Molecular determinants of permeation through the cation channel TRPV4

We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp(672) and Asp(682), as important determinants of the Ca(2+) sensitivity of the TRPV4 pore. Neutralization of either aspartate to alanine caused a moderate reduction of the relative permeability for divalent cations and of the degree of outward rectification. Neutralizing both aspartates simultaneously caused a much stronger reduction of Ca(2+) permeability and channel rectification and additionally altered the permeability order for monovalent cations toward Eisenman sequence II or I. Moreover, neutralizing Asp(682) but not Asp(672) strongly reduces the affinity of the channel for Ruthenium Red. Mutations to Met(680), which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca(2+) permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys(675), had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway.     

Identification of pore residues engaged in determining divalent cationic permeation in transient receptor potential melastatin subtype channel 2

The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca(2+) and Mg(2+) permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg(2+) permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca(2+) permeability along with concentration-dependent inhibition of Na(+)-carrying currents by Ca(2+). Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca(2+)-permeable TRPM channels, substantially increased Ca(2+) permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca(2+) permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.     

Mg2+-dependent gating and strong inward rectification of the cation channel TRPV6

TRPV6 (CaT1/ECaC2), a highly Ca(2+)-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular Mg(2+). Mg(2+) blocks the channel by binding to a site within the transmembrane electrical field where it interacts with permeant cations. The block is relieved at positive potentials, indicating that under these conditions Mg(2+) is able to permeate the selectivity filter of the channel. Although sizeable outward monovalent currents were recorded in the absence of intracellular Mg(2+), outward conductance is still approximately 10 times lower than inward conductance under symmetric, divalent-free ionic conditions. This Mg(2+)-independent rectification was preserved in inside-out patches and not altered by high intracellular concentrations of spermine, indicating that TRPV6 displays intrinsic rectification. Neutralization of a single aspartate residue within the putative pore loop abolished the Mg(2+) sensitivity of the channel, yielding voltage-independent, moderately inwardly rectifying monovalent currents in the presence of intracellular Mg(2+). The effects of intracellular Mg(2+) on TRPV6 are partially reminiscent of the gating mechanism of inwardly rectifying K(+) channels and may represent a novel regulatory mechanism for TRPV6 function in vivo.     

Outer pore architecture of a Ca2+-selective TRP channel

The TRP superfamily forms a functionally important class of cation channels related to the product of the Drosophila trp gene. TRP channels display an unusual diversity in activation mechanisms and permeation properties, but the basis of this diversity is unknown, as the structure of these channels has not been studied in detail. To obtain insight in the pore architecture of TRPV6, a Ca(2+)-selective member of the TRPV subfamily, we probed the dimensions of its pore and determined pore-lining segments using cysteine-scanning mutagenesis. Based on the permeability of the channel to organic cations, we estimated a pore diameter of 5.4 A. Mutating Asp(541), a residue involved in high affinity Ca(2+) binding, altered the apparent pore diameter, indicating that this residue lines the narrowest part of the pore. Cysteines introduced in a region preceding Asp(541) displayed a cyclic pattern of reactivity to Ag(+) and cationic methylthio-sulfanate reagents, indicative of a pore helix. The anionic methanethiosulfonate ethylsulfonate showed only limited reactivity in this region, consistent with the presence of a cation-selective filter at the outer part of the pore helix. Based on these data and on homology with the bacterial KcsA channel, we present the first structural model of a TRP channel pore. We conclude that main structural features of the outer pore, namely a selectivity filter preceded by a pore helix, are conserved between K(+) channels and TRPV6. However, the selectivity filter of TRPV6 is wider than that of K(+) channels and lined by amino acid side chains rather than main chain carbonyls.     

Molecular determinants of permeation through the cation channel TRPM6

TRPM6 and its closest relative TRPM7 are members of the Transient Receptor Potential Melastatin (TRPM) subfamily of cation channels and are known to be Mg2+ permeable. By aligning the sequence of the putative TRPM6 pore with the pore sequences of the other subfamily members, we located in the loop between the fifth and the sixth transmembrane domain, a stretch of amino acids residues, 1028GEIDVC1033, as the potential selectivity filter. Two negatively charged residues, E1024 (conserved in TRPM6, TRPM7, TRPM1 and TRPM3) and D1031 (conserved along the entire TRPM subfamily), were identified as important determinants of cation permeation through TRPM6, because neutralization of both residues into an alanine resulted in non-functional channels. Neutralization of E1029 (conserved in TRPM6, TRPM7, TRPM4 and TRPM5) resulted in channels with increased conductance for Ba2+ and Zn2+, decreased ruthenium red sensitivity and larger pore diameter compared to wild-type TRPM6. Changing the residue I1030 into methionine, resulted in channels with lower conductance for Ni2+, decreased sensitivity to ruthenium red block and reduced pore diameter. Thus, these data demonstrate that amino acid residues E1024, I1030 and D1031 are important for channel function and that subtle amino acid variation in the pore region accounts for TRPM6 permeation properties.     

