Role of Cav1.2 L-type Ca2+ channels in vascular tone: effects of nifedipine and Mg2+

Ca(2+) entry via L-type voltage-gated Ca(2+) channels (LVGCs) is a key factor in generating myogenic tone (MT), as dihydropyridines (DHPs) and other LVGC blockers, including Mg(2+), markedly reduce MT. Recent reports suggest, however, that elevated external Mg(2+) concentration and DHPs may also inhibit other Ca(2+)-entry pathways. Here, we explore the contribution of LVGCs to MT in intact, pressurized mesenteric small arteries using mutant mice (DHP(R/R)) expressing functional but DHP-insensitive Ca(v)1.2 channels. In wild-type (WT), but not DHP(R/R), mouse arteries, nifedipine (0.3-1.0 microM) markedly reduced MT and vasoconstriction induced by high external K(+) concentrations ([K(+)](o)), a measure of LVGC-mediated Ca(2+) entry. Blocking MT and high [K(+)](o)-induced vasoconstriction by <1 microM nifedipine in WT but not in DHP(R/R) arteries implies that Ca(2+) entry via Ca(v)1.2 LVGCs is obligatory for MT and that nifedipine inhibits MT exclusively by blocking LVGCs. We also examined the effects of Mg(2+) on MT and LVGCs. High external Mg(2+) concentration (10 mM) blocked MT, slowed the high [K(+)](o)-induced vasoconstrictions, and decreased their amplitude in WT and DHP(R/R) arteries. To verify that these effects of Mg(2+) are due to block of LVGCs, we characterized the effects of extracellular and intracellular Mg(2+) on LVGC currents in isolated mesenteric artery myocytes. DHP-sensitive LVGC currents are inhibited by both external and internal Mg(2+). The results indicate that Mg(2+) relaxes MT by inhibiting Ca(2+) influx through LVGCs. These data provide new information about the central role of Ca(v)1.2 LVGCs in generating and maintaining MT in mouse mesenteric small arteries.     

Mg(2+) block unmasks Ca(2+)/Ba(2+) selectivity of alpha1G T-type calcium channels

We have examined permeation by Ca(2+) and Ba(2+), and block by Mg(2+), using whole-cell recordings from alpha1G T-type calcium channels stably expressed in HEK 293 cells. Without Mg(o)(2+), inward currents were comparable with Ca(2+) and Ba(2+). Surprisingly, three other results indicate that alpha1G is actually selective for Ca(2+) over Ba(2+). 1) Mg(2+) block is approximately 7-fold more potent with Ba(2+) than with Ca(2+). With near-physiological (1 mM) Mg(o)(2+), inward currents were approximately 3-fold larger with 2 mM Ca(2+) than with 2 mM Ba(2+). The stronger competition between Ca(2+) and Mg(2+) implies that Ca(2+) binds more tightly than Ba(2+). 2) Outward currents (carried by Na(+)) are blocked more strongly by Ca(2+) than by Ba(2+). 3) The reversal potential is more positive with Ca(2+) than with Ba(2+), thus P(Ca) > P(Ba). We conclude that alpha1G can distinguish Ca(2+) from Ba(2+), despite the similar inward currents in the absence of Mg(o)(2+). Our results can be explained by a 2-site, 3-barrier model if Ca(2+) enters the pore 2-fold more easily than Ba(2+) but exits the pore at a 2-fold lower rate.     

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-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes

Effects of changing cytosolic free Mg(2+) concentration on L-type Ca(2+) (I(Ca)) and Ba(2+) currents (I(Ba)) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8mM [Mg(2+)] ([Mg(2+)](p)) buffered with 30mM citrate and 10mM ATP. Increasing [Mg(2+)](p) from 0.2 to 1.8mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current-voltage relationships were studied with two-pulse and tail current (I(T)) protocols, respectively. Increasing [Mg(2+)](p) shifted the V(M) for half inactivation by -20mV but dramatically decreased I(Ca) amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I(T) amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg(2+)](p) might also inhibit permeation through open Ca(2+) channels at positive V(M).     

