Cys Palmitoylation of the β Subunit Modulates Gating of the Epithelial Sodium Channel

The epithelial Na⁺ channel (ENaC) is comprised of three homologous subunits (α, β, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the β and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant β subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a β C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na⁺ self-inhibition, and reduced single channel P_o when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that β Cys-43 is in proximity to the first transmembrane α helix, whereas β Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that β subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.     

Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel

The L-type Ca²⁺ channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α_1s or Ca_V1.1, α₂, β_1a, δ1, and γ), we created transgenic mice expressing a recombinant β_1a subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α₂-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca_V1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the β subunit in a single docking possibility that defines the α1-β interaction. The β subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α_1s subunit to the membrane and its suggested role in excitation-contraction coupling.     

Structures and Functions of Calcium Channel β Subunits

Calcium channel subunits have profound effects on how ₁ subunits perform. In this article we summarize our present knowledge of the primary structures of subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with ₁ subunits, the effects of subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by subunits, such as long-lasting prepulse facilitation of _1C (absent in _1E) and inhibition by G protein dimer of _1E, absent in _1C. One subunit, a brain 2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of ₁ in oocytes requires a subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca²⁺ channels to account for ₁ alone channels seen in cells with limited subunit expression. In one model, dissociates from the mature ₁ after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of would then have subunit composition ₁. In the other model, the chaperoning remains associated with the mature channel and ₁ alone channels would in fact be ₁ channels. Upon co-expression of high levels of the regulated channels would have composition [₁].     

Orientation of the Calcium Channel β Relative to the α12.2 Subunit Is Critical for Its Regulation of Channel Activity

Background

The Ca_vβ subunits of high voltage-activated Ca²⁺ channels control the trafficking and biophysical properties of the α₁ subunit. The Ca_vβ-α₁ interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that βs regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the α-interaction domain (AID) be a rigid structure.

Methodology/Principal Findings

The present study tests this hypothesis by altering the flexibility and orientation of this region in α₁2.2, then testing for Ca_vβ regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished β2a and β3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of β2a to produce non-inactivating currents. Orientation of Ca_vβ with respect to α₁2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca_vβ subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca_vβ subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms β-sheet. The orientation of β with respect to α was confirmed by the bimolecular fluorescence complementation assay.

Conclusions/Significance

These results show that the orientation of the Ca_vβ subunit relative to the α₁2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca_vβ to regulate channel activity.     

Subunit Regulation of the Human Brain α1E Calcium Channel

The α₁ subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α₂δ and β subunits. α_1E channels directed with the expression of Ba²⁺ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α_1E with α_2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α_1E with β_2a shifted the peak current relationship by −10 mV, and strongly reduced Ba²⁺ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba²⁺ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β_2a and the neuronal α_2bδ subunits increased by ≈10-fold whole-cell Ba²⁺ currents although coinjection with either β_2a or α_2bδ alone failed to significantly increase α_1E peak currents. Coexpression with β_2a and α_2bδ yielded Ba²⁺ currents with inactivation kinetics similar to the β_2a induced currents, indicating that the neuronal α_2bδ subunit has little effect on α_1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit genes. The slower inactivation was unique to α_1E/β_2a currents. Coexpression with β_1a, β_1b, β₃, and β₄, yielded faster-inactivating Ba²⁺ currents than currents recorded from the α_1E subunit alone. Furthermore, α_1E/α_2bδ/β_1a; α_1E/α_2bδ/β_1b; α_1E/α_2bδ/β₃; α_1E/α_2bδ/β₄ channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The β subunit-induced changes in the properties of α_1E channel were comparable to modulation effects reported for α_1C and α_1A channels with β₃≈β_1b > β_1a≈β₄≫β_2a inducing fastest to slowest rate of whole-cell inactivation. Received: 27 March 1997/Revised: 10 July 1997     

Genetic Background Influences P/Q-type Ca2+ Channel α1A Subunit mRNA Expression in Olfactory Bulb and Reproductive Ability of N-type Ca2+ Channel α1B Subunit-Deficient Mice

The Ca²⁺ channel α_1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although the N-type Ca²⁺ channel plays a role in a variety of neuronal functions, α_1B-deficient mice did not show apparent behavioral abnormality. In a previous study, we observed a compensatory increase of mRNA expression of the P/Q-type Ca²⁺ channel α_1A subunit gene in olfactory bulb of α_1B-deficient mice with a CBA × C57BL/6 background; these mice showed a normal reproductive ability. In this study, we found that the mRNA expression level of the α_1A subunit was the same in olfactory bulb of wild, heterozygous, and homozygous α_1B-deficient mice with a CBA/JN background, and the homozygous male mice produced no offspring. These results suggest that the genetic background influences α_1A subunit mRNA expression and reproductive ability in α_1B-deficient mice.     