A helix-breaking mutation in the epithelial Ca(2+) channel TRPV5 leads to reduced Ca(2+)-dependent inactivation

TRPV5, a member of transient receptor potential (TRP) superfamily of ion channels, plays a crucial role in epithelial calcium transport in the kidney. This channel has a high selectivity for Ca(2+) and is tightly regulated by intracellular Ca(2+) concentrations. Recently it was shown that the molecular basis of deafness in varitint-waddler mouse is the result of hair cell death caused by the constitutive activity of transient receptor potential mucolipin 3 (TRPML3) channel carrying a helix breaking mutation, A419P, at the intracellular proximity of the fifth transmembrane domain (TM5). This mutation significantly elevates intracellular Ca(2+) concentration and causes rapid cell death. Here we show that substituting the equivalent location in TRPV5, the M490, to proline significantly modulates Ca(2+)-dependent inactivation of TRPV5. The single channel conductance, time constant of inactivation (τ) and half maximal inhibition constant (IC(50)) of TRPV5(M490P) were increased compared to TRPV5(WT). Moreover TRPV5(M490P) showed lower Ca(2+) permeability. Out of different point mutations created to characterize the importance of M490 in Ca(2+)-dependent inactivation, only TRPV5(M490P)-expressing cells showed apoptosis and extremely altered Ca(2+)-dependent inactivation. In conclusion, the TRPV5 channel is susceptible for helix breaking mutations and the proximal intracellular region of TM5 of this channel plays an important role in Ca(2+)-dependent inactivation.     

TRPM5 is a voltage-modulated and Ca(2+)-activated monovalent selective cation channel

The TRPM subfamily of mammalian TRP channels displays unusually diverse activation mechanisms and selectivities. One member of this subfamily, TRPM5, functions in taste receptor cells and has been reported to be activated through G protein-coupled receptors linked to phospholipase C. However, the specific mechanisms regulating TRPM5 have not been described. Here, we demonstrate that TRPM5 is a monovalent-specific cation channel with a 23 pS unitary conductance. TRPM5 does not display constitutive activity. Rather, it is activated by stimulation of a receptor pathway coupled to phospholipase C and by IP(3)-mediated Ca(2+) release. Gating of TRPM5 was dependent on a rise in Ca(2+) because it was fully activated by Ca(2+). Unlike any previously described mammalian TRP channel, TRPM5 displayed voltage modulation and rapid activation and deactivation kinetics upon receptor stimulation. The most closely related protein, the Ca(2+)-activated monovalent-selective cation channel TRPM4b, also showed voltage modulation, although with slower relaxation kinetics than TRPM5. Taken together, the data demonstrate that TRPM5 and TRPM4b represent the first examples of voltage-modulated, Ca(2+)-activated, monovalent cation channels (VCAMs). The voltage modulation and rapid kinetics provide TRPM5 with an excellent set of properties for participating in signaling in taste receptors and other excitable cells.     

Molecular determinants of ion permeation and selectivity in inositol 1,4,5-trisphosphate receptor Ca2+ channels

We tested the hypothesis that key residues in a putative intraluminal loop contribute to determination of ion permeation through the intracellular Ca(2+) release channel (inositol 1,4,5-trisphosphate receptors (IP(3)Rs)) that is gated by the second messenger inositol 1,4,5-trisphosphate (IP(3)). To accomplish this, we mutated residues within the putative pore forming region of the channel and analyzed the functional properties of mutant channels using a (45)Ca(2+) flux assay and single channel electrophysiological analyses. Two IP(3)R mutations, V2548I and D2550E, retained the ability to release (45)Ca(2+) in response to IP(3). When analyzed at the single channel level; both recombinant channels had IP(3)-dependent open probabilities similar to those observed in wild-type channels. The mutation V2548I resulted in channels that exhibited a larger K(+) conductance (489 +/- 13 picosiemens (pS) for V2548I versus 364 +/- 5 pS for wild-type), but retained a Ca(2+) selectivity similar to wild-type channels (P(Ca(2+)):P(K(+)) approximately 4:1). Conversely, D2550E channels were nonselective for Ca(2+) over K(+) (P(Ca(2+)):P(K(+)) approximately 0.6:1), while the K(+) conductance was effectively unchanged (391 +/- 4 pS). These results suggest that amino acid residues Val(2548) and Asp(2550) contribute to the ion conduction pathway. We propose that the pore of IP(3)R channels has two distinct sites that control monovalent cation permeation (Val(2548)) and Ca(2+) selectivity (Asp(2550)).     

