Studies on Calcium and Sodium in Uterine Smooth Muscle Excitation under Current-Clamp and Voltage-Clamp Conditions

The objective of these studies was to define the roles of calcium and sodium in uterine smooth muscle excitation. The double sucrose-gap technique was used for current-clamp and voltage-clamp experiments. It was shown that neither sodium nor calcium alone is capable of supporting excitation in estrogen-dominated uterine smooth muscle. Calcium dependence was explained in part by increased membrane "leakage" current in calcium-free solution and calcium control of the voltage dependence of the early transient conductance. High concentrations of TTX did not affect the magnitude of the peak transient current while La⁺⁺⁺, Mn⁺⁺, and Co⁺⁺ greatly reduced or abolished it and decreased the steady-state current. From these and other data it was concluded that the regenerative mechanism in uterine smooth muscle has the functional characteristics of a single transient conductance channel whose activation requires the presence of both sodium and calcium. Insensitivity to TTX indicates that the molecular structure of the channel is unlike that in certain sodium-dependent systems, while the effects of La⁺⁺⁺, Mn⁺⁺, Co⁺⁺, and Ca⁺⁺ reveal a similar dependence of conductances on extracellular polyvalent cations.     

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops fromChara australis

Summary Patch-clamp studies of cytoplasmic drops from the charophyteChara australis have previously revealed K⁺ channels combining high conductance (170 pS) with high selectivity for K⁺, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K⁺>Rb⁺>NH ₄ ⁺ Na⁺ and Cl^–. Divalent cytosolic ions reduce the K⁺ conductance of this channel and alter its K⁺ gating in a voltage-dependent manner. The order of blocking potency is Ba²⁺>Sr²⁺>Ca²⁺>Mg²⁺. The channel is activated by micromolar cytosolic Ca²⁺, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca²⁺ needed to open the channel. The possible role of the Ca²⁺-activated K⁺ channel in the tonoplast ofChara is discussed.     

Calcium Inhibits Paracellular Sodium Conductance through Claudin-2 by Competitive Binding

Claudins form paracellular pores at the tight junction in epithelial cells. Profound depletion of extracellular calcium is well known to cause loosening of the tight junction with loss of transepithelial resistance. However, moderate variations in calcium concentrations within the physiological range can also regulate transepithelial permeability. To investigate the underlying molecular mechanisms, we studied the effects of calcium on the permeability of claudin-2, expressed in an inducible MDCK I cell line. We found that in the physiological range, calcium acts as a reversible inhibitor of the total conductance and Na⁺ permeability of claudin-2, without causing changes in tight junction structure. The effect of calcium is enhanced at low Na⁺ concentrations, consistent with a competitive effect. Furthermore, mutation of an intrapore negatively charged binding site, Asp-65, to asparagine partially abrogated the inhibitory effect of calcium. This suggests that calcium competes with Na⁺ for binding to Asp-65. Other polyvalent cations had similar effects, including La³⁺, which caused severe and irreversible inhibition of conductance. Brownian dynamics simulations demonstrated that such inhibition can be explained if Asp-65 has a relatively high charge density, thus favoring binding of Ca²⁺ over that of Na⁺, reducing Ca²⁺ permeation by inhibiting its dissociation from this site, and decreasing Na⁺ conductance through repulsive electrostatic interaction with Ca²⁺. These findings may explain why hypercalcemia inhibits Na⁺ reabsorption in the proximal tubule of the kidney.     

An analysis of conductance changes in squid axon

The membrane of the squid axon is considered on the basis of a pore model in which the distribution of the pore sizes strongly favors K⁺ transfer when there is no potential. Electrical asymmetry causes non-penetrating ions on the membrane capacitor to exert a mechanical force on both membrane surfaces and this force results in a deformation of the membrane pore system such that it assumes a distribution of sizes favoring the ions exerting mechanical force. The ions involved appear to be Ca⁺⁺ on the outside of the membrane and isethionate^-, (i^-) on the inside; as Ca⁺⁺ is equivalent in size to Na⁺, the charged membrane is potentially able to transfer Na⁺, when the ions deforming the membrane pore distribution are removed. A depolarization of the membrane leads to an opening of pores that will allow Na⁺ penetration and a release of the membrane from deformation. The pores revert to the zero-potential pore size distribution hence the Na permeability change is a transient. Calculation shows that the potassium conductance vs. displacement of membrane potential curve for the squid axon and the "inactivation" function, h, can be obtained directly from the assumed membrane distortion without the introduction of arbitrary parameters. The sodium conductance, because it is a transient, requires assumptions about the time constants with which ions unblock pores at the outside and the inside of the membrane.     

