Acidic residues on the voltage-sensor domain determine the activation of the NaChBac sodium channel

The voltage-sensing domain of voltage-gated ion channels is characterized by specific, conserved, charged residues. Positively charged residues on segment S4 are the main contributors to voltage-sensing and negatively charged residues on the S2 and S3 segments are believed to participate to the process. However, their function in the voltage sensor is not well understood. To probe the role of three acidic residues in NaChBac (D-58 and E-68 in S2, and D-91 in S3), we employed site-directed mutagenesis to substitute native acidic residues with cysteine (neutral), lysine (positive charge), or either aspartate or glutamate (negative charge). We used a combination of the patch-clamp technique to record Na+ currents and molecular modeling to visualize interacting amino acid residues. We suggest that the acidic residues on the S2 and S3 segments form specific interactions with adjacent amino acids in the voltage-sensor domain. The main interactions in NaChBac are D-58 (S2) with A-97-G-98 (S3) and R-120 (S4), E-68 (S2) with R-129 (L4-5), and D-91 (S3) with R-72 (S2). Changing these acidic residues modified the interactions, which in turn altered the sensitivity of the voltage sensor.     

Molecular dissection of the contribution of negatively and positively charged residues in S2, S3, and S4 to the final membrane topology of the voltage sensor in the K+ channel,KAT1

Voltage-dependent ion channels control changes in ion permeability in response to membrane potential changes. The voltage sensor in channel proteins consists of the highly positively charged segment, S4, and the negatively charged segments, S2 and S3. The process involved in the integration of the protein into the membrane remains to be elucidated. In this study, we used in vitro translation and translocation experiments to evaluate interactions between residues in the voltage sensor of a hyperpolarization-activated potassium channel, KAT1, and their effect on the final topology in the endoplasmic reticulum (ER) membrane. A D95V mutation in S2 showed less S3-S4 integration into the membrane, whereas a D105V mutation allowed S4 to be released into the ER lumen. These results indicate that Asp(95) assists in the membrane insertion of S3-S4 and that Asp(105) helps in preventing S4 from being releasing into the ER lumen. The charge reversal mutation, R171D, in S4 rescued the D105R mutation and prevented S4 release into the ER lumen. A series of constructs containing different C-terminal truncations of S4 showed that Arg(174) was required for correct integration of S3 and S4 into the membrane. Interactions between Asp(105) and Arg(171) and between negative residues in S2 or S3 and Arg(174) may be formed transiently during membrane integration. These data clarify the role of charged residues in S2, S3, and S4 and identify posttranslational electrostatic interactions between charged residues that are required to achieve the correct voltage sensor topology in the ER membrane.     

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.     

Conserved motifs in voltage-sensing and pore-forming modules of voltage-gated ion channel proteins

Voltage-gated ion channels (VGCs) mediate selective diffusion of ions across cell membranes to enable many vital cellular processes. Three-dimensional structure data are lacking for VGC proteins; hence, to better understand their function, there is a need to identify the conserved motifs using sequence analysis methods. In this study, we have used a profile-to-profile alignment method to identify several new conserved motifs specific to each transmembrane segment (TMS) of the voltage-sensing and the pore-forming modules of Ca2+, Na+, and K+ channel subfamilies. For Ca2+ and Na+, the functional theme of motif conservation is similar in all segments while they differ with those of the K+ channel proteins. Nevertheless, the conservation is strikingly similar in the S4 segment of the voltage-sensing module across all subfamilies. In each subfamily and for each TMS, we have identified conserved motifs/residues and correlated their functional significance and disease associations in human, using mutational data from the literature.     

Role of charged residues in the S1-S4 voltage sensor of BK channels

The activation of large conductance Ca(2+)-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K(+) (K(V)) channels. Yet BK and K(V) channels share many conserved charged residues in transmembrane segments S1-S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (P(o)) and I(K) kinetics (tau(I(K))) over an extended voltage range in 0-50 microM [Ca(2+)](i). mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of P(O). The voltage dependence of P(O) and tau(I(K)) at extreme negative potentials was also reduced, implying that the closed-open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and K(V) channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to K(V) channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1-S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3-7 kcal mol(-1), indicating a strong contribution of non-voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.     

