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

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

Ca2+ modulation of Ca2+ release-activated Ca2+ channels is responsible for the inactivation of its monovalent cation current

The Ca(2+) release-activated Ca(2+) (CRAC) channel is the most well documented of the store-operated ion channels that are widely expressed and are involved in many important biological processes. However, the regulation of the CRAC channel by intracellular or extracellular messengers as well as its molecular identity is largely unknown. Specifically, in the absence of extracellular divalent cations it becomes permeable to monovalent cations with a larger conductance, however this monovalent cation current inactivates rapidly by an unknown mechanism. Here we found that Ca(2+) dissociation from a site on the extracellular side of the CRAC channel is responsible for the inactivation of its Na(+) current, and Ca(2+) occupancy of this site otherwise potentiates its Ca(2+) as well as Na(+) currents. This Ca(2+)-dependent potentiation is required for the normal functioning of CRAC channels.     

Permeation of monovalent cations through the non-capacitative arachidonate-regulated Ca2+ channels in HEK293 cells. Comparison with endogenous store-operated channels

In a manner similar to voltage-gated Ca(2+) channels and Ca(2+) release-activated Ca(2+) (CRAC) channels, the recently identified arachidonate-regulated Ca(2+) (ARC) channels display a large monovalent conductance upon removal of external divalent cations. Using whole-cell patch-clamp recording, we have characterized the properties of these monovalent currents in HEK293 cells stably transfected with the m3 muscarinic receptor and compared them with the corresponding currents through the endogenous store-operated Ca(2+) (SOC) channels in the same cells. Although the monovalent currents seen through these two channels displayed certain similarities, several marked differences were also apparent, including the magnitude of the monovalent current/Ca(2+) current ratio, the rate and nature of the spontaneous decline in the currents, and the effects of external monovalent cation substitutions and removal of internal Mg(2+). Moreover, monovalent ARC currents could be activated after the complete spontaneous inactivation of the corresponding SOC current in the same cell. We conclude that the non-capacitative ARC channels share, with voltage-gated Ca(2+) channels and store-operated Ca(2+) channels (e.g. SOC and CRAC the general property of monovalent ion permeation in the nominal absence of extracellular divalent ions. However, the clear differences between the properties of these currents through ARC and SOC channels in the same cell confirm that these represent distinct conductances.     

Intra and extracellular surface charges near Ca2+ channels in neurons and neuroblastoma cells.

The properties of low (LVA) and high (HVA) voltage-activated calcium currents were investigated in rat sensory neurons and a murine neuroblastoma cell line exposed to various concentrations of intra- or extracellular monovalent ([c+]i/o) and trivalent ([c3+]i/o) cations. In neurons, when [c+]i was changed from 150 to 20 mM, positive shifts of 18-28 mV were observed in activation curves of both LVA and HVA currents, as well as in LVA inactivation curves. Extracellularly, in divalent-free solutions, [c+]o of 20-50 mM produced medium (12-22 mV) negative shifts of the LVA channel properties. These data were used to estimate, by a "screening" model, a negative surface charge density around neuron's calcium channels of 1/1,000 and 1/1,325 eA-2 at the outside or inside face, respectively. In the presence of physiological concentrations of divalent cations, [c+]o of 20-60 mM caused smaller (4-11 mV) negative shifts of the activation and inactivation curves, which can be explained by assuming a partial neutralization of negative charges by divalent cations. By applying the above procedure to LVA channels of neuroblastoma cells, the ratio of extra- to intracellular surface charge density turned out to be more than tenfold higher than in neurons. Effects produced by [c3+]i/o were not in agreement with expectations based on screening or binding models.     

Effects of divalent cations on voltage-gated Ca2+ channels and depolarization-induced [Ca2+], transients of freshly isolated pyramidal cells of the rat dorsal cochlear nucleus

