Requirement of negative residues,Asp 95 and Asp 105, in S2 on membrane integration of a voltage-dependent K+ channel,KAT1

Voltage-dependent K+ channels consist of a voltage-sensing region and a pore-forming region. Here we have identified the negative residues of the second transmembrane segment in the plant voltage-dependent K+ channel, KAT1, which involves the function of voltage sensing. Point mutations at D95 and D105 but not D89 and D116 failed to complement the K+ uptake deficient properties of the mutant yeast. In vitro translation and translocation experiments showed that the membrane integration of the third and fourth segments involving voltage sensor were impaired by the replacement of D95 or D105 by serine. These data show that both the residues play a crucial role in the membrane topogenesis of the voltage sensor in KAT1.     

Membrane integration of the second transmembrane segment of band 3 requires a closely apposed preceding signal-anchor sequence

We have investigated the topogenic rules of multispanning membrane proteins using erythrocyte band 3. Here, the fine structural requirements for the correct disposition of its second transmembrane segment (TM2) were assessed. We made fusion proteins where TM1 and the loop sequence preceding TM2 were changed and fused to prolactin. They were expressed in a cell-free system supplemented with rough microsomal membrane, and their topologies on the membrane were assessed by protease sensitivity and N-glycosylation. TM1 was demonstrated to be a signal-anchor sequence that mediates translocation of the downstream portion, and thus TM2 should be responsible to halt the translocation to acquire TM topology. When the loop between TM1 and TM2 was elongated, however, TM2 was readily translocated through the membrane and not integrated. For the membrane integration of TM2, TM2 must be in close proximity to TM1. The TM1 can be replaced with another signal-anchor sequence with a long hydrophobic segment but not with a signal sequence with shorter hydrophobic stretch. The length of the hydrophobic segment affected final topology of TM2. We concluded that the two TM segments work synergistically within the translocon to acquire the correct topology and that the length of the preceding signal sequence is critical for stable transmembrane assembly of TM2. We propose that direct interaction among the TM segments is one of the critical factors for the transmembrane topogenesis of multispanning membrane proteins.     

Assembly of ROMK1 (Kir 1.1a) inward rectifier K+ channel subunits involves multiple interaction sites.

The ROMK1 (Kir 1.1a) channel is formed by a tetrameric complex of subunits, each characterized by cytoplasmic N- and C-termini and a core region of two transmembrane helices flanking a pore-forming segment. To delineate the general regions mediating the assembly of ROMK1 subunits we constructed epitope-tagged N-terminal, C-terminal, and transmembrane segment deletion mutants. Nonfunctional subunits with N-terminal, core region, and C-terminal deletions had dominant negative effects when coexpressed with wild-type ROMK1 subunits in Xenopus oocytes. In contrast, coexpression of these nonfunctional subunits with Kv 2.1 (DRK1) did not suppress Kv 2.1 currents in control oocytes. Interactions between epitope-tagged mutant and wild-type ROMK1 subunits were studied in parallel by immunoprecipitating [35S]-labeled oocyte membrane proteins. Complexes containing both wild-type and mutant subunits that retained H5, M2, and C-terminal regions were coimmunoprecipitated to a greater extent than complexes consisting of wild-type and mutant subunits with core region and/or C-terminal deletions. The present findings are consistent with the hypothesis that multiple interaction sites located in the core region and cytoplasmic termini of ROMK1 subunits mediate homomultimeric assembly.     

Structural Diversity in the Cytoplasmic Region of G Protein-Gated Inward Rectifier K+ Channels

Inward rectifier K+ (Kir) channels can be functionally categorized into two groups: those that are constitutively active and those that are constitutively inactive, with examples such as Kir2.x and Kir3.x, respectively. Their cytoplasmic regions are thought to be critical for control of channel gating, but a structural basis for this hypothesis is not known. In this study, we report a structure for the cytoplasmic region of a G protein-gated Kir channel, Kir3.2, and compare it with those of Kir3.1 and Kir2.1 channels. The isolated cytoplasmic region of Kir3.2 forms a tetrameric assembly in solution and also in the crystal. While the secondary structure arrangement and the subunit interface of the Kir3.2 crystal structure are found to be nearly identical to those of Kir3.1 and Kir2.1, it is quite different at and around loops between βC- and βD-strands and between βH- and βI-strands. These structural elements are located at the interface with the plasma membrane. Therefore, these structural elements could associate with the Kir channel transmembrane helices and be involved in the regulation of Kir channel gating.     