Magnesium Sensitizes Slow Vacuolar Channels to Physiological Cytosolic Calcium and Inhibits Fast Vacuolar Channels in Fava Bean Guard Cell Vacuoles

Vacuolar ion channels in guard cells play important roles during stomatal movement and are regulated by many factors including Ca(2+), calmodulin, protein kinases, and phosphatases. We report that physiological cytosolic and luminal Mg(2+) levels strongly regulate vacuolar ion channels in fava bean (Vicia faba) guard cells. Luminal Mg(2+) inhibited fast vacuolar (FV) currents with a K(i) of approximately 0.23 mM in a voltage-dependent manner at positive potentials on the cytoplasmic side. Cytosolic Mg(2+) at 1 mM also inhibited FV currents. Furthermore, in the absence of cytosolic Mg(2+), cytosolic Ca(2+) at less than 10 μM did not activate slow vacuolar (SV) currents. However, when cytosolic Mg(2+) was present, submicromolar concentrations of cytosolic Ca(2+) activated SV currents with a K(d) of approximately 227 nM, suggesting a synergistic Mg(2+)-Ca(2+) effect. The activation potential of SV currents was shifted toward physiological potentials in the presence of cytosolic Mg(2+) concentrations. The direction of SV currents could also be changed from outward to both outward and inward currents. Our data predict a model for SV channel regulation, including a cytosolic binding site for Ca(2+) with an affinity in the submicromolar range and a cytosolic low-affinity Mg(2+)-Ca(2+) binding site. SV channels are predicted to contain a third binding site on the vacuolar luminal side, which binds Ca(2+) and is inhibitory. In conclusion, cytosolic Mg(2+) sensitizes SV channels to physiological cytosolic Ca(2+) elevations. Furthermore, we propose that cytosolic and vacuolar Mg(2+) concentrations ensure that FV channels do not function as a continuous vacuolar K(+) leak, which would prohibit stomatal opening.     

S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca(2+) channels

Modulation of smooth muscle, L-type Ca(2+) channels (class C, Ca(V)1.2b) by thionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryonic kidney 293 expression system at the level of whole-cell and single-channel currents. Extracellular administration of GSNO (2 mM) rapidly reduced whole-cell Ba(2+) currents through channels derived either by expression of alpha1C-b or by coexpression of alpha1C-b plus beta2a and alpha2-delta. The non-thiol nitric oxide (NO) donors 2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which elevated cellular cGMP levels to a similar extent as GSNO, failed to affect Ba(2+) currents significantly. Intracellular administration of copper ions, which promote decomposition of the thionitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on alpha1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither alpha1C-b channel function nor their modulation by GSNO. Analysis of single-channel behavior revealed that GSNO inhibited Ca(2+) channels mainly by reducing open probability. The development of GSNO-induced inhibition was associated with the transient occurrence of a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca(2+) channels by an intracellular mechanism that is independent of NO release and stimulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca(2+) channel gating and conductance.     

Ni2+ block of CaV3.1 (alpha1G) T-type calcium channels

Ni(2+) inhibits current through calcium channels, in part by blocking the pore, but Ni(2+) may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni(2+) on Ca(V)3.1 is affected by permeant ions. We find two components to block by Ni(2+), a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent K(d) = 3 vs. 1 mM Ni(2+), with 2 mM Ca(2+) or Ba(2+)) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca(2+) or Ba(2+)). Slow block depends both on the concentration and on the identity of the permeant ion (Ca(2+) vs. Ba(2+) vs. Na(+)). Slow block is 2-3x faster in Ba(2+) than in Ca(2+) (2 or 110 mM), and is approximately 10x faster with 2 vs. 110 mM Ca(2+) or Ba(2+). Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations ( approximately 3 muM Ca(2+)). We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore. The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore. In contrast to the complex pore block observed for Ca(V)3.1, inhibition of Ca(V)3.2 by Ni(2+) was essentially independent of voltage, and was similar in 2 mM Ca(2+) vs. Ba(2+), consistent with inhibition by a different mechanism, at a site outside the pore.     

Amino acid substitutions in the pore of the Ca(V)1.2 calcium channel reduce barium currents without affecting calcium currents

