A consensus segment in the M2 domain of the hP2X7 receptor shows ion channel activity in planar lipid bilayers and in biological membranes

The P2X₇ receptor (P2X₇R) is an ATP-gated, cation-selective channel permeable to Na⁺, K⁺ and Ca²⁺. This channel has also been associated with the opening of a non-selective pore that allows the flow of large organic ions. However, the biophysical properties of the P2X₇R have yet to be characterized unequivocally. We investigated a region named ADSEG, which is conserved among all subtypes of P2X receptors (P2XRs). It is located in the M2 domain of hP2X₇R, which aligns with the H5 signature sequence of potassium channels. We investigated the channel forming ability of ADSEG in artificial planar lipid bilayers and in biological membranes using the cell-attached patch-clamp techniques. ADSEG forms channels, which exhibit a preference for cations. They are voltage independent and show long-term stability in planar lipid bilayers as well as under patch-clamping conditions. The open probability of the ADSEG was similar to that of native P2X₇R. The conserved part of the M2 domain of P2X₇R forms ionic channels in planar lipid bilayers and in biological membranes. Its electrophysiological characteristics are similar to those of the whole receptor. Conserved and hydrophobic part of the M2 domain forms ion channels.     

Transmembrane segment M2 of glycine receptor as a model system for the pore-forming structure of ion channels

The glycine receptor belongs to the ligand-gated ion channel superfamily. It is a chloride conducting channel composed of four transmembrane domains. It was previously shown that the second transmembrane domain (M2) of the glycine receptor forms an ion conduction pathway throughout lipid bilayers. The amino-acid sequence of the transmembrane segment M2 of the glycine receptor has a high homology to all receptors of the ligand-gated ion channel superfamily. In our report, we have used a synthetic M2 peptide. It was incorporated into a planar membrane of known lipid composition and currents induced by M2 were measured by the Black Lipid Membrane technique. When the planar lipid bilayer was composed of 75% phosphatidylethanolamine and 25% phosphatidylserine, the reversal potential measured in a 150/600 mM KCl (cis/trans) gradient was -19 mV suggesting that the examined >pore was preferential to anions, P(K)/P(Cl) = 0.25. In contrast, when 75% phosphatidylserine and 25% phosphatidylethanolamine was used, the reversal potential was +20 mV and the >pore was preferential to cations, P(K)/P(Cl) = 4.36. Single-channel currents were recorded with two predominant amplitudes corresponding to the main-conductance and sub-conductance states. Both conductance states (about 12 pS and 30 pS) were measured in a symmetric solution of 50 mM KCl. The observed single-channel properties suggest that the selectivity and conductance of the pore formed by the M2 peptide of the glycine receptor depend on the lipid composition of the planar bilayer.     

Electrophysiological evidence for heptameric stoichiometry of ion channels formed by Staphylococcus aureus alpha-toxin in planar lipid bilayers

Staphylococcal alpha-toxin forms homo-oligomeric channels in lipid bilayers and cell membranes. Here, we report that electrophysiological monitoring of single-channel function using a derivatized cysteine substitution mutant allows accurate determination of the subunit stoichiometry of the oligomer in situ. The electrophysiological phenotype of channels formed in planar lipid bilayers with the cysteine replacement mutant I7C is equal to that of the wild type. When pores were formed with I7C, alterations of several channel properties were observed upon modification with SH reagents. Decreases in conductance then occurred that were seen only as negative voltage was applied. At the level of single channels, these were manifest as stepwise changes in conductance, each step most probably reflecting modification of a single SH group within the oligomer. Because seven steps were observed, the functional channel formed by alpha-toxin in planar lipid membranes is a heptamer.     

Location of the permeation pathway in the recombinant type 1 inositol 1,4,5-trisphosphate receptor.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) forms ligand-regulated intracellular Ca(2+) release channels in the endoplasmic reticulum of all mammalian cells. The InsP(3)R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP(3)R pore. Mutant InsP(3)Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; P(Ca)/P(Cs) = 6.3). These mutant channels bound InsP(3), but ligand occupancy did not regulate the constitutively open pore (P(o) > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398-2589) near the COOH terminus of the protein forms the InsP(3)R pore. Further, we have produced a constitutively open InsP(3)R pore mutant that is ideal for future site-directed mutagenesis studies of the structure-function relationships that define Ca(2+) permeation through the InsP(3)R channel.     