Site-directed glycosylation tagging of functional Kir2.1 reveals that the putative pore-forming segment is extracellular

Inwardly rectifying K+ channels or Kirs are a large gene family and have been predicted to have two transmembrane segments, M1 and M2, intracellular N and C termini, and two extracellular loops, E1 and E2, separated by an intramembranous pore-forming segment, H5. H5 contains a stretch of eight residues that are similar in voltage-dependent K+ channels, Kvs, and this stretch is called the signature sequence of K+ channels. Because mutations in this sequence altered selectivity in Kvs, it has been designated as the selectivity filter. Previously, we used N-glycosylation substitution mutants to map the extracellular topology of a weak inwardly rectifying K+ channel, Kir1.1 or ROMK1, and found that the entire H5 segment was extracellular. We now report utilization of introduced N-glycosylation sites, NX(S/T), at positions Ser(128) in E1, and Gln(140), Ileu(143), and Phe(147) in the H5 sequence of a strong inwardly rectifying K+ channel, Kir2.1. Furthermore, we show that biotinylated channel proteins with N-linked oligosaccharides attached at positions 140 and 143 in the signature sequence are located at the cell surface. Mutant channels were functional as detected by whole-cell and single-channel recordings. Unlike Kir1.1, position Lys(117) was not occupied. We conclude that, for yet another K+ channel, the invariant G(Y/F)G sequence is extracellular rather than intramembranous.     

Identification of gamma-aminobutyric acid receptor-interacting factor 1 (TRAK2) as a trafficking factor for the K+ channel Kir2.1

To identify proteins that regulate potassium channel activity and expression, we performed functional screening of mammalian cDNA libraries in yeast that express the mammalian K(+) channel Kir2.1. Growth of Kir2.1-expressing yeast in media with low K(+) concentration is a function of K(+) uptake via Kir2.1 channels. Therefore, the host strain was transformed with a human cDNA library, and cDNA clones that rescued growth at low K(+) concentration were selected. One of these clones was identical to the protein of unknown function isolated previously as gamma-aminobutyric acid receptor-interacting factor 1 (GRIF-1) (Beck, M., Brickley, K., Wilkinson, H., Sharma, S., Smith, M., Chazot, P., Pollard, S., and Stephenson, F. (2002) J. Biol. Chem. 277, 30079-30090). GRIF-1 specifically enhanced Kir2.1-dependent growth in yeast and Kir2.1-mediated (86)Rb(+) efflux in HEK293 cells. Quantitative microscopy and flow cytometry analysis of immunolabeled surface Kir2.1 channel showed that GRIF-1 significantly increased the number of Kir2.1 channels in the plasma membrane of COS and HEK293 cells. Physical interaction of Kir2.1 channel and GRIF-1 was demonstrated by co-immunoprecipitation from HEK293 lysates and yeast two-hybrid assay. In vivo association of Kir2.1 and GRIF-1 was demonstrated by co-immunoprecipitation from brain lysate. Yeast two-hybrid assays showed that an N-terminal region of GRIF-1 interacts with a C-terminal region of Kir2.1. These results indicate that GRIF-1 binds to Kir2.1 and facilitates trafficking of this channel to the cell surface.     