Conserved residues within the putative S4-S5 region serve distinct functions among thermosensitive vanilloid transient receptor potential (TRPV) channels

The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat, or highly depolarizing voltages, and also by 2-aminoethoxydiphenyl borate (2-APB), a common activator of the related thermally gated vanilloid TRP channels TRPV1, TRPV2, and TRPV3. In these channels, the conserved charged residues in the intracellular S4-S5 region have been proposed to constitute part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. The molecular basis of this gating event is poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and identified four potential voltage-sensing residues (Arg(557), Glu(570), Asp(576), and Arg(579)) that, when specifically mutated, altered the functionality of the channel with respect to voltage, capsaicin, heat, 2-APB, and/or their interactions in different ways. The nonfunctional charge-reversing mutations R557E and R579E were partially rescued by the charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2-APB, revealing the specific role of Lys(571) in chemical sensitivity. Surprisingly, substitutions at homologous residues in TRPV2 or TRPV3 had no effect on temperature- and 2-APB-induced activity. Thus, the charged residues in S4 and the S4-S5 linker contribute to voltage sensing in TRPV1 and, despite their highly conserved nature, regulate the temperature and chemical gating in the various TRPV channels in different ways.     

Changes in local S4 environment provide a voltage-sensing mechanism for mammalian hyperpolarization-activated HCN channels

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions in the COOH-terminal half of S4 selectively reacted with internal MTSET when the channel was open. In the open state, the boundary between externally and internally accessible residues was remarkably narrow (approximately 3 residues). This suggests that S4 lies in a water-filled gating canal with a very narrow barrier between the external and internal solutions, similar to depolarization-gated channels. However, the pattern of reactivity is incompatible with either classical gating models, which postulate a large translational or rotational movement of S4 within a gating canal, or with a recent model in which S4 forms a peripheral voltage-sensing paddle (with S3b) that moves within the lipid bilayer (the KvAP model). Rather, we suggest that voltage sensing is due to a rearrangement in transmembrane segments surrounding S4, leading to a collapse of an internal gating canal upon channel closure that alters the shape of the membrane field around a relatively static S4 segment.     

Role of hydrophobic and ionic forces in the movement of S4 of the Shaker potassium channel

Abstract

Voltage-gated ion (K⁺, Na⁺, Ca²⁺) channels contain a pore domain (PD) surrounded by four voltage sensing domains (VSD). Each VSD is made up of four transmembrane helices, S1–S4. S4 contains 6–7 positively charged residues (arginine/lysine) separated two hydrophobic residues, whereas S1–S3 contribute to two negatively charged clusters. These structures are conserved among all members of the voltage-gated ion channel family and play essential roles in voltage gating. The role of S4 charged residues in voltage gating is well established: During depolarization, they move out of the membrane electric field, exerting a mechanical force on channel gates, causing them to open. However, the role of the intervening hydrophobic residues in voltage sensing is unclear. Here we studied the role of these residues in the prototypical Shaker potassium channel. We have altered the physicochemical properties of both charged and hydrophobic positions of S4 and examined the effect of these modifications on the gating properties of the channel. For this, we have introduced cysteines at each of these positions, expressed the mutants in Xenopus oocytes, and examined the effect of in situ addition of charge, via Cd²⁺, on channel gating by two-electrode voltage clamp. Our results reveal a face of the S4 helix (comprising residues L358, L361, R365 and R368) where introduction of charge at hydrophobic positions destabilises the closed state and removal of charges from charged positions has an opposite effect. We propose that hydrophobic residues play a crucial role in limiting gating to a physiological voltage range.     

Molecular mechanism of voltage sensor movements in a potassium channel

Voltage-gated potassium channels are six-transmembrane (S1-S6) proteins that form a central pore domain (4 x S5-S6) surrounded by four voltage sensor domains (S1-S4), which detect changes in membrane voltage and control pore opening. Upon depolarization, the S4 segments move outward carrying charged residues across the membrane field, thereby leading to the opening of the pore. The mechanism of S4 motion is controversial. We have investigated how S4 moves relative to the pore domain in the prototypical Shaker potassium channel. We introduced pairs of cysteines, one in S4 and the other in S5, and examined proximity changes between each pair of cysteines during activation, using Cd2+ and copper-phenanthroline, which crosslink the cysteines with metal and disulphide bridges, respectively. Modelling of the results suggests a novel mechanism: in the resting state, the top of the S3b-S4 voltage sensor paddle lies close to the top of S5 of the adjacent subunit, but moves towards the top of S5 of its own subunit during depolarization--this motion is accompanied by a reorientation of S4 charges to the extracellular phase.     

