Large Cytoplasmic Domain Research Articles

Structural basis for the activity regulation of Salt Overly Sensitive 1 in Arabidopsis salt tolerance.

The plasma membrane Na+/H+ exchanger Salt Overly Sensitive 1 (SOS1) is crucial for plant salt tolerance. Unlike typical sodium/proton exchangers, SOS1 contains a large cytoplasmic domain (CPD) that regulates Na+/H+ exchange activity. However, the underlying modulation mechanism remains unclear. Here we report the structures of SOS1 from Arabidopsis thaliana in two conformations, primarily differing in CPD flexibility. The CPD comprises an interfacial domain, a cyclic nucleotide-binding domain-like domain (CNBD-like domain) and an autoinhibition domain. Through yeast cell-based Na+ tolerance test, we reveal the regulatory role of the interfacial domain and the activation role of the CNBD-like domain. The CPD forms a negatively charged cavity that is connected to the ion binding site. The transport of Na+ may be coupled with the conformational change of CPD. These findings provide structural and functional insight into SOS1 activity regulation.

Bacterial cellulose: A highly versatile nanomaterial.

Bacterial cellulose: A highly versatile nanomaterial.

Defining the interactions between HCN4 and the regulatory proteins IRAG and LRMP.

Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are the molecular determinants of the funny (If) current, a mixed Na+/K+ current that controls pacemaking in cardiac cells. In the sinoatrial node (SAN), an increase in If in response to cAMP contributes to an increase in action potential (AP) firing rate and consequently heart rate. We recently discovered two related modulators of HCN4 function: lymphoid-restricted membrane protein (LRMP) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG). Despite homology between these two proteins, LRMP inhibits the depolarizing shift in the voltage dependence of activation of HCN4 in response to cAMP while IRAG shifts the basal voltage dependence in the depolarizing direction. Even with a clear picture of the functional effects that these two proteins exert on HCN4, the domains responsible for the interactions are completely unknown. Here, we pair a FRET two-hybrid approach with patch clamp electrophysiology to determine which domains of LRMP, IRAG, and HCN4 are involved in complex formation. Both LRMP and IRAG have small ER luminal domains, ER transmembrane segments, and a large cytoplasmic domain that likely interacts with an intracellular portion of HCN4 channels. By co-expressing cerulean-tagged cytosolic fragments of HCN4 with citrine-tagged fragments of LRMP and IRAG, we determine which regions on each protein are required for complex formation using FRET. We then test the functional effect of removing these critical domains on the regulation of the channel by each protein using patch clamp electrophysiology. Our preliminary results suggest that the N-terminus of HCN4 is critical for both LRMP and IRAG regulation of the channel. Moreover, the first 230 amino acids of LRMP is sufficient to bind HCN4. These results shed light on a novel amino terminus-dependent mechanism for regulating the gating of HCN4 channels.

Targeted deep sequencing analyses of long QT syndrome in a Japanese population.

Long QT syndrome (LQTS) is one of the most common inherited arrhythmias and multiple genes have been reported as causative. Presently, genetic diagnosis for LQTS patients is becoming widespread and contributing to implementation of therapies. However, causative genetic mutations cannot be detected in about 20% of patients. To elucidate additional genetic mutations in LQTS, we performed deep-sequencing of previously reported 15 causative and 85 candidate genes for this disorder in 556 Japanese LQTS patients. We performed in-silico filtering of the sequencing data and found 48 novel variants in 33 genes of 53 cases. These variants were predicted to be damaging to coding proteins or to alter the binding affinity of several transcription factors. Notably, we found that most of the LQTS-related variants in the RYR2 gene were in the large cytoplasmic domain of the N-terminus side. They might be useful for screening of LQTS patients who had no known genetic factors. In addition, when the mechanisms of these variants in the development of LQTS are revealed, it will be useful for early diagnosis, risk stratification, and selection of treatment.

Scavenger receptor class F member 2 (SCARF2) as a novel therapeutic target in glioblastoma.