Membrane topogenesis of the three amino-terminal transmembrane segments of glucose-6-phosphatase on endoplasmic reticulum

We investigated the membrane topogenesis of glucose-6-phosphatase (G6Pase), a multispanning membrane protein, on the endoplasmic reticulum. In COS-7 cells, the first transmembrane segment (TM1) with weak hydrophobicity is inserted into the membrane in the N-terminus-out/C-terminus-cytoplasm orientation. The following TM2 is inserted depending on TM3. TM3 has the same orientation as TM1. In contrast to data from living cells, the full-length molecule and N-terminal fusion constructs were not inserted into the membrane in a cell-free system. Addition of a signal recognition particle did not improve G6Pase insertion. When the 37-residue N-terminal segment was deleted, however, TM2 and TM3 were correctly inserted. We concluded that the three N-terminal TM segments are inserted into the membrane dependent on the two signal-anchor sequences of TM1 and TM3. TM1 is likely to be an unconventional signal sequence that barely functions in vitro. The 37-residue N-terminal segment inhibits the signal function of the following TM3 in cell-free systems.     

Molecular basis for K(ATP) assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter

K(ATP) channels are large heteromultimeric complexes containing four subunits from the inwardly rectifying K+ channel family (Kir6.2) and four regulatory sulphonylurea receptor subunits from the ATP-binding cassette (ABC) transporter family (SUR1 and SUR2A/B). The molecular basis for interactions between these two unrelated protein families is poorly understood. Using novel trafficking-based interaction assays, coimmunoprecipitation, and current measurements, we show that the first transmembrane segment (M1) and the N terminus of Kir6.2 are involved in K(ATP) assembly and gating. Additionally, the transmembrane domains, but not the nucleotide-binding domains, of SUR1 are required for interaction with Kir6.2. The identification of specific transmembrane interactions involved in K(ATP) assembly may provide a clue as to how ABC proteins that transport hydrophobic substrates evolved to regulate other membrane proteins.     

Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence

Uncleaved signal-anchor sequences of membrane proteins inserted into the endoplasmic reticulum initiate the translocation of either the amino-terminal or the carboxyl-terminal polypeptide segment across the bilayer. Which topology is acquired is not determined by the apolar segment of the signal but rather by the hydrophilic sequences flanking it. To study the role of charged residues in determining the membrane topology, the insertion of mutants of the asialoglycoprotein receptor H1, a single-spanning protein with a cytoplasmic amino terminus, was analyzed in transfected COS-7 cells. When the charged amino acids flanking the hydrophobic signal were mutated to residues of opposite charge, half the polypeptides inserted with the inverted orientation. When, in addition, the amino-terminal domain of the mutant protein was truncated, approximately 90% of the polypeptides acquired the inverted topology. The transmembrane orientation appears to be primarily determined by the charges flanking the signal sequence but is modulated by the domains to be translocated.     

Manipulation of membrane protein topology on the endoplasmic reticulum by a specific ligand in living cells

Almost all integral membrane proteins in the secretory pathway are cotranslationally inserted into the endoplasmic reticulum membrane. Their membrane topology is determined by their amino acid sequences. Here we show that the topology can be manipulated by a factor other than the amino acid sequence. A dihydrofolate reductase (DHFR) domain was fused to the N-terminus of the type I signal-anchor sequence of synaptotagmin II, which mediates translocation of the preceding portion. The DHFR domain was translocated through the membrane in COS7 cells and a transmembrane (TM) topology was achieved. When a DHFR ligand, methotrexate, was added to the culture medium, translocation of the DHFR domain was suppressed and both ends of the signal-anchor sequence remained on the cytoplasmic side. In contrast, translocation of the DHFR domain fused after the signal peptide, which translocates the following region, was not affected by the ligand. The topology-altered fusion protein was anchored to the membrane in a high salt-resistant state, and partially extracted from the membrane under alkali conditions. We concluded that the topology of membrane proteins can be manipulated by a trans-acting factor, even in living cells.     

Transmembrane orientation of signal-anchor proteins is affected by the folding state but not the size of the N-terminal domain.