Movement and crevices around a sodium channel S3 segment

Voltage sensing is due mainly to the movement of positively charged S4 segments through the membrane electric field during changes of membrane potential. The roles of other transmembrane segments are under study. The S3 segment of domain 4 (D4/S3) in the sodium channel Na(v)1.4 carries two negatively charged residues and has been implicated in voltage-dependent gating. We substituted cysteines into nine putative "high impact" sites along the complete length of D4/S3 and evaluated their accessibilities to extracellular sulfhydryl reagents. Only the four outermost substituted cysteines (L1433C, L1431C, G1430C, and S1427C) are accessible to extracellular sulfhydryl reagents. We measured the voltage-dependent modification rates of the two cysteines situated at the extreme ends of this accessible region, L1433C and S1427C. Independent of the charge on the sulfhydryl reagents, depolarization increases the reactivity of both of these residues. Thus, the direction of the voltage dependence is opposite to that expected for a negatively charged voltage sensor, namely an inward translational movement in response to depolarization. Intrinsic electrostatic potentials were probed by charged sulfhydryl reagents and were either negative or positive, respectively, near L1433C and S1427C. The magnitude of the electrostatic potential near S1427C decreases with depolarization, suggesting that the extracellular crevice next to it widens during depolarization. S1427C experiences 44% of the electric field, as probed by charged cysteine reagents. To further explore movements around D4/S3, we labeled cysteines with the photoactivatable cross-linking reagent benzophenone-4-carboxamidocysteine methanethiosulfonate and examined the effects of UV irradiation on channel gating. After labeling with this reagent, all accessible cysteine mutants show altered gating upon brief UV irradiation. In each case, the apparent insertion efficiency of the photoactivated benzophenone increases with depolarization, indicating voltage-dependent movement near the extracellular end of D4/S3.     

Voltage-dependent gating of hyperpolarization-activated, cyclic nucleotide-gated pacemaker channels: molecular coupling between the S4-S5 and C-linkers

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels have a transmembrane topology that is highly similar to voltage-gated K(+) channels, yet HCN channels open in response to membrane hyperpolarization instead of depolarization. The structural basis for the "inverted" voltage dependence of HCN gating and how voltage sensing by the S1-S4 domains is coupled to the opening of the intracellular gate formed by the S6 domain are unknown. Coupling could arise from interaction between specific residues or entire transmembrane domains. We previously reported that the mutation of specific residues in the S4-S5 linker of HCN2 (i.e. Tyr-331 and Arg-339) prevented normal channel closure presumably by disruption of a crucial interaction with the activation gate. Here we hypothesized that the C-linker, a carboxyl terminus segment that connects S6 to the cyclic nucleotide binding domain, interacts with specific residues of the S4-S5 linker to mediate coupling. The recently solved structure of the C-linker of HCN2 indicates that an alpha-helix (the A'-helix) is located near the end of each S6 domain, the presumed location of the activation gate. Ala-scanning mutagenesis of the end of S6 and the A'-helix identified five residues that were important for normal gating as mutations disrupted channel closure. However, partial deletion of the C-linker indicated that the presence of only two of these residues was required for normal coupling. Further mutation analyses suggested that a specific electrostatic interaction between Arg-339 of the S4-S5 linker and Asp-443 of the C-linker stabilizes the closed state and thus participates in the coupling of voltage sensing and activation gating in HCN channels.     

A unique role for the S4 segment of domain 4 in the inactivation of sodium channels

Sodium channels have four homologous domains (D1-D4) each with six putative transmembrane segments (S1-S6). The highly charged S4 segments in each domain are postulated voltage sensors for gating. We made 15 charge-neutralizing or -reversing substitutions in the first or third basic residues (arginine or lysine) by replacement with histidine, glutamine, or glutamate in S4 segments of each domain of the human heart Na+ channel. Nine of the mutations cause shifts in the conductance-voltage (G-V) midpoints, and all but two significantly decrease the voltage dependence of peak Na+ current, consistent with a role of S4 segments in activation. The decreases in voltage dependence of activation were equivalent to a decrease in apparent gating charge of 0.5-2.1 elementary charges (eo) per channel for single charge- neutralizing mutations. Three charge-reversing mutations gave decreases of 1.2-1.9 eo per channel in voltage dependence of activation. The steady-state inactivation (h infinity) curves were fit by single- component Boltzmann functions and show significant decreases in slope for 9 of the 15 mutants and shifts of midpoints in 9 mutants. The voltage dependence of inactivation time constants is markedly decreased by mutations only in S4D4, providing further evidence that this segment plays a unique role in activation-inactivation coupling.     

Structure and function of the voltage sensor of sodium channels probed by a beta-scorpion toxin

Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-scorpion toxin binding but alter voltage dependence of activation and enhance beta-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.     