Potentiation of TRPM7 inward currents by protons

TRPM7 is unique in being both an ion channel and a protein kinase. It conducts a large outward current at +100 mV but a small inward current at voltages ranging from -100 to -40 mV under physiological ionic conditions. Here we show that the small inward current of TRPM7 was dramatically enhanced by a decrease in extracellular pH, with an approximately 10-fold increase at pH 4.0 and 1-2-fold increase at pH 6.0. Several lines of evidence suggest that protons enhance TRPM7 inward currents by competing with Ca(2+) and Mg(2+) for binding sites, thereby releasing blockade of divalent cations on inward monovalent currents. First, extracellular protons significantly increased monovalent cation permeability. Second, higher proton concentrations were required to induce 50% of maximal increase in TRPM7 currents when the external Ca(2+) and Mg(2+) concentrations were increased. Third, the apparent affinity for Ca(2+) and Mg(2+) was significantly diminished at elevated external H(+) concentrations. Fourth, the anomalous-mole fraction behavior of H(+) permeation further suggests that protons compete with divalent cations for binding sites in the TRPM7 pore. Taken together, it appears that at physiological pH (7.4), Ca(2+) and Mg(2+) bind to TRPM7 and inhibit the monovalent cationic currents; whereas at high H(+) concentrations, the affinity of TRPM7 for Ca(2+) and Mg(2+) is decreased, thereby allowing monovalent cations to pass through TRPM7. Furthermore, we showed that the endogenous TRPM7-like current, which is known as Mg(2+)-inhibitable cation current (MIC) or Mg nucleotide-regulated metal ion current (MagNuM) in rat basophilic leukemia (RBL) cells was also significantly potentiated by acidic pH, suggesting that MIC/MagNuM is encoded by TRPM7. The pH sensitivity represents a novel feature of TRPM7 and implies that TRPM7 may play a role under acidic pathological conditions.     

Cation channels: homing in on the elusive CAN channels

The molecular identity of Ca(2+)-activated, non-selective (CAN) cation channels has been unclear, but a member of the TRP family, TRPM4, has now been shown to be a CAN channel without significant Ca(2+) permeability.     

A calcium-permeable non-selective cation channel in the thick ascending limb apical membrane of the mouse kidney

Non-selective cation channels have been described in the basolateral membrane of the renal tubule, but little is known about functional channels on the apical side. Apical membranes of microdissected fragments of mouse cortical thick ascending limbs were searched for ion channels using the cell-free configuration of the patch-clamp technique. A cation channel with a linear current-voltage relationship (19pS) that was permeable both to monovalent cations [P(NH4)(1.7)>P(Na) (1.0)=P(K) (1.0)] and to Ca(2+) (P(Ca)/P(Na)≈0.3) was detected. Unlike the basolateral TRPM4 Ca(2+)-impermeable non-selective cation channel, this non-selective cation channel was insensitive to internal Ca(2+), pH and ATP. The channel was already active after patch excision, and its activity increased after reduced pressure was applied via the pipette. External gadolinium (10(-5)M) decreased the channel-open probability by 70% in outside-out patches, whereas external amiloride (10(-4)M) had no effect. Internal flufenamic acid (10(-4)M) inhibited the channel in inside-out patches. Its properties suggest that the current might be supported by the TRPM7 protein that is expressed in the loop of Henle. The conduction properties of the channel suggest that it could be involved in Ca(2+) signaling.     