Upon insertion of a signal-anchor protein into the endoplasmic reticulum membrane, either the C-terminal or the N-terminal domain is translocated across the membrane. Charged residues flanking the transmembrane domain are important determinants for this decision, but are not necessarily sufficient to generate a unique topology. Using a model protein that is inserted into the membrane to an equal extent in either orientation, we have tested the influence of the size and the folding state of the N-terminal domain on the insertion process. A small zinc finger domain or the full coding sequence of dihydrofolate reductase were fused to the N-terminus. These stably folding domains hindered or even prevented their translocation. Disruption of their structure by destabilizing mutations largely restored transport across the membrane. Translocation efficiency, however, did not depend on the size of the N-terminal domain within a range of 40-237 amino acids. The folding behavior of the N-terminal domain is thus an important factor in the topogenesis of signal-anchor proteins.     

The selectivity filter may act as the agonist-activated gate in the G protein-activated Kir3.1/Kir3.4 K+ channel

The Kir3.1/Kir3.4 channel is activated by Gbetagamma subunits released on binding of acetylcholine to the M2 muscarinic receptor. A mechanism of channel opening, similar to that for the KcsA and Shaker K+ channels, has been suggested that involves translocation of pore lining transmembrane helices and the opening of an intracellular gate at the "bundle crossing" region. However, in the present study, we show that an extracellular gate at the selectivity filter is critical for agonist activation of the Kir3.1/Kir3.4 channel. Increasing the flexibility of the selectivity filter, by disrupting a salt bridge that lies directly behind the filter, abolished both selectivity for K+ and agonist activation of the channel. Other mutations within the filter that altered selectivity also altered agonist activation. In contrast, mutations within the filter that did not affect selectivity had little if any effect on agonist activation. Interestingly, mutation of bulky side chain phenylalanine residues at the bundle crossing also altered both agonist activation and selectivity. These results demonstrate a significant correlation between agonist activation and selectivity, which is determined by the selectivity filter, and suggests, therefore, that the selectivity filter may act as the agonist-activated gate in the Kir3.1/Kir3.4 channel.     

Direct modulation of Kir channel gating by membrane phosphatidylinositol 4,5-bisphosphate

Multiple ion channels have now been shown to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) at the cytoplasmic face of the membrane. However, direct evidence for a specific interaction between phosphoinositides and ion channels is critically lacking. We reconstituted pure KirBac1.1 and KcsA protein into liposomes of defined composition (3:1 phosphatidylethanolamine:phosphatidylglycerol) and examined channel activity using a 86Rb+ uptake assay. We demonstrate direct modulation by PIP2 of KirBac1.1 but not KcsA activity. In marked contrast to activation of eukaryotic Kir channels by PIP2, KirBac1.1 is inhibited by PIP2 incorporated in the membrane (K(1/2) = 0.3 mol %). The dependence of inhibition on the number of phosphate groups and requirement for a lipid tail matches that for activation of eukaryotic Kir channels, suggesting a fundamentally similar interaction mechanism. The data exclude the possibility of indirect modulation via cytoskeletal or other intermediary elements and establish a direct interaction of the channel with PIP2 in the membrane.     

Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis

During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61α. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.     

The signal-anchor sequence of CYP2C1 inserts into the membrane as a hairpin structure

Microsomal cytochrome P450s (CYPs) are anchored to the endoplasmic reticulum membrane by the N-terminal signal-anchor sequence which is predicted to insert into the membrane as a type 1 transmembrane helix with a luminally located N-terminus. We have mapped amino acids of the CYP2C1 signal-anchor, fused to Cys-free glutathione S-transferase, within the membrane by Cys-specific labeling with membrane-impermeant maleimide polyethylene glycol. At the C-terminal end of the signal-anchor, Trp-20 was mapped to the membrane–cytosol interface and Leu-19 was within the membrane. Unexpectedly, at the N-terminal end, Glu-2 and Pro-3 were mapped to the cytoplasmic side of the membrane rather than the luminal side as expected of a type 1 transmembrane helix. Similar results were observed for the N-terminal amino acids of the signal-anchor sequences of CYP3A4 and CYP2E1. These observations indicate that contrary to the current model of the signal-anchor of CYPs as a type 1 transmembrane helix, CYP2C1, CYP2E1, and CYP3A4 are monotopic membrane proteins with N-terminal signal-anchors that have a hairpin or wedge orientation in the membrane.     