Ba(2+) currents through Ca(V)1.2 Ca(2+) channels are typically twice as large as Ca(2+) currents. Replacing Phe-1144 in the pore-loop of domain III with glycine and lysine, and Tyr-1152 with lysine, reduces whole-cell G(Ba)/G(Ca) from 2.2 (wild-type) to 0.95, 1.21, and 0.90, respectively. Whole-cell and single-channel measurements indicate that reductions in G(Ba)/G(Ca) result specifically from a decrease in Ba(2+) conductance and not changes in V(h) or P(O). Half-maximal block of I(Li) is increased by 3.2-, 3.8-, and 1.6-fold in Ca(2+), and 3.8-, 4.2-, and 1.8-fold in Ba(2+) for F1144G, Y1152K, and F1144K, respectively. High affinity interactions of individual divalent cations to the pore are not important for determining G(Ba)/G(Ca), because the fold increases in IC(50) values for Ba(2+) and Ca(2+) are similar. On the contrary, conductance-concentration curves indicate that G(Ba)/G(Ca) is reduced because the interactions of multiple Ba(2+) ions in the mutant pores are altered. The complexity of these interactions is exemplified by the anomalous mole fraction effect, which is flattened for F1144G and FY/GK but accentuated for F1144K. In summary, the physicochemical properties of the amino acid residues at positions 1144 and 1152 are crucial to the pore's ability to distinguish between multiple Ba(2+) ions and Ca(2+) ions.     

Effect of low external calcium on the ERG current of NG108-15 cells

K(+) currents through ERG (ether-à-go-go related gene) channels were recorded in whole-cell voltage clamped NG108-15 neuroblastomaxglioma hybrid cells. The channels were fully activated by low holding potential (V(H)=-20 mV) and long depolarizing prepulses. Hyperpolarizing pulses elicited inward currents which deactivated after reaching a peak. Lowering [Ca(2+)](o) from 5 to 1. 5 or 0.5 mM decreased tau(-1), the rate constant of deactivation. The effect can be explained by a shift of the tau(-1)(V) curve to more negative potentials caused by an increase in surface charge density. Plotting tau(-1) against [Ca(2+)](o) for different potentials yielded straight lines; their slope was independent of potential at -140 to -120 mV and decreased at more positive potentials. The time to peak curve and the maximum of the steady-state inward current were also shifted to more negative potentials. In addition, peak ERG inward current increased. Raising [Ca(2+)](o) from 5 to 10 mM accelerated deactivation and decreased the peak current. 5 mM Ba(2+) affected tau(-1) similarly and inhibited peak current more strongly whereas 5 mM Mg(2+) was less potent. As found by Faravelli et al. (J. Physiol. 496 (1996) 13), bath solutions devoid of divalent cations (0 Ca(2+), 0 Mg(2+), 0.1 or 1.1 mM EGTA) abolished deactivation almost completely. The phenomenon was seen with bath containing either 40 or 6.5 mM K(+). Its occurrence was favored by raising the temperature to 34 degrees C. It suggests a particular requirement of channel closing for Ca(2+).     

Characteristics of anion channels in the tonoplast of the liverwort Conocephalum conicum

Isolated vacuoles of the liverwort Conocephalum conicum thallus cells were investigated using the patch-clamp technique. At high cytosolic Ca(2+) activities, slowly activating currents were evoked by positive potentials. The currents were conducted by the SV (slow-vacuolar) channel. When isolation of vacuoles was carried out at high Mg(2+) and low Ca(2+) concentration and the same proportion of the cations was kept in the bath, currents were recorded at negative potentials. Once activated, these currents persisted even after replacing Mg(2+) with K(+) in the bath. Sr(2+) and Ba(2+) were also effective activators of the currents. With a Cl(-) gradient, 10 mM in the bath and 100 mM in the lumen, currents were significantly reduced and the current-voltage characteristics shifted towards the reversal potential of Cl(-), indicating Cl(-) selectivity. Currents almost vanished after substituting Cl(-) with gluconate. They were strongly reduced by anion channel inhibitors 4,4'-diisothicyanatostilbene-2,2'-disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (A9C; 2 mM) and ethacrinic acid (0.5 mM). Single-channel recordings revealed a 32 pS channel activating at negative voltages. It is concluded that the currents at negative potentials are carried by anion channels suitable for conducting anions from the cytosol to the vacuole. The anion channels were weakly calcium dependent, remaining active at physiological calcium concentration. The channels were almost equally permeable to Cl(-), NO3(-) and SO4(2-), and much less permeable to malate(2-). Anion channels did not respond to ATP addition. cAMP (10 microM) had a weak effect on anion channels. Protein kinase A (0.4 U) added to the medium caused no significant effect on anion channels.     