New scorpion toxins with a very high affinity for Na+ channels. Biochemical characterization and use for the purification of Na+ channels

Biochemical characterization of the Tityus gamma toxin receptor associated with the voltage-sensitive Na+ channel was carried out in different tissue preparations with the use of an iodinated toxin derivative. The affinity of the toxin for the receptor is high with a dissociation constant of 4 X 10(-12) M for rat synaptosomes. The density of binding sites is in the range of 0.3 to 2 pmol/mg of protein. Toxin gamma does not seem to bind to Na+ channels located on transverse-tubule membranes of skeletal muscle, but only to Na+ channels located on the sarcolemma. Both affinity labelling and radiation inactivation analysis indicate a molecular weight for the toxin receptor of 270 000 daltons. The same molecular weight is found using the tetrodotoxin. Only one single major protein component of the Na+ channel was purified from Electrophorus electroplax, rat brain membranes and chick heart membrane using the toxin gamma as a marker. The molecular weight of this component is 230 000-270 000 daltons. Reconstitution of the purified Na+ channel into planar lipid bilayers has been carried out. Two different types of electrically excitable channels with conductances of 150 and 25 pS were detected. The activity of both channels is blocked by saxitoxin.     

Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex

The purinergic receptor P2X(7) is part of a complex signaling mechanism participating in a variety of physiological and pathological processes. Depending on the activation scheme, P2X(7) receptors in vivo are non-selective cation channels or form large pores that can mediate apoptotic cell death. Expression of P2X(7)R in Xenopus oocytes results exclusively in formation of a non-selective cation channel. However, here we show that co-expression of P2X(7)R with pannexin1 in oocytes leads to the complex response seen in many mammalian cells, including cell death with prolonged ATP application. While the cation channel activity is resistant to carbenoxolone treatment, this gap junction and hemichannel blocking drug suppressed the currents induced by ATP in pannexin1/P2X(7)R co-expressing cells. Thus, pannexin1 appears to be the molecular substrate for the permeabilization pore (or death receptor channel) recruited into the P2X(7)R signaling complex.     

Beticolins, nonpeptidic, polycyclic molecules produced by the phytopathogenic fungus Cercospora beticola, as a new family of ion channel-forming toxins

Beticolins are toxins produced by Cercospora beticola, a phytopathogenic fungus responsible for the leaf spot disease of sugar beet. They form a family of 20 nonpeptidic compounds (named B0 to B19) that share the same polycyclic skeleton but differ by isomeric configuration (ortho- or para-) and by a variable residue R (bridging two carbons in one of the six cycles). It has been previously shown that B0 assembles itself into a multimeric structure and forms ion channels into planar lipid bilayers (C. Goudet, A.-A. Very, M.-L. Milat, M. Ildefonse, J.-B. Thibaud, H. Sentenac, and J.-P. Blein, Plant J. 14:359-364, 1998). In the present work, we investigate pore formation by three ortho-beticolins, B0, B2, and B4, and their related (i.e., same R) para-isomers, B13, B1, and B3, respectively, using planar lipid bilayers. All beticolins were able to form ion channels with multiple conductance states, although the type of cyclization (ortho- or para-) and residue (R) result in variations of channel conductance and ionic permeability, respectively. Channel formation by beticolins is likely to be involved in the biological activity of these toxins.     

Heterogeneity of calcium channels from a purified dihydropyridine receptor preparation.

Dihydropyridine receptors were purified from rabbit skeletal muscle transverse tubule membranes and incorporated into planar lipid bilayers. Calcium channels from both the purified dihydropyridine receptor preparation and the intact transverse tubule membranes exhibited two sizes of unitary currents, corresponding to conductances of 7 +/- 1 pS and 16 +/- 3 pS in 80 mM BaCl2. Both conductance levels were selective for divalent cations over monovalent cations and anions. Cadmium, an inorganic calcium channel blocker, reduced the single channel conductance of calcium channels from the purified preparation. The organic calcium channel antagonist nifedipine reduced the probability of a single channel being open with little effect on the single channel conductance. The presence of two conductance levels in both the intact transverse tubule membranes and the purified dihydropyridine receptor preparation suggests that the calcium channel may have multiple conductance levels or that multiple types of calcium channels with closely related structures are present in transverse tubule membranes.     