Scavenger receptor class F member 2 (SCARF2) is expressed by endothelial cells with very large cytoplasmic domains and is the second isotype, also known as scavenger receptor expressed by endothelial cells 2 (SREC-2). SREC-1 plays an important role in the binding and endocytosis of various endogenous and exogenous ligands. Many studies have been carried out on modified low-density lipoprotein internalization activity, but there have been few studies on SCARF2. Higher expression of SCARF2 has been found in glioblastoma (GBM) than normal brain tissue. Through analysis of The Cancer Genome Atlas database, it was confirmed that SCARF2 is widely expressed in GBM, and increased SCARF2 expression correlated with a poor prognosis in patients with glioma. The results of this study showed that the expression of SCARF2 is increased in GBM cell lines and patients, suggesting that SCARF2 may be a potential diagnostic marker and therapeutic molecule for cancers including glioma.

How Structural Biology Has Directly Impacted Our Understanding of P2X Receptor Function and Gating.

P2X receptors are ATP-gated ion channels expressed in a wide variety of eukaryotic cells. They play key roles in diverse processes such as platelet activation, smooth muscle contraction, synaptic transmission, nociception, cell proliferation, and inflammation making this receptor family an important pharmacological target. Structures of P2X receptors solved by X-ray crystallography have been instrumental in helping to define mechanisms of molecular P2X receptor function. In 2009, the first X-ray structure of the P2X4 receptor subtype confirmed a trimeric stoichiometry and revealed the overall architecture of the functional ion channel. Subsequent X-ray structures have provided the molecular details to define the orthosteric ATP binding pocket, the orthosteric antagonist binding pocket, an allosteric antagonist binding pocket, and the pore architecture in each of the major conformational states of the receptor gating cycle. Moreover, the unique gating mechanism by which P2X receptor subtypes desensitize at differing rates, referred to as the helical recoil model of receptor desensitization, was discovered directly from X-ray structures of the P2X3 receptor. However, structures of P2X receptors solved by X-ray crystallography have only been able to provide limited information on the cytoplasmic domain of this receptor family, as this domain was always truncated to varying degrees in order to facilitate crystallization. Because the P2X7 receptor subtype has a significantly larger cytoplasmic domain that has been shown to be necessary for its ability to initiate apoptosis, an absence of structural information on the P2X7 receptor cytoplasmic domain has limited our understanding of its complex signaling pathways as well as its unusual ability to remain open without undergoing desensitization. This absence of cytoplasmic structural information for P2X7 receptors was recently overcome when the first full-length P2X7 receptor structures were solved by single-particle cryogenic electron microscopy. These structures finally provide insight into the large and unique P2X7 receptor cytoplasmic domain and revealed two novel structural elements and several surprising findings: first, a cytoplasmic structural element called the cytoplasmic ballast was identified that contains a dinuclear zinc ion complex and a high affinity guanosine nucleotide binding site and second, a palmitoylated membrane proximal structural element called the C-cys anchor was identified which prevents P2X7 receptor desensitization. This chapter will highlight the major structural and functional aspects of P2X receptors discovered through structural biology, with a key emphasis on the most recent cryogenic electron microscopy structures of the full-length, wild-type P2X7 receptor.

A Simplified Protocol to Incorporate the Fluorescent Unnatural Amino Acid ANAP into Xenopus laevis Oocyte-Expressed P2X7 Receptors.

The long intracellular P2X7 C-terminus accounts for diverse downstream effects of P2X7 activation. Although the recent determination of the cryo-EM structure of the full-length P2X7 receptor finally revealed the structure and several unexpected features of the large cytoplasmic domain, its molecular function remains enigmatic. Incorporation of unnatural amino acids (UAA) via an amber Stop codon has been a powerful tool for structure-function analysis of proteins. Voltage clamp fluorometry (VCF) with the fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) provides a means to study intracellular domain movements of ion channel receptors. In the Xenopus laevis oocyte expression system, site-specific introduction of this environment-sensitive fluorophore can be achieved by the nuclear injection of cDNA encoding an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair and subsequent cytoplasmic injection of ANAP together with the respective cRNA containing the amber Stop codon. Here, we describe this protocol for expression of ANAP-labeled P2X7. In addition, we provide a simplified alternative protocol, in which we coinject cRNAs encoding the tRNA synthetase and mutant P2X7 together with the synthesized amber suppressor tRNA and ANAP in one step into the cytosol. We found that the new protocol yielded more reproducible results and was less harmful for the oocytes. By selective fluorescence labeling of the ANAP-labeled P2X7 protein in the oocyte plasma membrane and VCF recordings, we show that this method results in comparable levels of functional ANAP-labeled P2X7 protein.