The nociceptor ion channel TRPA1 is potentiated and inactivated by permeating calcium ions

The transient receptor potential A1 (TRPA1) channel is the molecular target for environmental irritants and pungent chemicals, such as cinnamaldehyde and mustard oil. Extracellular Ca(2+) is a key regulator of TRPA1 activity, both potentiating and subsequently inactivating it. In this report, we provide evidence that the effect of extracellular Ca(2+) on these processes is indirect and can be entirely attributed to entry through TRPA1 and subsequent elevation of intracellular calcium. Specifically, we found that in a pore mutant of TRPA1, D918A, in which Ca(2+) permeability was greatly reduced, extracellular Ca(2+) produced neither potentiation nor inactivation. Both processes were restored by reducing intracellular Ca(2+) buffering, which allowed intracellular Ca(2+) levels to become elevated upon entry through D918A channels. Application of Ca(2+) to the cytosolic face of excised patches was sufficient to produce both potentiation and inactivation of TRPA1 channels. Moreover, in whole cell recordings, elevation of intracellular Ca(2+) by UV uncaging of 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA-potentiated TRPA1 currents. In addition, our data show that potentiation and inactivation are independent processes. TRPA1 currents could be inactivated by Mg(2+), Ba(2+), and Ca(2+) but potentiated only by Ba(2+) and Ca(2+). Saturating activation by cinnamaldehyde or mustard oil occluded potentiation but did not interfere with inactivation. Last, neither process was affected by mutation of a putative intracellular Ca(2+)-binding EF-hand motif. In conclusion, we have further clarified the mechanisms of potentiation and inactivation of TRPA1 using the D918A pore mutant, an important tool for investigating the contribution of Ca(2+) influx through TRPA1 to nociceptive signaling.     

Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium

The intermediate (IK(Ca)) and small (SK(Ca)) conductance Ca(2+)-sensitive K(+) channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IK(Ca) and SK(Ca) channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 microM free [Ca(2+)](i) and 1 mM free [Mg(2+)](i), membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectification at positive membrane potentials. Blockers of large-conductance, Ca(2+)-sensitive potassium (BK(Ca)) and strong inward rectifier potassium (K(ir)) channels did not affect the membrane current. However, blockers of IK(Ca) channels, charybdotoxin (ChTX), and of SK(Ca) channels, apamin (Ap), significantly reduced the whole-cell current. Although IK(Ca) and SK(Ca) channels are intrinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg(2+) significantly increased these currents. Moreover, concomitant reduction of the [Ca(2+)](i) to 1 microM caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectification. Block of IK(Ca) and SK(Ca) channels caused a significant endothelial membrane potential depolarization (approximately 11 mV) and decrease in [Ca(2+)](i) in mesenteric arteries in the absence of an agonist. These results indicate that [Ca(2+)](i) can both activate and block IK(Ca) and SK(Ca) channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium.     

Characterization of tescalcin, a novel EF-hand protein with a single Ca2+-binding site: metal-binding properties, localization in tissues and cells, and effect on calcineurin

The tescalcin gene is preferentially expressed during mouse testis differentiation. Here, we demonstrate that this gene encodes a 24 kDa Ca(2+)- and Mg(2+)-binding protein with one consensus EF-hand and three additional domains with EF-hand homology. Equilibrium dialysis with (45)Ca(2+) revealed that recombinant tescalcin binds approximately one Ca(2+) ion at physiological concentrations (pCa 4.5). The intrinsic tryptophan fluorescence of tescalcin was significantly reduced by Ca(2+), indicative of a conformational change. The apparent K(d) for Ca(2+) was 0.8 microM. A point mutation in the consensus EF-hand (D123A) abolished (45)Ca(2+) binding and prevented the fluorescence quenching, demonstrating that the consensus EF-hand alone mediates the Ca(2+)-induced conformational change. Tescalcin also binds Mg(2+) (K(d) 73 microM), resulting in a much smaller fluorescence decrease. In the presence of 1 mM Mg(2+), tescalcin's Ca(2+) affinity is shifted to 3.5 microM. These results illustrate that tescalcin should bind Mg(2+) constitutively in a quiescent cell, replacing it with Ca(2+) during stimulation. We also show that tescalcin is most abundant in adult mouse heart, brain, and stomach, as well as in HeLa and HL-60 cells. Immunofluorescence microscopy revealed that tescalcin is present in the cytoplasm and nucleus, with concentration in membrane ruffles and lamellipodia in the presence of serum, where it colocalizes with the small guanosine triphosphatase Rac-1. Tescalcin shares sequence and functional homology with calcineurin-B homologous protein (CHP), and we found that tescalcin, like CHP, can inhibit the phosphatase activity of calcineurin A. Hence, tescalcin is a novel calcineurin B-like protein that binds a single Ca(2+) ion.     