Molecular determinant for specific Ca/Ba selectivity profiles of low and high threshold Ca2+ channels

Voltage-gated Ca(2+) channels (VGCC) play a key role in many physiological functions by their high selectivity for Ca(2+) over other divalent and monovalent cations in physiological situations. Divalent/monovalent selection is shared by all VGCC and is satisfactorily explained by the existence, within the pore, of a set of four conserved glutamate/aspartate residues (EEEE locus) coordinating Ca(2+) ions. This locus however does not explain either the choice of Ca(2+) among other divalent cations or the specific conductances encountered in the different VGCC. Our systematic analysis of high- and low-threshold VGCC currents in the presence of Ca(2+) and Ba(2+) reveals highly specific selectivity profiles. Sequence analysis, molecular modeling, and mutational studies identify a set of nonconserved charged residues responsible for these profiles. In HVA (high voltage activated) channels, mutations of this set modify divalent cation selectivity and channel conductance without change in divalent/monovalent selection, activation, inactivation, and kinetics properties. The Ca(V)2.1 selectivity profile is transferred to Ca(V)2.3 when exchanging their residues at this location. Numerical simulations suggest modification in an external Ca(2+) binding site in the channel pore directly involved in the choice of Ca(2+), among other divalent physiological cations, as the main permeant cation for VGCC. In LVA (low voltage activated) channels, this locus (called DCS for divalent cation selectivity) also influences divalent cation selection, but our results suggest the existence of additional determinants to fully recapitulate all the differences encountered among LVA channels. These data therefore attribute to the DCS a unique role in the specific shaping of the Ca(2+) influx between the different HVA channels.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca(2+) channels are important structural determinants for the passage of Ca(2+) across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a 1S subunit of the skeletal L-type channel (Ca(v)1.1) to lysine virtually eliminates passage of Ca(2+) during step depolarizations. In this study, we examined the ability of this mutant Ca(v)1.1 channel (SkEIIIK) to conduct inward Na(+) current. When 150 mM Na(+) was present as the sole monovalent cation in the bath solution, dysgenic (Ca(v)1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na(+). Ca(2+) block of SkEIIIK-mediated Na(+) current was revealed by the substantial enhancement of Na(+) current amplitude after reduction of Ca(2+) in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na(+) currents through the mutant Ca(v)1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na(+) channel when Na(+) is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca(2+) permeability mediated by Ca(v) channels on physiological processes that extend beyond channel gating and permeability.     

Sodium permeability of a cloned small-conductance calcium-activated potassium channel

Small-conductance Ca2+-activated potassium channels (SK(Ca) channels) are heteromeric complexes of pore-forming main subunits and constitutively bound calmodulin. SK(Ca) channels in neuronal cells are activated by intracellular Ca2+ that increases during action potentials, and their ionic currents have been considered to underlie neuronal afterhyperpolarization. However, the ion selectivity of neuronal SK(Ca) channels has not been rigorously investigated. In this study, we determined the monovalent cation selectivity of a cloned rat SK(Ca) channel, rSK2, using heterologous expression and electrophysiological measurements. When extracellular K+ was replaced isotonically with Na+, ionic currents through rSK2 reversed at significantly more depolarized membrane potentials than the value expected for a Nernstian relationship for K+. We then determined the relative permeability of rSK2 for monovalent cations and compared them with those of the intermediate- and large-conductance Ca2+-activated K+ channels, IK(Ca) and BK(Ca) channels. The relative permeability of the rSK2 channel was determined as K+(1.0)>Rb+(0.80)>NH(4)+(0.19) approximately Cs+(0.19)>Li+(0.14)>Na+(0.12), indicating substantial permeability of small ions through the channel. Although a mutation near the selectivity filter mimicking other K+-selective channels influenced the size-selectivity for permeant ions, Na+ permeability of rSK2 channels was still retained. Since the reversal potential of endogenous SK(Ca) current is determined by Na+ permeability in a physiological ionic environment, the ion selectivity of native SK(Ca) channels should be reinvestigated and their in vivo roles may need to be restated.     

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.     

CRACM1 multimers form the ion-selective pore of the CRAC channel

Receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) is often followed by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC) channels in the plasma membrane . RNAi screens have identified STIM1 as the putative ER Ca(2+) sensor and CRACM1 (Orai1; ) as the putative store-operated Ca(2+) channel. Overexpression of both proteins is required to reconstitute CRAC currents (I(CRAC); ). We show here that CRACM1 forms multimeric assemblies that bind STIM1 and that acidic residues in the transmembrane (TM) and extracellular domains of CRACM1 contribute to the ionic selectivity of the CRAC-channel pore. Replacement of the conserved glutamate in position 106 of the first TM domain of CRACM1 with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na(+), Ba(2+), and Sr(2+) permeation relative to Ca(2+). Mutating E190Q in TM3 also affects channel selectivity, suggesting that glutamate residues in both TM1 and TM3 face the lumen of the pore. Furthermore, mutating a putative Ca(2+) binding site in the first extracellular loop of CRACM1 (D110/112A) enhances monovalent cation permeation, suggesting that these residues too contribute to the coordination of Ca(2+) ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca(2+)-selective CRAC-channel pore.     