The effects of divalent cations on voltage-activated Ca2+ channels and depolarization-evoked cytoplasmic [Ca2+] elevations were studied in pyramidal neurones isolated from the dorsal cochlear nucleus of the rat. Ca2+ currents were recorded using the whole-cell configuration of the patch-clamp technique. 10 micromol x l(-1) Cd2+ exerted a greater blocking effect on the high-voltage activated (HVA) currents than on the low-voltage activated (LVA) ones (decrease to 26.6+/-2.5% and to 87.8+/-2.1%, respectively). The blocking effect of 200 micromol x l(-1) Cd2+ was more pronounced and the difference between the effect on the HVA and LVA currents became smaller (decrease to 11.7+/-2.1% and to 32.4+/-2.7%, respectively). 200 micromol x l(-1) Ni2+ reduced the LVA component more effectively (to 77.6+/-5.4%) than the HVA one (to 86.9+/-2.6%). Cytoplasmic [Ca2+] changes were measured applying a fluorimetric technique (Fura-2). 10 micromol x l(-1) Cd2+ decreased the peak values of 50 mmol x l(-1) K+ depolarization-induced [Ca2]+i transients to 30.4+/-1.4% while 200 micromol x l(-1) Cd2+ caused a drop to 2.5+/-0.2%. 200 micromol x l(-1) Ni2+ decreased the peak of the transients to 69.6+/-2.9%. Comparison of the blocking effects of divalent cations on Ca2+ currents and [Ca2+]i transients supports further the conclusion that the depolarization-induced [Ca2+]i changes are produced mainly by the activation of the HVA Ca2+ channels.     

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.     

Molecular determinants of sensitivity and conductivity of human TRPM7 to Mg2+ and Ca2+

It is known that extracellular Mg(2+) and Ca(2+) can permeate TRPM7 and at the same time block the permeation by monovalent cations. In the present study, we examined the molecular basis for the conductivity and sensitivity of human TRPM7 to these divalent cations. Extracellular acidification to pH 4.0 markedly reduced the blocking effects of Mg(2+) and Ca(2+) on the Cs(+) currents, decreasing their binding affinities: their IC(50) values increased 510- and 447-fold, respectively. We examined the effects of neutralizing each of four negatively charged amino acid residues, Glu-1047, Glu-1052, Asp-1054 and Asp-1059, within the putative pore-forming region of human TRPM7. Mutating Glu-1047 to alanine (E1047A) resulted in non-functional channels, whereas mutating any of the other residues resulted in functionally expressed channels. Cs(+) currents through D1054A and E1052A were less sensitive to block by divalent cations; the IC(50) values were increased 5.5- and 3.9-fold, respectively, for Mg(2+) and 10.5- and 6.7-fold, respectively, for Ca(2+). D1059A also had a significant reduction, though less marked compared to the reductions seen for D1054A and E1052A, in sensitivity to Mg(2+) (1.7-fold) and Ca(2+) (3.9-fold). The D1054A mutation largely abolished inward currents conveyed by Mg(2+) and Ca(2+). In the E1052A and D1059A mutants, inward Mg(2+) and Ca(2+) currents were sizable but significantly diminished. Thus, it is concluded that in human TRPM7, (1) both Asp-1054 and Glu-1052, which are located near the narrowest portion in the pore's selectivity filter, may provide the binding sites for Mg(2+) and Ca(2+), (2) Asp-1054 is an essential determinant of Mg(2+)and Ca(2+) conductivity, and (3) Glu-1052 and Asp-1059 facilitate the conduction of divalent cations.     

Ca2+ selectivity of a chemically modified OmpF with reduced pore volume

We studied an E. coli OmpF mutant (LECE) containing both an EEEE-like locus, typical of Ca(2+) channels, and an accessible and reactive cysteine. After chemical modification with the cysteine-specific, negatively charged (-1e) reagents MTSES or glutathione, this LECE mutant was tested for Ca(2+) versus alkali metal selectivity. Selectivity was measured by conductance and zero-current potential. Conductance measurements showed that glutathione-modified LECE had reduced conductance at Ca(2+) mole fractions <10(-3). MTSES-modified LECE did not. Apparently, the LECE protein is (somehow) a better Ca(2+) chelator after modification with the larger glutathione. Zero-current potential measurements revealed a Ca(2+) versus monovalent cation selectivity that was highest in the presence of Li(+) and lowest in the presence of Cs(+). Our data clearly show that after the binding of Ca(2+) the LECE pore (even with the bulky glutathione present) is spacious enough to allow monovalent cations to pass. Theoretical computations based on density functional theory combined with Poisson-Nernst-Planck theory and a reduced pore model suggest a functional separation of ionic pathways in the pore, one that is specific for small and highly charged ions, and one that accepts preferentially large ions, such as Cs(+).     

Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels

Although store-operated calcium release-activated Ca(2+) (CRAC) channels are highly Ca(2+)-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na(+) and Cs(+) with a unitary conductance of approximately 40 pS, and that intracellular Mg(2+) modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg(2+)-inhibited cation (MIC) channels that open as Mg(2+) is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8-10 mM intracellular Mg(2+) to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca(2+) to divalent-free extracellular solution evokes Na(+) current through open CRAC channels (Na(+)-I(CRAC)) that is initially eightfold larger than the preceding Ca(2+) current and declines by approximately 80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs(+) (P(Cs)/P(Na) = 0.13 vs. 1.2 for MIC). Neither the decline in Na(+)-I(CRAC) nor its low Cs(+) permeability are affected by intracellular Mg(2+) (90 microM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca(2+) channels.     

Block by extracellular divalent cations of Drosophila big brain channels expressed in Xenopus oocytes

Drosophila Big Brain (BIB) is a transmembrane protein encoded by the neurogenic gene big brain (bib), which is important for early development of the fly nervous system. BIB expressed in Xenopus oocytes is a monovalent cation channel modulated by tyrosine kinase signaling. Results here demonstrate that the BIB conductance shows voltage- and dose-dependent block by extracellular divalent cations Ca(2+) and Ba(2+) but not by Mg(2+) in wild-type channels. Site-directed mutagenesis of negatively charged glutamate (Glu(274)) and aspartate (Asp(253)) residues had no effect on divalent cation block. However, mutation of a conserved glutamate at position 71 (Glu(71)) in the first transmembrane domain (M1) altered channel properties. Mutation of Glu(71) to Asp introduced a new sensitivity to block by extracellular Mg(2+); substitutions with asparagine or glutamine decreased whole-cell conductance; and substitution with lysine compromised plasma membrane expression. Block by divalent cations is important in other ion channels for voltage-dependent function, enhanced signal resolution, and feedback regulation. Our data show that the wild-type BIB conductance is attenuated by external Ca(2+), suggesting that endogenous divalent cation block might be relevant for enhancing signal resolution or voltage dependence for the native signaling process in neuronal cell fate determination.     

Identification of an aspartic residue in the P-loop of the vanilloid receptor that modulates pore properties

Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed in Xenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu(648) and Glu(651) rendered minor effects on VR1 pore attributes, while Glu(636) slightly modulated pore blockade. In contrast, replacement of Asp(646) by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg(2+) with respect to Na(+) without changing the selectivity of monovalent cations. At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results suggest that Asp(646) is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway.     

Distinct properties of CRAC and MIC channels in RBL cells

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.     

The selectivity filter of the cation channel TRPM4

Transient receptor potential channel melastatin subfamily (TRPM) 4 and its close homologue, TRPM5, are the only two members of the large transient receptor potential superfamily of cation channels that are impermeable to Ca(2+). In this study, we located the TRPM4 selectivity filter and investigated possible structural elements that render it Ca(2+)-impermeable. Based on homology with known cation channel pores, we identified an acidic stretch of six amino acids in the loop between transmembrane helices TM5 and TM6 ((981)EDMDVA(986)) as a potential selectivity filter. Substitution of this six-amino acid stretch with the selectivity filter of TRPV6 (TIIDGP) resulted in a functional channel that combined the gating hallmarks of TRPM4 (activation by Ca(2+), voltage dependence) with TRPV6-like sensitivity to block by extracellular Ca(2+) and Mg(2+) as well as Ca(2+) permeation. Neutralization of Glu(981) resulted in a channel with normal permeability properties but a strongly reduced sensitivity to block by intracellular spermine. Neutralization of Asp(982) yielded a functional channel that exhibited extremely fast desensitization (tau < 5 s), possibly indicating destabilization of the pore. Neutralization of Asp(984) resulted in a non-functional channel with a dominant negative phenotype when coexpressed with wild type TRPM4. Combined neutralization of all three acidic residues resulted in a functional channel whose voltage dependence was shifted toward very positive potentials. Substitution of Gln(977) by a glutamate, the corresponding residue in divalent cation-permeable TRPM channels, altered the monovalent cation permeability sequence and resulted in a pore with moderate Ca(2+) permeability. Our findings delineate the selectivity filter of TRPM channels and provide the first insight into the molecular basis of monovalent cation selectivity.     