Mg2+ enhances voltage sensor/gate coupling in BK channels

BK (Slo1) potassium channels are activated by millimolar intracellular Mg(2+) as well as micromolar Ca(2+) and membrane depolarization. Mg(2+) and Ca(2+) act in an approximately additive manner at different binding sites to shift the conductance-voltage (G(K)-V) relation, suggesting that these ligands might work through functionally similar but independent mechanisms. However, we find that the mechanism of Mg(2+) action is highly dependent on voltage sensor activation and therefore differs fundamentally from that of Ca(2+). Evidence that Ca(2+) acts independently of voltage sensor activation includes an ability to increase open probability (P(O)) at extreme negative voltages where voltage sensors are in the resting state; 2 microM Ca(2+) increases P(O) more than 15-fold at -120 mV. However 10 mM Mg(2+), which has an effect on the G(K)-V relation similar to 2 microM Ca(2+), has no detectable effect on P(O) when voltage sensors are in the resting state. Gating currents are only slightly altered by Mg(2+) when channels are closed, indicating that Mg(2+) does not act merely to promote voltage sensor activation. Indeed, channel opening is facilitated in a voltage-independent manner by Mg(2+) in a mutant (R210C) whose voltage sensors are constitutively activated. Thus, 10 mM Mg(2+) increases P(O) only when voltage sensors are activated, effectively strengthening the allosteric coupling of voltage sensor activation to channel opening. Increasing Mg(2+) from 10 to 100 mM, to occupy very low affinity binding sites, has additional effects on gating that more closely resemble those of Ca(2+). The effects of Mg(2+) on steady-state activation and I(K) kinetics are discussed in terms of an allosteric gating scheme and the state-dependent interactions between Mg(2+) and voltage sensor that may underlie this mechanism.     

A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine

Native smooth muscle L-type Ca(v)1.2 calcium channels have been shown to support a fraction of Ca(2+) currents with a window current that is close to resting potential. The smooth muscle L-type Ca(2+) channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Ca(v)1.2 channels exhibiting hyperpolarized shift in steady-state inactivation would contribute to larger inhibition by DHP, in addition to structural differences of the channels generated by alternative splicing that modulate DHP sensitivities. In addition, it has also been shown that alternative splicing modulates DHP sensitivities by generating structural differences in the Ca(v)1.2 channels. Here, we report a smooth muscle L-type Ca(v)1.2 calcium channel splice variant, Ca(v)1.2SM (1/8/9(*)/32/Delta33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Ca(v)1.2b clone (1/8/9(*)/32/33). This variant activates from more negative potentials and generates a window current closer to resting membrane potential. We also identified the predominant cardiac isoform Ca(v)1.2CM clone (1a/8a/Delta9(*)/32/33) that is different from the established Ca(v)1.2a (1a/8a/Delta9(*)/31/33). Importantly, Ca(v)1.2SM channels were shown to be more sensitive to nifedipine blockade than Ca(v)1.2b and cardiac Ca(v)1.2CM channels when currents were recorded in either 5 mM Ba(2+) or 1.8 mM Ca(2+) external solutions. This is the first time that a smooth muscle Ca(v)1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP.     

Low-affinity Ca(2+) and Ba(2+) binding sites in the pore of alpha7 nicotinic acetylcholine receptors

alpha7 nicotinic receptors are highly permeable to Ca(2+) as well as monovalent cations. We extended the characterization of the Ca(2+) permeation of non-desensitizing chick alpha7 receptors (S240T/L247T alpha7 nAChRs) expressed in Xenopus oocytes by (1) measuring the concentration dependence of conductance under conditions in which Ca(2+) or Ba(2+) were the only permeant cations in the extracellular solution, and (2) measuring the concentration dependence of Ca(2+) block of K(+) currents through the receptors. The first set of experiments yielded an apparent affinity of 0.96 mM Ca(2+) activity (2.4 mM concentration) for Ca(2+) permeation and an apparent affinity of 0.65 mM Ba(2+) activity (1.7 mM concentration) for Ba(2+) permeation. The apparent affinity of Ca(2+) inhibition of K(+) currents was 0.49 mM activity (1.5 mM concentration). The similarity of these apparent affinities in the millimolar range suggests that the pore of alpha7 receptors has one or more low-affinity Ca(2+) binding sites and no high-affinity sites.     