Modulation of Ca2+ entry and plasma membrane potential by human TRPM4b

TRPM4b is a Ca(2+)-activated, voltage-dependent monovalent cation channel that has been shown to act as a negative regulator of Ca(2+) entry and to be involved in the generation of oscillations of Ca(2+) influx in Jurkat T-lymphocytes. Transient overexpression of TRPM4b as an enhanced green fluorescence fusion protein in human embryonic kidney (HEK) cells resulted in its localization in the plasma membrane, as demonstrated by confocal fluorescence microscopy. The functionality and plasma membrane localization of overexpressed TRPM4b was confirmed by induction of Ca(2+)-dependent inward and outward currents in whole cell patch clamp recordings. HEK-293 cells stably overexpressing TRPM4b showed higher ionomycin-activated Ca(2+) influx than wild-type cells. In addition, analysis of the membrane potential using the potentiometric dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol and by current clamp experiments in the perforated patch configuration revealed a faster initial depolarization after activation of Ca(2+) entry with ionomycin. Furthermore, TRPM4b expression facilitated repolarization and thereby enhanced sustained Ca(2+) influx. In conclusion, in cells with a small negative membrane potential, such as HEK-293 cells, TRPM4b acts as a positive regulator of Ca(2+) entry.     

Permeation through store-operated CRAC channels in divalent-free solution: potential problems and implications for putative CRAC channel genes

CRAC channels are key calcium conduits in both physiological and pathological states. Understanding how these channels are controlled is important as this will not only provide insight into a novel signal transduction pathway coupling intracellular stores to the channels in the plasma membrane, but might also be of clinical relevance. Determining the molecular identity of the CRAC channels will certainly be a major step forward. Like all Ca(2+)-selective channels, CRAC channels lose their selectivity in divalent-free external solution to support large monovalent Na(+) currents. This approach has provided new insight into channel permeation and selectivity, and identifies some interesting differences between CRAC channels and voltage-operated calcium channels (VOCCs). Studies in divalent-free solution are a double-edged sword, however. Electrophysiologists need to be wary because some of the conditions used to study I(CRAC) in divalent-free external solution, notably omission of Mg(2+)/Mg-ATP from the recording pipette solution, activates an additional current permeating through Mg(2+)-nucleotide-regulated metal ion current (MagNuM; TRPM7) channels. This channel underlies the large single-channel events that have been attributed to CRAC channels in the past and which have been used to as a tool to identify store-operated channels in native cells and recombinant expression systems.Are we any closer to identifying the elusive CRAC channel gene(s)? TRPV6 seemed a very attractive candidate, but one of the main arguments supporting it was a single-channel conductance in divalent-free solution similar to that for CRAC reported under conditions where MagNuM is active. We now know that the conductance of TRPV6 is approximately 200-fold larger than that of CRAC in native tissue. Moreover, it is unclear if TRPV6 is store-operated. Further work on TRPV6, particularly whether its single-channel conductance is still high under conditions where it apparently forms multimers with endogenous store-operated channels, and whether it is activated by a variety of store depletion protocols, will be helpful in finally resolving this issue.     

Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation.

The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel.     

Role and regulation of TRP channels in neutrophil granulocytes

Members of the transient receptor potential (TRP) family for which mRNA can be demonstrated in neutrophil granulocytes with RT-PCR include TRPC6 (as only "short" TRP), TRPM2, TRPV1, TRPV2, TRPV5 and TRPV6. When these are analyzed in heterologous overexpression experiments, TRPM2 is the only cation channel with characteristic properties that can be used as fingerprint to provide functional evidence for its expression in neutrophil granulocytes. As cells transfected with TRPM2, neutrophil granulocytes display non-selective cation currents and typical channel activity evoked by intracellular ADP-ribose and NAD. Thus, stimulation of TRPM2 is likely to occur after activation of CD38 (producing ADP-ribose) and during the oxidative burst (enhancing the NAD concentration). This novel mode of cation entry regulation may be of particular importance for the response of granulocytes to chemoattractants. TRPV6 is a likely but not exclusive candidate as subunit of the channels mediating store-operated Ca2+ entry (SOCE). Evidence for SOCE in granulocytes has been presented with the fura-2 technique but not with electrophysiological methods although Ca2+-selective store-operated currents can be demonstrated in HL-60 cells, a cell culture model of neutrophil granulocytes.