Specific residues in the cytoplasmic domain modulate photocurrent kinetics of channelrhodopsin from Klebsormidium nitens

Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. All currently known ChRs comprise a large cytoplasmic domain, whose function is elusive. Here, we report the cation channel properties of KnChR, one of the photoreceptors from a filamentous terrestrial alga Klebsormidium nitens, and demonstrate that the cytoplasmic domain of KnChR modulates the ion channel properties. KnChR is constituted of a 7-transmembrane domain forming a channel pore, followed by a C-terminus moiety encoding a peptidoglycan binding domain (FimV). Notably, the channel closure rate was affected by the C-terminus moiety. Truncation of the moiety to various lengths prolonged the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. We propose that electrostatic interaction between the rhodopsin domain and the C-terminus domain accelerates the channel kinetics. Additionally, maximal sensitivity was exhibited at 430 and 460 nm, the former making KnChR one of the most blue-shifted ChRs characterized thus far, serving as a novel prototype for studying the molecular mechanism of color tuning of the ChRs. Furthermore, KnChR would expand the optogenetics tool kit, especially for dual light applications when short-wavelength excitation is required.

Isoform-specific regulation of HCN4 channels by a family of endoplasmic reticulum proteins

Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, and Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage dependence of HCN4 in response to the binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage dependence in the absence of cAMP. The mechanisms of action of LRMP and IRAG are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 current. Our results suggest important roles for LRMP and IRAG in the regulation of cellular excitability, as tools for advancing mechanistic understanding of HCN4 channel function, and as possible scaffolds for coordination of signaling pathways.

Coupling of Ca2+ and voltage activation in BK channels through the αB helix/voltage sensor interface

Large-conductance Ca2+ and voltage-activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high-affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved.

The SRP signal sequence of KdpD

KdpD is a four-spanning membrane protein that has two large cytoplasmic domains at the amino- and at the carboxyterminus, respectively. During its biogenesis KdpD binds to the signal recognition particle (SRP) of Escherichia coli that consists of a 48-kDa protein Ffh and a 4.5S RNA. The protein is targeted to the inner membrane surface and is released after contacting the SRP receptor protein FtsY. The information within the KdpD protein that confers SRP interaction was found in the amino-terminal cytoplasmic domain of KdpD, particularly at residues 22–48. Within this sequence a Walker A motif is present at residues 30–38. To determine the actual sequence specificity to SRP, a collection of mutants was constructed. When the KdpD peptides (residues 22–48) were fused to sfGFP the targeting to the membrane was observed by fluorescence microscopy. Further, nascent chains of KdpD bound to ribosomes were purified and their binding to SRP was analysed by microscale thermophoresis. We found that the amino acid residues R22, K24 and K26 are important for SRP interaction, whereas the residues G30, G34 and G36, essential for a functional Walker A motif, can be replaced with alanines without affecting the affinity to SRP-FtsY and membrane targeting.