Membrane proteins involved in potassium shifts during muscle activity and fatigue

Muscle activity is associated with potassium displacements, which may cause fatigue. It was reported previously that the density of the large-conductance Ca2+-dependent K+ (BK(Ca)) channel is higher in the T tubule membrane than in the sarcolemmal membrane and that the opposite is the case for the ATP-sensitive K+ (K(ATP)) channel. In the present experiments, we investigated the subcellular localizations of the strong inward rectifier 2.1 K+ (Kir2.1) channel and the Na+-K+-2Cl- (NKCC)1 cotransporter with Western blot analysis of different muscle fractions. Furthermore, muscle function was studied while trying to manipulate the opening probability or transport capacity of these proteins during electrical stimulation of isolated soleus muscles. All experiments were made with excised muscle from male Wistar rats. Kir2.1 channels were almost undetectable in the sarcolemmal membrane but present in the T tubule membrane, whereas NKCC1 cotransporters were present in the sarcolemmal membrane. For muscles incubated in a buffer containing pinacidil, NS1619, Ba2+, or bumetanide, there was a faster reduction in peak force (P < 0.05). Furthermore, bumetanide incubation reduced the peak force at the onset of electrical stimulation (P < 0.05). Thus the effects on muscle force indicate that these drugs can affect K+-transporting proteins and thereby influence K+ accumulation, especially in the T tubules, suggesting that K(ATP) and BK(Ca) channels are responsible for K+ release and decrease in force during repeated muscle contractions, whereas Kir2.1 and NKCC1 may have a role in K+ reuptake.     

Role of conserved glycines in pH gating of Kir1.1 (ROMK)

Gating of inward rectifier Kir1.1 potassium channels by internal pH is believed to occur when large hydrophobic leucines, on each of the four subunits, obstruct the permeation path at the cytoplasmic end of the inner transmembrane helices (TM2). In this study, we examined whether closure of the channel at this point involves bending of the inner helix at one or both of two highly conserved glycine residues (corresponding to G134 and G143 in KirBac1.1) that have been proposed as putative "gating hinges" for potassium channels. Replacement of these conserved inner helical glycines by less flexible alanines did not abolish gating but shifted the apparent pKa from 6.6 +/- 0.01 (wild-type) to 7.1 +/- 0.01 for G157A-Kir1.1b, and to 7.3 +/- 0.01 for G148A-Kir1.1b. When both glycines were mutated the effect was additive, shifting the pKa by 1.2 pH units to 7.8 +/- 0.04 for the double mutant: G157A+G148A. At this pKa, the double mutant would remain completely closed under physiological conditions. In contrast, when the glycine at G148 was replaced by a proline, the pKa was shifted in the opposite direction from 6.6 +/- 0.01 (wild-type) to 5.7 +/- 0.01 for G148P. Although conserved glycines at G148 and G157 made it significantly easier to open the channel, they were not an absolute requirement for pH gating in Kir1.1. In addition, none of the glycine mutants produced more than small changes in either the cell-attached or excised single-channel kinetics which, in this channel, argues against changes in the selectivity filter. The putative pH sensor at K61-Kir1.1b, (equivalent to K80-Kir1.1a) was also examined. Mutation of this lysine to an untitratable methionine did not abolish pH gating, but shifted the pKa into an acid range from 6.6 +/- 0.01 to 5.4 +/- 0.04, similar to pH gating in Kir2.1. Hence K61-Kir1.1b cannot function as the exclusive pH sensor for the channel, although it may act as one of multiple pH sensors, or as a link between a cytoplasmic sensor and the channel gate. K61-Kir1.1b also interacted differently with the two glycine mutations. Gating of the double mutant: K61M+G148A was indistinguishable from K61M alone, whereas gating of K61M+G157A was midway between the alkaline pKa of G157A and the acid pKa of K61M. Finally, closure of ROMK, G148A, G157A, and K61M all required the same L160-Kir1.1b residue at the cytoplasmic end of the inner transmembrane helix. Hence in wild-type and mutant channels, closure occurs by steric occlusion of the permeation path by four leucine side chains (L160-Kir1.1b) at the helix bundle crossing. This is facilitated by the conserved glycines on TM2, but pH gating in Kir1.1 does not absolutely require glycine hinges in this region.     