The Phytotoxic Lipodepsipeptide Syringopeptin 25A from Pseudomonas syringae pv syringae Forms Ion Channels in Sugar Beet Vacuoles

Syringopeptin 25A (SP(25)A) belongs to a family of cyclic lipodepsipeptides (LDPs) produced by the gram-negative bacterium Pseudomonas syringae, a phytopathogenic organism that affects several plants of agronomic interest. LDPs increase the permeability of plasma and, possibly, intracellular membranes in plant cells. Consistently, SP(25)A forms ion channels in planar lipid bilayers and other model membranes. Here we used sugar beet tonoplasts as a new biological model system to study toxin action. When applied to the vacuoles by a fast perfusion procedure, SP(25)A increases membrane permeability by forming discrete ion channels even at low applied potentials. The SP(25)A channel displays anion selectivity (with a Cl-/K+ permeability ratio of 6.7 +/- 1.3) and has intrinsic rectification properties that derive from a different channel conductance at negative and positive voltages, presumably owing to an asymmetric distribution of fixed charges on the pore. Substitution of chloride with different anions reveals the following selectivity sequence NO3- approximately Cl-> F- > gluconate-, suggesting that the permeation pore is filled with water. The properties of the SP(25)A channels in vacuolar membranes are similar to those observed in planar lipid membranes prepared with asolectin. This work provides a direct demonstration of toxin effects on a native plant membrane, extending to a biological system previous results obtained on artificial planar lipid membranes.     

Regulation of epithelial Na(+) channels by actin in planar lipid bilayers and in the Xenopus oocyte expression system

The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na(+) channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na(+) versus K(+) permselectivity, and a substantial increase in mean open and closed times of wild-type alphabetagamma-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the alpha subunit was deleted (alpha(R613X)betagamma-rENaC). 2) When alpha(R613X)betagamma-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either alpha(R613X)betagamma-rENaC or wild-type alphabetagamma-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that alpha-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.     

Cutting edge: a natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor

The P2X7 receptor (P2X(7)R) is an ATP-gated channel that mediates apoptosis of cells of the immune system. The capacity of P2X(7)R to form large pores depends on its large cytoplasmic tail, which harbors a putative TNFR-related death domain. Previous transfection studies indicated that mouse P2X(7)R forms pores much less efficiently than its counterparts from humans and rats. In this study, we demonstrate that an allelic mutation (P451L) in the predicted death domain of P2X(7)R confers a drastically reduced sensitivity to ATP-induced pore formation in cells from some commonly used strains of mice, i.e., C57BL/6 and DBA/2. In contrast, most other strains of mice, including strains derived from wild mice, carry P451 at this position as do rats and humans. The effects of the P451L mutation resemble those of the E496A mutation in human P2X(7)R. These P2X(7)R mutants may provide useful tools to decipher the molecular mechanisms leading to pore formation.     

Magainin 2 channel formation in planar lipid membranes: the role of lipid polar groups and ergosterol

Magainin 2, a polycationic peptide, displays bactericidal and tumoricidal activity, presumably interacting with negatively charged phospholipids in the membrane hosts. In this work, we investigate the role played by the lipid head-group in the interactions and self-association of magainin 2 during pore formation in lipid bilayers. Two methods are used: single-channel and macroscopic incorporation into planar lipid membranes. Single-channel incorporation showed that magainin 2 did not interact with zwitterionic membranes, while the addition of negatively charged dioleoylphosphatidylglycerol to the membrane leads to channel formation. On the other hand, magainin 2 did not form channels in membranes made up of dioleoylphosphatidylserine (DOPS), although the addition of ergosterol to DOPS membranes leads to channel formation. This finding could indicate that ergosterol may be a possible target of magainin 2 in fungal membranes. Further support for this hypothesis comes from experiments in which the addition of ergosterol to palmitoyloleoylphosphatidylcholine membranes induced channel formation. Besides the role of negatively charged membranes, this study has shown that magainin 2 also forms channels in membranes lacking heads, such as monoolein and oxidized cholesterol, indicating an interaction of magainin 2 with acyl chains and cholesterol, respectively. This finding provides further evidence that peptide binding and assembly in lipid membranes is a complex process driven by electrostatic and/or hydrophobic interactions, depending on the structure of the peptide and the membrane composition.     