Characterization of Temperature-Dependent Gating in Archaebacterial Calcium Activated Potassium Channel

MthK is an archaebacterial K+ channel, which is gated by large cytoplasmic RCK domains that facilitate Ca2+-activation of the channel. This bacterial channel is a good model for studying the mechanisms underlying calcium dependent gating in ion channels because it tractable to both structural and functional studies. An earlier study has also suggested that the calcium-dependent gating of MthK is also regulated by temperature (Parfenova, L. V. et al. (2006). J Biol Chem 281, 21131-21138). Here, using inside-out patch-clamping recording of inactivation removed MthK from spheroplasts, I have characterized the temperature-dependence of MthK channel at varying Ca2+ concentrations. Our preliminary data shows that the activation of this inactivation-removed channel is highly temperature-dependent. We have also purified and reconstituted this inactivation removed MthK in soybean polar lipids and found similar temperature-dependence albeit at lower concentrations of calcium. This suggests that the temperature-dependence of MthK is intrinsic. Furthermore, allosteric analysis of electrophysiological data shows that temperature unexpectedly modulates coupling between Ca2+ sensor and the pore rather than acting through a completely independent allosteric domain.

Length of KASH Domains Affect Linc Complex Functions

The linker of the nucleoskeleton and cytoskeleton (LINC) complexes are composed of inner nuclear membrane Sad1/UNC-84 (SUN, and outer nuclear membrane Klarsicht/ANC-1/SYNE homology (KASH) proteins. LINC complexes span the physical barrier of the nuclear envelope and regulate the physical connection between the interior of the nucleus and the cytoplasm during various cellular functions. The large cytoplasmic domains of KASH proteins bind to various elements of the cytoskeleton, whereas their 10-30 amino acid KASH domains reside in the perinuclear space (PNS) where they bind to SUN proteins. Interestingly, different KASH domain proteins can independently bind to the same SUN protein to mediate distinct cellular functions. For example, in mammals the SUN protein SUN1 can transiently associate with KASH5 in the PNS to mediate meiotic chromosome movement. Alternatively, SUN1 can bind to Nesprin1/2 to mediate nuclear movement. Similarly, in Caenorhabditis elegans (C. elegans) SUN protein UNC-84 transiently binds to KASH protein UNC-83 in embryonic hypodermal cells to mediate nuclear migration. Later, the same SUN protein UNC-84 independently binds to a different KASH protein ANC-1 to anchor of the nucleus in place for several days. In this work, we combined in vivo C. elegans genetics and in silico molecular dynamics simulations to explore the role of KASH domain length on the function of the SUN-KASH complex and its response to mechanical forces. Our results imply that longer KASH domains can withstand and transfer higher forces across the SUN-KASH complex. Additionally, our results suggest that longer KASH domains can interact with the membrane through a conserved membrane proximal EEDY domain that is unique to longer KASH domains. Finally, we show that swapping the KASH domains of ANC-1 and UNC-83, or shortening the KASH domain of ANC-1, both result in a nuclear anchorage defect in C. elegans.

Function, Structure, and Transport Aspects of ZIP and ZnT Zinc Transporters in Immune Cells.

Zinc is an important trace metal in immune systems, and zinc transporters are involved in many immune responses. Recent advances have revealed the structural and biochemical bases for zinc transport across the cell membrane, with clinical implications for the regulation of zinc homeostasis in immune cells like dendritic cells, T cells, B cells, and mast cells. In this review, we discuss the function, structure, and transport aspects of two major mammalian zinc transporter types, importers and exporters. First, Zrt-/Irt-like proteins (ZIPs) mediate the zinc influx from the extracellular or luminal side into the cytoplasm. There are 14 ZIP family members in humans. They form a homo- or heterodimer with 8 transmembrane domains and extra-/intracellular domains of various lengths. Several ZIP members show specific extracellular domains composed of two subdomains, a helix-rich domain and proline-alanine-leucine (PAL) motif-containing domain. Second, ZnT (zinc transporter) was initially identified in early studies of zinc biology; it mediates zinc efflux as a counterpart of ZIPs in zinc homeostasis. Ten family members have been identified. They show a unique architecture characterized by a Y-shaped conformation and a large cytoplasmic domain. A precise, comprehensive understanding of the structures and transport mechanisms of ZIP and ZnT in combination with mice experiments would provide promising drug targets as well as a basis for identifying other transporters with therapeutic potential.