Effects of permeant ion concentrations on the gating of L-type Ca2+ channels in hair cells

We determined the gating and permeation properties of single L-type Ca(2+) channels, using hair cells and varying concentrations (5-70 mM) of the charge carriers Ba(2+) and Ca(2+). The channels showed distinct gating modes with high- and low-open probability. The half-activation voltage (V(1/2)) shifted in the hyperpolarizing direction from high to low permeant ion concentrations consistent with charge screening effects. However, the differences in the slope of the voltage shifts (in VM(-1)) between Ca(2+) (0.23) and Ba(2+) (0.13), suggest that channel-ion interaction may also contribute to the gating of the channel. We examined the effect of mixtures of Ba(2+) and Ca(2+) on the activation curve. In 5 mM Ca(2+), the V(1/2) was, -26.4 +/- 2.0 mV compared to Ba(2+), -34.7 +/- 2.9 mV, as the charge carrier. However, addition of 1 mM Ba(2+) in 4 mM Ca(2+), a molar ratio, which yielded an anomalous-mole fraction effect, was sufficient to shift the V(1/2) to -34.7 +/- 1.5 mV. Although Ca(2+)-dependent inactivation of the L-type channels in hair cells can yield the present findings, we provide evidence that the anomalous gating of the channel may stem from the closed interaction between ion permeation and gating.     

Block of CaV1.2 channels by Gd3+ reveals preopening transitions in the selectivity filter

Using the lanthanide gadolinium (Gd(3+)) as a Ca(2+) replacing probe, we investigated the voltage dependence of pore blockage of Ca(V)1.2 channels. Gd(+3) reduces peak currents (tonic block) and accelerates decay of ionic current during depolarization (use-dependent block). Because diffusion of Gd(3+) at concentrations used (<1 microM) is much slower than activation of the channel, the tonic effect is likely to be due to the blockage that occurred in closed channels before depolarization. We found that the dose-response curves for the two blocking effects of Gd(3+) shifted in parallel for Ba(2+), Sr(2+), and Ca(2+) currents through the wild-type channel, and for Ca(2+) currents through the selectivity filter mutation EEQE that lowers the blocking potency of Gd(3+). The correlation indicates that Gd(3+) binding to the same site causes both tonic and use-dependent blocking effects. The apparent on-rate for the tonic block increases with the prepulse voltage in the range -60 to -45 mV, where significant gating current but no ionic current occurs. When plotted together against voltage, the on-rates of tonic block (-100 to -45 mV) and of use-dependent block (-40 to 40 mV) fall on a single sigmoid that parallels the voltage dependence of the gating charge. The on-rate of tonic block by Gd(3+) decreases with concentration of Ba(2+), indicating that the apparent affinity of the site to permeant ions is about 1 mM in closed channels. Therefore, we propose that at submicromolar concentrations, Gd(3+) binds at the entry to the selectivity locus and that the affinity of the site for permeant ions decreases during preopening transitions of the channel.     

Magnesium permeation through mechanosensitive channels： single-current measurements

Compelling evidence shows that intracellular free magnesium [Mg^2＋]i may be a critical regulator of cell activity in eukaryotes. However, membrane transport mechanisms mediating Mg^2＋ influx in mammalian cells are poorly understood. Here, we show that mechanosensitive （MS） cationic channels activated by stretch are permeable for Mg^2＋ ions at different extracellular concentrations including physiological ones. Single-channel currents were recorded from cell-attached and inside-out patches on K562 leukaemia cells at various concentrations of MgCl2 when Mg^2＋ was the only available carrier of inward currents. At 2 mM Mg^2＋, inward mechanogated currents representing Mg^2＋ influx through MS channels corresponded to the unitary conductance of about 5 pS. At higher Mg^2＋ levels, only slight increase of single-channel currents and conductance occurred, implying that Mg^2＋ permeation through MS channels is characterized by strong saturation. At 20 and 90 mM Mg^2＋, mean conductance values for inward currents carried by Mg^2＋ were rather similar, being equal to 6.8 ± 0.5 and 6.4 ± 0.5 pS, respectively. The estimation of the channel-selective permeability according to constant field equation is obviously limited due to saturation effects. We conclude that the detection of single currents is the main evidence for Mg^2＋ permeation through membrane channels activated by stretch. Our single-current measurements document Mg^2＋ influx through MS channels in the plasma membrane of leukaemia cells.     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.