Mapping of the Mouse Actin Capping Protein α Subunit Genes and Pseudogenes

Capping protein (CP), a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filamentsin vitroand controls actin assembly and cell motilityin vivo.Vertebrates have three α isoforms (α1, α2, α3) produced from different genes, whereas lower organisms have only one gene and one isoform. We isolated genomic clones corresponding to the α subunits of mouse CP and found three α1 genes, two of which are pseudogenes, and a single gene for both α2 and α3. Their chromosomal locations were identified by interspecies backcross mapping. The α1 gene (Cappa1) mapped to Chromosome 3 betweenD3Mit11andD3Mit13.The α1 pseudogenes (Cappa1-ps1andCappa1-ps2) mapped to Chromosomes 1 and 9, respectively. The α2 gene (Cappa2) mapped to Chromosome 6 nearPtn.The α3 gene (Cappa3) also mapped to Chromosome 6, approximately 68 cM distal fromCappa2nearKras2.One mouse mutation,de,maps in the vicinity of the α1 gene. No known mouse mutations map to regions near the α2 or α3 genes.     

How Does KCNE1 Regulate the Kv7.1 Potassium Channel? Model-Structure,Mutations, and Dynamics of the Kv7.1-KCNE1 Complex

The voltage-gated potassium channel Kv7.1 and its auxiliary subunit KCNE1 are expressed in the heart and give rise to the major repolarization current. The interaction of Kv7.1 with the single transmembrane helix of KCNE1 considerably slows channel activation and deactivation, raises single-channel conductance, and prevents slow voltage-dependent inactivation. We built a Kv7.1-KCNE1 model-structure. The model-structure agrees with previous disulfide mapping studies and enables us to derive molecular interpretations of electrophysiological recordings that we obtained for two KCNE1 mutations. An elastic network analysis of Kv7.1 fluctuations in the presence and absence of KCNE1 suggests a mechanistic perspective on the known effects of KCNE1 on Kv7.1 function: slow deactivation is attributed to the low mobility of the voltage-sensor domains upon KCNE1 binding, abolishment of voltage-dependent inactivation could result from decreased fluctuations in the external vestibule, and amalgamation of the fluctuations in the pore region is associated with enhanced ion conductivity.     

Recognition Mechanism of a Novel Gabapentinoid Drug,Mirogabalin, for Recombinant Human α2δ1, a Voltage-Gated Calcium Channel Subunit

Mirogabalin is a novel gabapentinoid drug with a hydrophobic bicyclo substituent on the γ-aminobutyric acid moiety that targets the voltage-gated calcium channel subunit α₂δ1. Here, to reveal the mirogabalin recognition mechanisms of α₂δ1, we present structures of recombinant human α₂δ1 with and without mirogabalin analyzed by cryo-electron microscopy. These structures show the binding of mirogabalin to the previously reported gabapentinoid binding site, which is the extracellular dCache_1 domain containing a conserved amino acid binding motif. A slight conformational change occurs around the residues positioned close to the hydrophobic group of mirogabalin. Mutagenesis binding assays identified that residues in the hydrophobic interaction region, in addition to several amino acid binding motif residues around the amino and carboxyl groups of mirogabalin, are critical for mirogabalin binding. The A215L mutation introduced to decrease the hydrophobic pocket volume predictably suppressed mirogabalin binding and promoted the binding of another ligand, L-Leu, with a smaller hydrophobic substituent than mirogabalin. Alterations of residues in the hydrophobic interaction region of α₂δ1 to those of the α₂δ2, α₂δ3, and α₂δ4 isoforms, of which α₂δ3 and α₂δ4 are gabapentin-insensitive, suppressed the binding of mirogabalin. These results support the importance of hydrophobic interactions in α₂δ1 ligand recognition.     

Cloning of the Fatty Acid Synthetase β Subunit from Fission Yeast, Coexpression with the α Subunit, and Purification of the Intact Multifunctional Enzyme Complex

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe)fas1⁺gene, which encodes the fatty acid synthetase (FAS) β subunit, by applying a PCR technique to conserved regions in the β subunit of the α₆β₆types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (M_r= 230,616), exhibits the 48.1% identity with the β subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the α subunit, the sequence of which was previously reported by Saitohet al.(S. Saitohet al.,1996,J. Cell Biol.134, 949–961), carries three domains. We have developed a co-expression system of the FAS α and β subunits by cotransformation of two expression vectors, containing thelsd1⁺/fas2⁺gene and thefas1⁺gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1–2.4 × 10⁶, and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex fromS. pombeforms a heterododecameric α₆β₆structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.     