Loss of cytosolic Mg2+ binding sites in the Thermotoga maritima CorA Mg2+ channel is not sufficient for channel opening

The CorA Mg2+ channel is a homopentamer with five-fold symmetry. Each monomer consists of a large cytoplasmic domain and two transmembrane helices connected via a short periplasmic loop. In the Thermotoga maritima CorA crystal structure, a Mg2+ is bound between D89 of one monomer and D253 of the adjacent monomer (M1 binding site). Release of Mg2+ from these sites has been hypothesized to cause opening of the channel. We generated mutants to disrupt Mg2+ interaction with the M1 site. Crystal structures of the D89K/D253K and D89R/D253R mutants, determined to 3.05 and 3.3 Å, respectively, showed no significant structural differences with the wild type structure despite absence of Mg2+ at the M1 sites. Both mutants still appear to be in the closed state. All three mutant CorA proteins exhibited transport of 63Ni2+, indicating functionality. Thus, absence of Mg2+ from the M1 sites neither causes channel opening nor prevents function. We also provide evidence that the T. maritima CorA is a Mg2+ channel and not a Co2+ channel.

Structural basis for the regulation of inositol trisphosphate receptors by Ca2+ and IP3.

Inositol trisphosphate receptors (IP3R) are ubiquitous Ca2+-permeable channels that mediate release of Ca2+ from the endoplasmic reticulum to regulate numerous processes including cell division, cell death, differentiation and fertilization. IP3R is activated by both IP3 and its permeant ion Ca2+. At high concentrations, however, Ca2+ inhibits activity ensuring precise spatiotemporal control over intracellular Ca2+. Despite extensive characterization of IP3R, the mechanisms by which these molecules control channel gating have remained elusive. Here, we present structures of full-length human type 3 IP3R in ligand-bound and ligand-free states. Multiple IP3-bound structures demonstrate that the large cytoplasmic domain provides a platform for propagation of long-range conformational changes to the ion conduction gate. Structures in the presence of Ca2+ reveal two Ca2+ binding sites that induce the disruption of numerous interactions between subunits, thereby inhibiting IP3R. These structures thus begin to provide a mechanistic basis for understanding the regulation of IP3R.

In vivo assembly of a truncated H subunit mutant of the Rhodobacter sphaeroides photosynthetic reaction centre and direct electron transfer from the QA quinone to an electrode.

We address a challenge in the engineering of proteins to redirect electron transfer pathways, using the bacterial photosynthetic reaction centre (RC) pigment-protein complex. Direct electron transfer is shown to occur from the QA quinone of the Rhodobacter sphaeroides RC containing a truncated H protein and bound on the quinone side to a gold electrode. In previous reports of binding to the quinone side of the RC, electron transfer has relied on the use of a soluble mediator between the RC and an electrode, in part because the probability of QB quinone reduction is much greater than that of direct electron transfer through the large cytoplasmic domain of the H subunit, presenting a ~ 25Å barrier. A series of C-terminal truncations of the H subunit were created to expose the quinone region of the RC L and M proteins, and all truncated RC H mutants assembled in vivo. The 45M mutant was designed to contain only the N-terminal 45 amino acid residues of the H subunit including the membrane-spanning α-helix; the mutant RC was stable when purified using the detergent N-dodecyl-β-D-maltoside, contained a near-native ratio of bacteriochlorophylls to bacteriopheophytins, and showed a charge-separated state of [Formula: see text]. The 45M-M229 mutant RC had a Cys residue introduced in the vicinity of the QA quinone on the newly exposed protein surface for electrode attachment, decreasing the distance between the quinone and electrode to ~ 12Å. Steady-state photocurrents of up to around 200nA/cm2 were generated in the presence of 20mM hydroquinone as the electron donor to the RC. This novel configuration yielded photocurrents orders of magnitude greater than previous reports of electron transfer from the quinone region of RCs bound in this orientation to an electrode.

A Cytoplasmic Antiholin Is Embedded In Frame with the Holin in a Lactobacillus fermentum Bacteriophage.