A mutation that increases a novel calcium-activated potassium conductance ofParamecium tetraurelia

Summary Under two-electrode voltage clamp, a mutant ofP. tetraurelia, restless (rst/rst), showed a large increase in induced current and an outward tail current when compared to the wildtype cell for hyperpolarizing voltage steps. An increase in the induced and tail currents is also observed for depolarizing voltage steps. The larger current during voltage steps and tail in the mutant were eliminated by the use of CsCl-filled electrodes and tetraethylammonium ion (TEA⁺) in the bath solution, characterizing the lesion as affecting a K⁺ conductance. Ionophoretic injection of ethylene glycol bis-(beta-aminoethyl ether) n,n,n,n-tetraacetic acid (EGTA) to buffer internal Ca²⁺ concentration reduced the increased K⁺ current and tail of therestless cell, indicating Ca²⁺ activation of the K⁺ current. Time course and amplitude of remaining currents after blockage of K⁺ conductances with Cs⁺ and TEA⁺ were similar in wild-type andrestless cells suggesting norestless defect in entry of calcium. The Ca²⁺-activated sodium current was similar in the mutant to that in wild type arguing against a defect in calcium regulation activating the K⁺ channel in therestless cell. We conclude that therestless mutation alters a Ca²⁺-activated potassium conductance other than the one previously described. The multiplicity of Ca²⁺-activated potassium conductances inParamecium is discussed.     

Prolonged calcium influx after termination of light-induced calcium release in invertebrate photoreceptors

In microvillar photoreceptors, light stimulates the phospholipase C cascade and triggers an elevation of cytosolic Ca²⁺ that is essential for the regulation of both visual excitation and sensory adaptation. In some organisms, influx through light-activated ion channels contributes to the Ca²⁺ increase. In contrast, in other species, such as Lima, Ca²⁺ is initially only released from an intracellular pool, as the light-sensitive conductance is negligibly permeable to calcium ions. As a consequence, coping with sustained stimulation poses a challenge, requiring an alternative pathway for further calcium mobilization. We observed that after bright or prolonged illumination, the receptor potential of Lima photoreceptors is followed by the gradual development of an after-depolarization that decays in 1–4 minutes. Under voltage clamp, a graded, slow inward current (I_slow) can be reproducibly elicited by flashes that saturate the photocurrent, and can reach a peak amplitude in excess of 200 pA. I_slow obtains after replacing extracellular Na⁺ with Li⁺, guanidinium, or N-methyl-d-glucamine, indicating that it does not reflect the activation of an electrogenic Na/Ca exchange mechanism. An increase in membrane conductance accompanies the slow current. I_slow is impervious to anion replacements and can be measured with extracellular Ca²⁺ as the sole permeant species; Ba can substitute for Ca²⁺ but Mg²⁺ cannot. A persistent Ca²⁺ elevation parallels I_slow, when no further internal release takes place. Thus, this slow current could contribute to sustained Ca²⁺ mobilization and the concomitant regulation of the phototransduction machinery. Although reminiscent of the classical store depletion–operated calcium influx described in other cells, I_slow appears to diverge in some significant aspects, such as its large size and insensitivity to {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 and lanthanum; therefore, it may reflect an alternative mechanism for prolonged increase of cytosolic calcium in photoreceptors.     