Detection of caveolin-3/caveolin-1/P2X7R complexes in mice atrial cardiomyocytes in vivo and in vitro

Caveolae and caveolins, structural components of caveolae, are associated with specific ion channels in cardiac myocytes. We have previously shown that P2X purinoceptor 7 (P2X7R), a ligand-gated ion channel, is increased in atrial cardiomyocytes of caveolin-1 knockout mice; however, the specific biochemical relationship of P2X7R with caveolins in the heart is not clear. The aim of this work was to study the presence of the P2X7R in atrial cardiomyocytes and its biochemical relationship to caveolin-1 and caveolin-3. Caveolin isoforms and P2X7R were predominantly localized in buoyant membrane fractions (lipid rafts/caveolae) prepared from hearts using detergent-free sucrose gradient centrifugation. Caveolin-1 knockout mice showed normal distribution of caveolin-3 and P2X7R to buoyant membranes indicating the importance of caveolin-3 to formation of caveolae. Using clear native-PAGE, we showed that caveolin-1, -3 and P2X7R contribute to the same protein complex in the membranes of murine cardiomyocytes and in the immortal cardiomyocyte cell line HL-1. Western blot analysis revealed increased caveolin-1 and -3 proteins in tissue homogenates of P2X7R knockout mice. Finally, tissue homogenates of atrial tissues from caveolin-3 knockout mice showed elevated mRNA for P2X7R in atria. The colocalization of caveolins with P2X7R in a biochemical complex and compensated upregulation of P2X7R or caveolins in the absence of any component of the complex suggests P2X7R and caveolins may serve an important regulatory control point for disease pathology in the heart.     

Refolded outer membrane protein A of Escherichia coli forms ion channels with two conductance states in planar lipid bilayers

Outer membrane protein A (OmpA), a major structural protein of the outer membrane of Escherichia coli, consists of an N-terminal 8-stranded beta-barrel transmembrane domain and a C-terminal periplasmic domain. OmpA has served as an excellent model for studying the mechanism of insertion, folding, and assembly of constitutive integral membrane proteins in vivo and in vitro. The function of OmpA is currently not well understood. Particularly, the question whether or not OmpA forms an ion channel and/or nonspecific pore for uncharged larger solutes, as some other porins do, has been controversial. We have incorporated detergent-purified OmpA into planar lipid bilayers and studied its permeability to ions by single channel conductance measurements. In 1 M KCl, OmpA formed small (50-80 pS) and large (260-320 pS) channels. These two conductance states were interconvertible, presumably corresponding to two different conformations of OmpA in the membrane. The smaller channels are associated with the N-terminal transmembrane domain, whereas both domains are required to form the larger channels. The two channel activities provide a new functional assay for the refolding in vitro of the two respective domains of OmpA. Wild-type and five single tryptophan mutants of urea-denatured OmpA are shown to refold into functional channels in lipid bilayers.     

Single-channel function of recombinant type 2 inositol 1,4, 5-trisphosphate receptor

A full-length rat type 2 inositol 1,4,5-trisphosphate (InsP(3)) receptor cDNA construct was generated and expressed in COS-1 cells. Targeting of the full-length recombinant type 2 receptor protein to the endoplasmic reticulum was confirmed by immunocytochemistry using isoform specific affinity-purified antibodies and InsP(3)R-green fluorescent protein chimeras. The receptor protein was solubilized and incorporated into proteoliposomes for functional characterization. Single-channel recordings from proteoliposomes fused into planar lipid bilayers revealed that the recombinant protein formed InsP(3)- and Ca(2+)-sensitive ion channels. The unitary conductance ( approximately 250 pS; 220/20 mM Cs(+) as charge carrier), gating, InsP(3), and Ca(2+) sensitivities were similar to those previously described for the native type 2 InsP(3)R channel. However, the maximum open probability of the recombinant channel was slightly lower than that of its native counterpart. These data show that our full-length rat type 2 InsP(3)R cDNA construct encodes a protein that forms an ion channel with functional attributes like those of the native type 2 InsP(3)R channel. The possibility of measuring the function of single recombinant type 2 InsP(3)R is a significant step toward the use of molecular tools to define the determinants of isoform-specific InsP(3)R function and regulation.     