Gating of inward-rectifier K+ channels by intracellular pH.

Inward rectifier K+ channels of the Kir1.1 (ROMK) and Kir4.1 subtype are predominantly expressed in epithelial cells where they are responsible for K+ transport across the plasma membrane. Uniquely among the members of the Kir family, these channels are gated by intracellular pH in the physiological range. pH-gating involves structural rearrangements in cytoplasmic domains and the P-loop of the Kir protein. The energy for the gating transition is delivered by protonation of a lysine residue that is located prior to the first transmembrane segment and serves as a 'pH sensor'. The anomalous titration required for lysine operating in the neutral pH range results from its close interaction with two positively charged arginines from the distant N- and C-termini termed the R/K/R triad. Disturbance of this triad as results from a number of point mutations found in patients with hyperprostaglandin E syndrome (HPS) increases the pKa of the pH sensor and results in channels being permanently inactivated under physiological conditions. This article will focus on the mechanism of pH-gating, its implications for the tertiary structure of Kir proteins and on its significance for the pathogenesis of HPS.     

The adenovirus E3-6.7K protein adopts diverse membrane topologies following posttranslational translocation

The E3 region of adenovirus codes for several membrane proteins, most of which are involved in immune evasion and prevention of host cell apoptosis. We explored the topology and targeting mechanisms of E3-6.7K, the most recently described member of this group, by using an in vitro translation system supplemented with microsomes. Here, we present evidence that E3-6.7K, one of the smallest signal-anchor proteins known, translocates across the membrane of the endoplasmic reticulum in a posttranslational, ribosome-independent, yet ATP-dependent manner, reminiscent of the translocation of tail-anchored proteins. Our analysis also demonstrated that E3-6.7K could achieve several distinct topological fates. In addition to the previously postulated type III orientation (N-luminal/C-cytoplasmic, termed NtmE3-6.7K), we detected a tail-anchored form adopting the opposite orientation (N-cytoplasmic/C-luminal, termed CtmE3-6.7K) as well as the possibility of a fully translocated form (N and C termini are both translocated, termed NCE3-6.7K). Due to the translocation of a positively charged domain, both the CtmE3-6.7K and NCE3-6.7K topologies of E3-6.7K constitute exceptions to the "positive inside" rule. The NtmE3-6.7K and NCE3-6.7K are the first examples of posttranslationally translocated proteins in higher eukaryotes that are not tail anchored. Distinct topological forms were also found in transfected cells, as both N and C termini of E3-6.7K were detected on the extracellular surface of transfected cells. The demonstration of unexpected topological forms and translocation mechanisms for E3-6.7K defies conventional thinking about membrane protein topogenesis and advises that both the mode of targeting and topology of signal-anchor proteins should be determined experimentally.     

Cholesterol sensitivity of KIR2.1 is controlled by a belt of residues around the cytosolic pore

Kir channels play an important role in setting the resting membrane potential and modulating membrane excitability. A common feature of several Kir channels is that they are regulated by cholesterol. Yet, the mechanism by which cholesterol affects channel function is unclear. We recently showed that the cholesterol sensitivity of Kir2 channels depends on several CD-loop residues. Here we show that this cytosolic loop is part of a regulatory site that also includes residues in the G-loop, the N-terminus, and the connecting segment between the C-terminus and the inner transmembrane helix. Together, these residues form a cytosolic belt that surrounds the pore of the channel close to its interface with the transmembrane domain, and modulate the cholesterol sensitivity of the channel. Furthermore, we show that residues in this cluster are correlated with residues located in the most flexible region of the G-loop, the major cytosolic gate of Kir2.1, implying that the importance of these residues extends beyond their effect on the channel's cholesterol sensitivity. We suggest that the residues of the cholesterol sensitivity belt are critical for channel gating.