In double-stranded DNA bacteriophages, infection cycles are ended by host cell lysis through the action of phage-encoded endolysins and holins. The precise timing of lysis is regulated by the holin inhibitors, named antiholins. Sequence analysis has revealed that holins with a single transmembrane domain (TMD) are prevalent in Lactobacillus bacteriophages. A temperate bacteriophage of Lactobacillus fermentum, ϕPYB5, has a two-component lysis cassette containing endolysin Lyb5 and holin Hyb5. The hyb5 gene is 465 bp long, encoding 154 amino acid residues with an N-terminal TMD and a large cytoplasmic C-terminal domain. However, the N terminus contains no dual-start motif, suggesting that Hyb5 oligomerization could be inhibited by a specific antiholin. Two internal open reading frames in hyb5, hyb5157-465 and hyb5209-328, were identified as genes encoding putative antiholins for Hyb5 and were coexpressed in trans with lyb5-hyb5 in Escherichia coli Surprisingly, host cell lysis was delayed by Hyb5157-465 but accelerated by abolishment of the translation initiation site of this protein, indicating that Hyb5157-465 acts as an antiholin to holin Hyb5. Moreover, deletion of 45 amino acid residues at the C terminus of Hyb5 resulted in early cell lysis, even in the presence of Hyb5157-465, implying that the interaction between Hyb5157-465 and Hyb5 occurs at the C terminus of the holin. In vivo and in vitro, Hyb5157-465 and Hyb5 were detected in the cytoplasmic and membrane fractions, respectively, and pulldown assays confirmed direct interaction between Hyb5157-465 and Hyb5. All the results suggest that Hyb5157-465 is an antiholin of Hyb5 that is involved in lysis timing.IMPORTANCE Phage-encoded holins are considered to be the "molecular clock" of phage infection cycles. The interaction between a holin and its inhibitor antiholin precisely regulates the timing of lysis of the host cells. As a prominent biological group in dairy processes, phages of lactic acid bacteria (LAB) have been extensively genome sequenced. However, little is known about the antiholins of LAB phage holins and the holin-antiholin interactions. In this work, we identified an in-frame antiholin against the class III holin of Lactobacillus fermentum phage ϕPYB5, Hyb5, and demonstrated its interaction with the cognate holin, which occurred in the bacterial cytoplasm.

Steric exclusion and protein conformation determine the localization of plasma membrane transporters

The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to cytosolic and luminal Ca2+

The ryanodine receptor ion channel RyR1 is present in skeletal muscle and has a large cytoplasmic N-terminal domain and smaller C-terminal pore-forming domain comprising six transmembrane helices, a pore helix, and a selectivity filter. The RyR1 S6 pore-lining helix has two conserved glycines, Gly-4934 and Gly-4941, that facilitate RyR1 channel gating by providing S6 flexibility and minimizing amino acid clashes. Here, we report that substitution of Gly-4941 with Asp or Lys results in functional channels as indicated by caffeine-induced Ca2+ release response in HEK293 cells, whereas a low response of the corresponding Gly-4934 variants suggested loss of function. Following purification, the RyR1 mutants G4934D, G4934K, and G4941D did not noticeably conduct Ca2+ in single-channel measurements. Gly-4941 replacement with Lys resulted in channels having reduced K+ conductance and reduced selectivity for Ca2+ compared with wildtype. RyR1-G4941K did not fully close at nanomolar cytosolic Ca2+ concentrations and nearly fully opened at 2 μm cytosolic or sarcoplasmic reticulum luminal Ca2+, and Ca2+- and voltage-dependent regulation of RyR1-G4941K mutant channels was demonstrated. Computational methods and single-channel recordings indicated that the open G4941K variant results in the formation of a salt bridge to Asp-4938. In contrast, wildtype RyR1 was closed and not activated by luminal Ca2+ at low cytosolic Ca2+ levels. A model suggested that luminal Ca2+ activates RyR1 by accessing a recently identified cytosolic Ca2+-binding site in the open channel as the Ca2+ ions pass through the pore.