An Arg307 to Gln polymorphism within the ATP-binding site causes loss of function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X receptor have been observed, explained in part by (7)loss-of-function polymorphisms that change Glu(496) to Ala (E496A) and Ile(568) to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg(307) (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X(7) carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X(7) and blocks P2X(7) function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X(7) function in cells heterozygous for the R307Q to a value 10-40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X(7), and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X(7) receptor, and since the affected Arg(307) is homologous to those amino acids essential for ATP binding to P2X(1) and P2X(2), it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X(7).     

Single channel function of recombinant type-1 inositol 1,4,5-trisphosphate receptor ligand binding domain splice variants.

In this study we describe the expression and function of the two rat type-1 inositol 1,4,5-trisphosphate receptor (InsP3R) ligand binding domain splice variants (SI+/-/SII+). Receptor protein from COS-1 cells transfected with the type-1 InsP3R expression plasmids (pInsP3R-T1, pInsP3R-T1ALT) or control DNA were incorporated into planar lipid bilayers and the single channel properties of the recombinant receptors were defined. The unitary conductance of the two splice variants were approximately 290 pS with Cs+ as charge carrier and approximately 65 pS with Ca2+ as charge carrier. Both InsP3R expression products consistently behaved like those of the native type-1 receptor isoform isolated from cerebellum in terms of their InsP3, Ca2+, and heparin sensitivity. An InsP3 receptor ligand binding domain truncation lacking the 310 amino-terminal amino acids (pInsP3R-DeltaT1ALT) formed tetrameric complexes but failed to bind InsP3 with high affinity, and did not form functional Ca2+ channels when reconstituted in lipid bilayers. These data suggest that 1) the ligand binding alternative splice site is functionally inert in terms of InsP3 binding and single channel function, and 2) the single channel properties of the expressed recombinant type-1 channel are essentially identical to those of the native channel. This work establishes a foundation from which molecular/biophysical approaches can be used to define the structure-function properties of the InsP3 receptor channel family.     

Purification and characterization of two voltage-dependent anion channel isoforms from plant seeds

Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 M urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates.     

Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.     

Regulation of calcium channel activity by lipid domain formation in planar lipid bilayers

The sarcoplasmic reticulum channel (ryanodine receptor) from cardiac myocytes was reconstituted into planar lipid bilayers consisting of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) in varying ratios. The channel activity parameters, i.e., open probability and average open time and its resolved short and long components, were determined as a function of POPE mole fraction (X(PE)) at 22.4 degrees C. Interestingly, all of these parameters exhibited a narrow and pronounced peak at X(PE) approximately 0.80. Differential scanning calorimetric measurements on POPE/POPC liposomes with increasing X(PE) indicated that the lipid bilayer enters a composition-driven transition from the liquid-crystalline state to the gel state at 22.4 degrees C when X(PE) approaches 0.80. Thus, the peaking of the reconstituted channel activity at X(PE) approximately 0.80 in the planar bilayer could result from the appearance of gel/liquid-crystalline domain boundaries at this POPE content. Lipid packing at domain boundaries is known to be looser as compared to the homogenous gel or liquid-crystalline state. We propose that the attractive potential of packing defects at lipid domain boundaries and entropic excluded-volume effects could result in the direct interactions of the transmembrane region of the channel protein with the lipid-packing defects at the lipid/protein interface, which could thus provide a favorable environment for the open state of the protein. The present findings indicate that the activity of the sarcoplasmic reticulum calcium channel could be modulated by lipid domain formation upon slight changes in membrane lipid composition in vivo.