enables these mitochondria to reduce added NADP+ by malate to form free NADPH. Half-maximum activation of the mitochondrial malic enzyme requires 0.3 mM Ca2+ and 1 mM Mg2+. Solubilized mitochondrial malic enzymes is independent of Ca2+ and has a K M of 0.2 mM for Mg2+. The Ca2+ effect is dependent on an initial period of active Ca2+ uptake which also causes other changes in respiratory properties similar to those observed with mitochondria from other tissues. After Ca2+ accumulation has taken place, free Ca2+, but not additional accumulation, is still required for malic enzyme activity. The requirement for Mg2+ can be met by Mn2+ (1 mM). This concentration of Mn2+ alone yielded only a slight activation of mitochondrial malic enzyme while higher concentrations of Mn2+ alone gave good activation of the mitochondrial malic enzy.e The NADPH generated by the Ca2+-Mg2+ activated malic enzyme effectively supports the 11beta-hydroxylation of deoxycorticosterone, whereas in the presence of malate, or malate plus Mg2+ but absence of Ca2+, the energy linked transhydrogenase supplies all the required NADPH. The activated malic enzyme appears to be more efficient than transhydrogenase in generating NADPH to support 11beta-hydroxylation. Cyanide and azide have been found to inhibit solubilized mitochondrial malic enzyme.     

Coupled gating of skeletal muscle ryanodine receptors is modulated by Ca2+, Mg2+, and ATP

Coupled gating (synchronous openings and closures) of groups of skeletal muscle ryanodine receptors (RyR1), which mimics RyR1-mediated Ca(2+) release underlying Ca(2+) sparks, was first described by Marx et al. (Marx SO, Ondrias K, Marks AR. Science 281: 818-821, 1998). The nature of the RyR1-RyR1 interactions for coupled gating still needs to be characterized. Consequently, we defined planar lipid bilayer conditions where ～25% of multichannel reconstitutions contain mixtures of coupled and independently gating RyR1. In ～10% of the cases, all RyRs (2-10 channels; most frequently 3-4) gated in coupled fashion, allowing for quantification. Our results indicated that coupling required cytosolic solutions containing ATP/Mg(2+) and high (50 mM) luminal Ca(2+) (Ca(lum)) or Sr(2+) solutions. Bursts of coupled activity (events) started and ended abruptly, with all channels activating/deactivating within ～300 μs. Coupled RyR1 were heterogeneous, where highly active RyR1 ("drivers") seemed open during the entire coupled event (P(o) = 1), while other RyR1s ("followers") displayed abundant flickering and smaller amplitude. Drivers mean open time increased with cytosolic Ca(2+) (Ca(cyt)) or caffeine, whereas followers flicker frequency was Ca(cyt) independent and more sensitive to inhibition by cytosolic Mg(2+). Coupled events were insensitive to varying lumen-to-cytosol Ca(2+) fluxes from ～1 to 8 pA, which does not corroborate coupling of neighboring RyR1 by local Ca(2+)-induced Ca(2+) release. However, coupling requires specific Ca(lum) sites, as it was lost when Ca(lum) was replaced by luminal Ba(2+) or Mg(2+). In summary, coupled events reveal complex interactions among heterogeneous RyR1, differentially modulated by cytosolic ATP/Mg(2+), Ca(cyt), and Ca(lum,) which under cell-like ionic conditions may parallel synchronous RyR1 gating during Ca(2+) sparks.     

The single pore residue Asp542 determines Ca2+ permeation and Mg2+ block of the epithelial Ca2+ channel

The epithelial Ca(2+) channel (ECaC), which was recently cloned from rabbit kidney, exhibits distinctive properties that support a facilitating role in transcellular Ca(2+) (re)absorption. ECaC is structurally related to the family of six transmembrane-spanning ion channels with a pore-forming region between S5 and S6. Using point mutants of the conserved negatively charged amino acids present in the putative pore, we have identified a single aspartate residue that determines Ca(2+) permeation of ECaC and modulation by extracellular Mg(2+). Mutation of the aspartate residue, D542A, abolishes Ca(2+) permeation and Ca(2+)-dependent current decay as well as block by extracellular Mg(2+), whereas monovalent cations still permeate the mutant channel. Variation of the side chain length in mutations D542N, D542E, and D542M attenuated Ca(2+) permeability and Ca(2+)-dependent current decay. Block of monovalent currents through ECaC by Mg(2+) was decreased. Exchanging the aspartate residue for a positively charged amino acid, D542K, resulted in a nonfunctional channel. Mutations of two neighboring negatively charged residues, i.e. Glu(535) and Asp(550), had only minor effects on Ca(2+) permeation properties.     

Cytochemical localization of calcium and Ca2+,Mg2(+)-adenosine triphosphatase in colchicine-altered rat incisor ameloblasts

Colchicine is known to affect secretory, transport, and degradative functions of ameloblasts. The effects of colchicine on membrane-associated calcium and Ca2+,Mg2(+)-ATPase in secretory and maturation ameloblasts were investigated cytochemically. The pyroantimonate (PPA) method was used for localizing calcium and a modified Wachstein-Meisel medium was used to localize Ca2+,Mg2(+)-ATPase. Sections representing secretory and early maturation stages were examined by transmission electron microscopy. Morphological changes induced by colchicine included dislocated organelles and other well-established reactions to such anti-microtubule drugs. Calcium pyroantimonate (Ca-PA) deposits in most ameloblast types were markedly reduced, with the greater reduction occurring in those cells more severely altered morphologically. However, the cell membranes of both control and experimental smooth-ended maturation ameloblasts were essentially devoid of Ca-PA. The normal distribution and intensity of Ca2+,Mg2(+)-ATPase was not affected by colchicine. Because the observed reduction of membrane-associated calcium is apparently not mediated by Ca2+,Mg2(+)-ATPase in this case, other aspects of the calcium regulating system of ameloblasts are apparently targeted by colchicine.     

Regulation of (Ca2+, Mg2+)-ATPase in human erythrocytes dependent on calcium and calmodulin

The enzymatic basis for active Ca2+ transport in erythrocytes, and probably in many other cells, is a Mg2+-dependent Ca2+-ATPase located in the plasma membrane. The Ca2+-ATPase can be prepared in two different forms, a calmodulin-deficient (A-state) and a calmodulin-saturated (B-state). The ATPase is regulated by Ca2+-, K+, and ATP, and these effectors interact in a cooperative way, especially in the B-state. Both A-state and B-state can be solubilized by treatment with Triton X-100. The detergent activates the A-state but reduces the cooperative interactions in both states. The membrane-bound A and B state contain 5 and 25 nmoles calmodulin/g membrane protein, respectively, and the amount of Ca2+-ATPase was estimated at 10--20 nmoles/g membrane protein. It is discussed whether both A and B state represent natives states of the pump-enzymes.