Fluorescent sensors based on BODIPY derivatives for aluminium ion recognition: an experimental and theoretical study

Two BODIPY derivative sensors for metal ion recognition containing 10-(4-hydroxyphenyl) (L1) and 10-(3,4-dihydroxyphenyl) (L2) were synthesized in a one-pot reaction of benzaldehyde derivative and 2,4-dimethylpyrrole in the presence of trifluoroacetic acid as catalyst. The binding abilities between these sensors and 50 equivalents of Na⁺, K⁺, Ag⁺, Ca²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Pb²⁺, Al³⁺ and Cr³⁺ ions were studied using UV–vis and fluorescent spectroscopic methods. Of all the metal ions tested, Al³⁺ ion showed the greatest decrease in intensity in the spectra of the sensors, and therefore Al³⁺ ion forms the strongest complex. The binding abilities of BODIPY receptors with Na⁺, Ag⁺, Ca²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ and Al³⁺ ions were also investigated using density functional theory (DFT) calculations at B3LYP/LanL2DZ theoretical level. The calculated results point to the same conclusion. DFT calculations also provided the HOMO–LUMO energy levels, which can explain the spectrum change upon complexation.

Differential effects of heavy metal ions on Ca2+-dependent K+ channels

Summary 1. The ability of various divalent metal ions to substitute for Ca²⁺ in activating distinct types of Ca²⁺-dependent K⁺ [K⁺(Ca²⁺] channels has been investigated in excised, inside-out membrane patches of human erthrocytes and of clonal N1E-115 mouse neuroblastoma cells using the patch clamp technique. The effects of the various metal ions have been compared and related to the effects of Ca²⁺.2. At concentrations between 1 and 100 µM Pb²⁺, Cd²⁺ and Co²⁺ activate intermediate conductance K⁺(Ca²⁺) channels in erythrocytes and large conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Pb²⁺ and Co²⁺, but not Cd²⁺, activate small conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Mg²⁺ and Fe²⁺ do not activate any of the K⁺(Ca²⁺) channels.3. Rank orders of the potencies for K⁺(Ca²⁺) activation are Pb²⁺, Cd²⁺>Ca²⁺, Co²⁺>>Mg²⁺, Fe²⁺ for the intermediate erythrocyte K⁺(Ca²⁺) channel, and Pb²⁺, Cd²⁺>Ca²⁺>Co²⁺>>Mg²⁺, Fe²⁺ for the small, and Pb²⁺>Ca²⁺>Co²⁺>>Cd²⁺, Mg²⁺, Fe²⁺ for the large K⁺(Ca²⁺) channel in neuroblastoma cells.4. At high concentrations Pb²⁺, Cd²⁺, and Co²⁺ block K⁺(Ca²⁺) channels in erythrocytes by reducing the opening frequency of the channels and by reducing the single channel amplitude. The potency orders of the two blocking effects are Pb²⁺>Cd²⁺, Co²⁺>>Ca²⁺, and Cd²⁺>Pb²⁺, Co²⁺>>Ca²⁺, respectively, and are distinct from the potency orders for activation.5. It is concluded that the different subtypes of K⁺(Ca²⁺) channels contain distinct regulatory sites involved in metal ion binding and channel opening. The K⁺(Ca²⁺) channel in erythrocytes appears to contain additional metal ion interaction sites involved in channel block.     

A Voltage-Dependent Ca<Superscript>2+</Superscript> Influx Pathway Regulates the Ca<Superscript>2+</Superscript>-Dependent Cl<Superscript>−</Superscript> Conductance of Renal IMCD-3 Cells

We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca²⁺-dependent Cl⁻ current (I_CLCA). Here we report that I_CLCA activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al³⁺-sensitive, voltage-dependent, Ca²⁺ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (−60 mV), I_CLCA was found to be inactive and resting currents were predominantly K⁺ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of I_CLCA (T _0.5 ~ 500 s), while repolarization in turn resulted in a monoexponential decay in I_CLCA (T _0.5 ~ 100 s). The activation of I_CLCA by depolarization was reduced by lowering extracellular Ca²⁺ and completely inhibited by buffering cytosolic Ca²⁺ with EGTA, suggesting a role for Ca²⁺ influx in the activation of I_CLCA. However, raising bulk cytosolic Ca²⁺ at −60 mV did not produce sustained I_CLCA activity. Therefore I_CLCA is dependent on both an increase in intracellular Ca²⁺ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca²⁺ influx pathway that displays equal permeability to Ca²⁺ and Ba²⁺ ions and that is blocked by extracellular Al³⁺ and La³⁺. Furthermore, Al³⁺ completely and reversibly inhibited depolarization-induced activation of I_CLCA, thereby directly linking Ca²⁺ influx to activation of I_CLCA. We speculate that during sustained membrane depolarization, calcium influx activates I_CLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.