Cloning and Characterization of Yeast LEU4, One of Two Genes Responsible for α-Isopropylmalate Synthesis

By complementation of an alpha-isopropylmalate synthase-negative mutant of Saccharomyces cerevisiae (leu4 leu5), a plasmid was isolated that carried a structural gene for alpha-isopropylmalate synthase. Restriction mapping and subcloning showed that sequences sufficient for complementation of the leu4 leu5 strain were located within a 2.2-kilobase SalI-PvuII segment. Southern transfer hybridization indicated that the cloned DNA was derived intact from the yeast genome. The cloned gene was identified as LEU4 by integrative transformation that caused gene disruption at the LEU4 locus. When this transformation was performed with a LEU4fbr LEU5 strain, the resulting transformants had lost the 5',5',5'-trifluoro-D,L-leucine resistance of the recipient strain but were still Leu+. When it was performed with a LEU4 leu5 recipient, the resulting transformants were Leu-. The alpha-isopropylmalate synthase of a transformant that carried the LEU4 gene on a multicopy plasmid (in a leu5 background) was characterized biochemically. The transformant contained about 20 times as much alpha-isopropylmalate synthase as wild type. The enzyme was sensitive to inhibition by leucine and coenzyme A, was inactivated by antibody generated against alpha-isopropylmalate synthase purified from wild type and was largely confined to the mitochondria. The subunit molecular weight was 65,000-67,000. Limited proteolysis generated two fragments with molecular weights of about 45,000 and 23,000. Northern transfer hybridization showed that the transformant produced large amounts of LEU4-specific RNA with a length of about 2.1 kilonucleotides. The properties of the plasmid-encoded enzyme resemble those of a previously characterized alpha-isopropylmalate synthase that is predominant in wild-type cells. The existence in yeast of a second alpha-isopropylmalate synthase activity that depends on the presence of an intact LEU5 gene is discussed.     

Molecular Evolution and Nucleotide Sequences of the Maize Plastid Genes for the α Subunit of Cf1 (atpA) and the Proteolipid Subunit of Cf0 (atpH)

The nucleotide sequences of the maize plastid genes for the alpha subunit of CF1 (atpA) and the proteolipid subunit of CF0 (atpH) are presented. The evolution of these genes among higher plants is characterized by a transition mutation bias of about 2:1 and by rates of synonymous and nonsynonymous substitution which are much lower than similar rates for genes from other sources. This is consistent with the notion that the plastid genome is evolving conservatively in primary sequence. Yet, the mode and tempo of sequence evolution of these and other plastid-encoded coupling factor genes are not the same. In particular, higher rates of nonsynonymous substitution in atpE (the gene for the epsilon subunit of CF1) and higher rates of synonymous substitution in atpH in the dicot vs. monocot lineages of higher plants indicate that these sequences are likely subject to different evolutionary constraints in these two lineages. The 5'- and 3'-transcribed flanking regions of atpA and atpH from maize, wheat and tobacco are conserved in size, but contain few putative regulatory elements which are conserved either in their spatial arrangement or sequence complexity. However, these regions likely contain variable numbers of "species-specific" regulatory elements. The present studies thus suggest that the plastid genome is not a passive participant in an evolutionary process governed by a more rapidly changing, readily adaptive, nuclear compartment, but that novel strategies for the coordinate expression of genes in the plastid genome may arise through rapid evolution of the flanking sequences of these genes.     

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca²⁺ channel complexes. Ca_Vα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type Ca_V1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant Ca_Vα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of Ca_Vα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the Ca_Vα2δ1-mediated increase in the peak current density and voltage-dependent gating of Ca_V1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored Ca_Vα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of Ca_Vα2δ1 as well as the Ca_Vα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of Ca_Vα2δ1, and furthermore that N-glycosylation of Ca_Vα2δ1 is essential to produce functional L-type Ca²⁺ channels.     

Structural Basis for Calcium and Magnesium Regulation of a Large Conductance Calcium-activated Potassium Channel with β1 Subunits

Large conductance Ca²⁺- and voltage-activated potassium (BK) channels, composed of pore-forming α subunits and auxiliary β subunits, play important roles in diverse physiological activities. The β1 is predominately expressed in smooth muscle cells, where it greatly enhances the Ca²⁺ sensitivity of BK channels for proper regulation of smooth muscle tone. However, the structural basis underlying dynamic interaction between BK mSlo1 α and β1 remains elusive. Using macroscopic ionic current recordings in various Ca²⁺ and Mg²⁺ concentrations, we identified two binding sites on the cytosolic N terminus of β1, namely the electrostatic enhancing site (mSlo1(K392,R393)-β1(E13,T14)), increasing the calcium sensitivity of BK channels, and the hydrophobic site (mSlo1(L906,L908)-β1(L5,V6,M7)), passing the physical force from the Ca²⁺ bowl onto the enhancing site and S6 C-linker. Dynamic binding of these sites affects the interaction between the cytosolic domain and voltage-sensing domain, leading to the reduction of Mg²⁺ sensitivity. A comprehensive structural model of the BK(mSlo1 α-β1) complex was reconstructed based on these functional studies, which provides structural and mechanistic insights for understanding BK gating.