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

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

Voltage-dependent cation channels permeable to NH(+)(4), K(+), and Ca(2+) in the symbiosome membrane of the model legume Lotus japonicus

The symbiosome of nitrogen fixing root nodules mediates metabolite exchange between endosymbiotic rhizobia bacteria and the legume host. In the present study, the ion currents of the symbiosome membrane of the model legume Lotus japonicus were analyzed by patch-clamp recording. Both excised and symbiosome-attached patches exhibited a large inward (toward the cytosolic side of the membrane) current that is activated in a time-dependent manner by negative (on the cytosolic side) potentials. Based on reversal potential determinations and recordings with the impermeant cation N-methyl-glucamine, this current shows a high permeability for monovalent cations with no apparent permeability for anions. The current also showed a finite Ca(2+) permeability. However, the currents were predominantly carried by univalent cations with a slightly greater selectivity for NH(4)(+) over K(+). Increased Ca(2+) concentration inhibited the current with a K(0.5) for inhibition of 0.317 mM. The current showed strong rectification that is mediated by divalent cations (either Mg(2+) or Ca(2+)). The influence of divalent cations is symmetrical in nature, because rectification can be exerted in either direction depending upon which side of the membrane has the highest concentration of divalent cations. However, based on observations with symbiosome-attached patches, the direction of the current in vivo is proposed to be toward the cytosol with cytosolic Mg(2+) acting as the putative gating regulator. The findings suggest that L. japonicus possesses a voltage-dependent cation efflux channel that is capable of exporting fixed NH(4)(+), and may also play an additional role in Ca(2+) transport.     

Self-incompatibility in Papaver rhoeas activates nonspecific cation conductance permeable to Ca2+ and K+

Cellular responses rely on signaling. In plant cells, cytosolic free calcium is a major second messenger, and ion channels play a key role in mediating physiological responses. Self-incompatibility (SI) is an important genetically controlled mechanism to prevent self-fertilization. It uses interaction of matching S-determinants from the pistil and pollen to allow "self" recognition, which triggers rejection of incompatible pollen. In Papaver rhoeas, the S-determinants are PrsS and PrpS. PrsS is a small novel cysteine-rich protein; PrpS is a small novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates S-specific increases in cytosolic free calcium and alterations in the actin cytoskeleton, resulting in programmed cell death in incompatible but not compatible pollen. Here, we have used whole-cell patch clamping of pollen protoplasts to show that PrsS stimulates SI-specific activation of pollen grain plasma membrane conductance in incompatible but not compatible pollen grain protoplasts. The SI-activated conductance does not require voltage activation, but it is voltage sensitive. It is permeable to divalent cations (Ba(2+) ≥ Ca(2+) > Mg(2+)) and the monovalent ions K(+) and NH(4)(+) and is enhanced at voltages negative to -100 mV. The Ca(2+) conductance is blocked by La(3+) but not by verapamil; the K(+) currents are tetraethylammonium chloride insensitive and do not require Ca(2+). We propose that the SI-stimulated conductance may represent a nonspecific cation channel or possibly two conductances, permeable to monovalent and divalent cations. Our data provide insights into signal-response coupling involving a biologically important response. PrsS provides a rare example of a protein triggering alterations in ion channel activity.     

Localization of divalent cation-binding site in the pore of a small conductance Ca(2+)-activated K(+) channel and its role in determining current-voltage relationship

In our previous study, we proposed that the inwardly rectifying current-voltage (I-V) relationship of small-conductance Ca(2+)-activated K(+) channels (SK(Ca) channels) is the result of voltage-dependent blockade of K(+) currents by intracellular divalent cations. We expressed a cloned SK(Ca) channel, rSK2, in Xenopus oocytes and further characterized the nature of the divalent cation-binding site by electrophysiological means. Using site-directed substitution of hydrophilic residues in K(+)-conducting pathway and subsequent functional analysis of mutations, we identified an amino acid residue, Ser-359, in the pore-forming region of rSK2 critical for the strong rectification of the I-V relationship. This residue interacts directly with intracellular divalent cations and determines the ionic selectivity. Therefore, we confirmed our proposition by localizing the divalent cation-binding site within the conduction pathway of the SK(Ca) channel. Because the Ser residue unique for the subfamily of SK(Ca) channels is likely to locate closely to the selectivity filter of the channels, it may also contribute to other permeation characteristics of SK(Ca) channels.     

Classification of voltage-dependent Ca2+ channels in trigeminal ganglion neurons from neonatal rats

We examined the subtypes and characteristics of the Ca(2+) channel in small (diameter < 30 microm) trigeminal ganglion (TG) neurons from neonatal rats by means of whole cell patch clamp techniques. There were two current components, low-voltage activated (LVA) and high-voltage activated (HVA) I(Ba), with different activation ranges and waveforms. LVA I(Ba) elicited from a depolarizing step pulse at a holding potential (HP) of -80 mV was inhibited by 0.25 mM amiloride (62%), which did not produce any significant inhibition of the peak amplitude of HVA I(Ba). The application of 0.5 mM amiloride inhibited 10% of the HVA I(Ba). The LVA I(Ba) was also reduced by changing the HP from -80 to -60 mV (61%), and under these conditions the peak amplitude of HVA I(Ba) did not change significantly. In addition, HVA I(Ba) and LVA I(Ba) showed marked differences in their inactivation properties. Experiments with several Ca(2+) channel blockers revealed that on average, 26% of the HVA I(Ba) was nifedipine (10 microM) sensitive, 55% was sensitive to omega-conotoxinGVIA (1 microM), 4% was blocked by omega-agatoxinIVA (1 microM), and the remainder of the current that was resistant to the co-application of all three Ca(2+) channel blockers was 15% of the total current. These results suggest that the application of amiloride and the alteration of the holding potential level can discriminate between HVA and LVA Ba(2+) currents in TG neurons, and that TG neurons expressed T-, L-, N-, P-/Q- and R-type Ca(2+) channels.     

Regulation of CRAC channel activity by recruitment of silent channels to a high open-probability gating mode

CRAC (calcium release-activated Ca(2+)) channels attain an extremely high selectivity for Ca(2+) from the blockade of monovalent cation permeation by Ca(2+) within the pore. In this study we have exploited the blockade by Ca(2+) to examine the size of the CRAC channel pore, its unitary conductance for monovalent cations, and channel gating properties. The permeation of a series of methylammonium compounds under divalent cation-free conditions indicates a minimum pore diameter of 3.9 A. Extracellular Ca(2+) blocks monovalent flux in a manner consistent with a single intrapore site having an effective K(i) of 20 microM at -110 mV. Block increases with hyperpolarization, but declines below -100 mV, most likely due to permeation of Ca(2+). Analysis of monovalent current noise induced by increasing levels of block by extracellular Ca(2+) indicates an open probability (P(o)) of approximately 0.8. By extrapolating the variance/mean current ratio to the condition of full blockade (P(o) = 0), we estimate a unitary conductance of approximately 0.7 pS for Na(+), or three to fourfold higher than previous estimates. Removal of extracellular Ca(2+) causes the monovalent current to decline over tens of seconds, a process termed depotentiation. The declining current appears to result from a reduction in the number of active channels without a change in their high open probability. Similarly, low concentrations of 2-APB that enhance I(CRAC) increase the number of active channels while open probability remains constant. We conclude that the slow regulation of whole-cell CRAC current by store depletion, extracellular Ca(2+), and 2-APB involves the stepwise recruitment of silent channels to a high open-probability gating mode.     

Ca2+ dependence of the Ca2+-selective TRPV6 channel

Microfluorimetry and patch-clamp experiments were performed on TRPV6-expressing HEK cells to determine whether this Ca(2+)-sensing Ca(2+) channel is constitutively active. Intact cells loaded with fura-2 had an elevated intracellular free Ca(2+) concentration ([Ca(2+)](i)), which decreased to the same level such as in non-transfected cells if external Ca(2+) was chelated by EGTA. Whole cell recordings from non-transfected HEK cells and cells expressing human TRPV6 revealed the presence of a basal inward current in both types of cells when the internal solution contained 0.1 mm EGTA and 100 nm [Ca(2+)](i) or if the cytosolic Ca(2+) buffering remained undisturbed in perforated patch-clamp experiments. If recombinantly expressed TRPV6 forms open channels, one would expect Ca(2+)-induced current inhibition, because TRPV6 is negatively regulated by internal Ca(2+). However, dialyzing solutions with high [Ca(2+)] such as 1 microm into TRPV6-expressing cells did not block the basal inward current, which was not different from the recordings from non-transfected cells. In contrast, dialyzing 0.5 mm EGTA into TRPV6-expressing cells readily activated Ca(2+) inward currents, which were undetectable in non-transfected cells. Interestingly, monovalent cations permeated the TRPV6 channels under conditions where no Ca(2+) permeation was detectable, indicating that divalent cations block TRPV6 channels from the extracellular side. Like human TRPV6, the truncated human TRPV6(Delta695-725), which lacks the C-terminal domain required for Ca(2+)-calmodulin binding, does not form constitutive active channels, whereas the human TRPV6(D542A), carrying a point mutation in the presumed pore region, does not function as a channel. In summary, no constitutive open TRPV6 channels were detected in patch-clamp experiments from transfected HEK cells. However, channel activity is highly regulated by intracellular and extracellular divalent cations.