Molecular dynamics investigation of the ω-current in the Kv1.2 voltage sensor domains

Voltage sensor domains (VSD) are transmembrane proteins that respond to changes in membrane voltage and modulate the activity of ion channels, enzymes, or in the case of proton channels allow permeation of protons across the cell membrane. VSDs consist of four transmembrane segments, S1-S4, forming an antiparallel helical bundle. The S4 segment contains several positively charged residues, mainly arginines, located at every third position along the helix. In the voltage-gated Shaker K(+) channel, the mutation of the first arginine of S4 to a smaller uncharged amino acid allows permeation of cations through the VSD. These currents, known as ω-currents, pass through the VSD and are distinct from K(+) currents passing through the main ion conduction pore. Here we report molecular dynamics simulations of the ω-current in the resting-state conformation for Kv1.2 and for four of its mutants. The four tested mutants exhibit various degrees of conductivity for K(+) and Cl(-) ions, with a slight selectivity for K(+) over Cl(-). Analysis of the ion permeation pathway, in the case of a highly conductive mutant, reveals a negatively charged constriction region near the center of the membrane that might act as a selectivity filter to prevent permeation of anions through the pore. The residues R1 in S4 and E1 in S2 are located at the narrowest region of the ω-pore for the resting state conformation of the VSD, in agreement with experiments showing that the largest increase in current is produced by the double mutation E1D and R1S.     

Molecular Template for a Voltage Sensor in a Novel K+ Channel. III. Functional Reconstitution of a Sensorless Pore Module from a Prokaryotic Kv Channel

KvLm is a prokaryotic voltage-gated K⁺ (Kv) channel from Listeria monocytogenes. The sequence of the voltage-sensing module (transmembrane segments S1-S4) of KvLm is atypical in that it contains only three of the eight conserved charged residues known to be deterministic for voltage sensing in eukaryotic Kv's. In contrast, the pore module (PM), including the S4-S5 linker and cytoplasmic tail (linker-S5-P-S6-C-terminus) of KvLm, is highly conserved. Here, the full-length (FL)-KvLm and the KvLm-PM only proteins were expressed, purified, and reconstituted into giant liposomes. The properties of the reconstituted FL-KvLm mirror well the characteristics of the heterologously expressed channel in Escherichia coli spheroplasts: a right-shifted voltage of activation, micromolar tetrabutylammonium-blocking affinity, and a single-channel conductance comparable to that of eukaryotic Kv's. Conversely, ionic currents through the PM recapitulate both the conductance and blocking properties of the FL-KvLm, yet the KvLm-PM exhibits only rudimentary voltage dependence. Given that the KvLm-PM displays many of the conduction properties of FL-KvLm and of other eukaryotic Kv's, including strict ion selectivity, we conclude that self-assembly of the PM subunits in lipid bilayers, in the absence of the voltage-sensing module, generates a conductive oligomer akin to that of the native KvLm, and that the structural independence of voltage sensing and PMs observed in eukaryotic Kv channels was initially implemented by nature in the design of prokaryotic Kv channels. Collectively, the results indicate that this robust functional module will prove valuable as a molecular template for coupling new sensors and to elucidate PM residue–specific contributions to Kv conduction properties.     

Movement of the S4 segment in the hERG potassium channel during membrane depolarization

Abstract

The hERG potassium channel is a member of the voltage gated potassium (Kv) channel family, comprising a pore domain and four voltage sensing domains (VSDs). Like other Kv channels, the VSD senses changes in membrane voltage and transmits the signal to gates located in the pore domain; the gates open at positive potentials (activation) and close at negative potentials, thereby controlling the ion flux. hERG, however, differs from other Kv channels in that it is activated slowly but inactivated rapidly – a property that is crucial for the role it plays in the repolarization of the cardiac action potential. Voltage-gating requires movement of gating charges across the membrane electric field, which is accomplished by the transmembrane movement of the fourth transmembrane segment, S4, of the VSD containing the positively charged arginine or lysine residues. Here we ask if the functional differences between hERG and other Kv channels could arise from differences in the transmembrane movement of S4. To address this, we have introduced single cysteine residues into the S4 region of the VSD, expressed the mutant channels in Xenopus oocytes and examined the effect of membrane impermeable para-chloromercuribenzene sulphonate on function by the two-electrode voltage clamp technique. Our results show that depolarization results in the accessibility of seven consecutive S4 residues, including the first two charged residues, K525 and R528, to extracellularly applied reagent. These data indicate that the extent of S4 movement in hERG is similar to other Kv channels, including the archabacterial KvAP and the Shaker channel of Drosophila.     

The orientation and molecular movement of a k(+) channel voltage-sensing domain

Voltage-gated channels operate through the action of a voltage-sensing domain (membrane segments S1-S4) that controls the conformation of gates located in the pore domain (membrane segments S5-S6). Recent structural studies on the bacterial K(v)AP potassium channel have led to a new model of voltage sensing in which S4 lies in the lipid at the channel periphery and moves through the membrane as a unit with a portion of S3. Here we describe accessibility probing and disulfide scanning experiments aimed at determining how well the K(v)AP model describes the Drosophila Shaker potassium channel. We find that the S1-S3 helices have one end that is externally exposed, S3 does not undergo a transmembrane motion, and S4 lies in close apposition to the pore domain in the resting and activated state.     

Structural insight into the transmembrane segments 3 and 4 of the hERG potassium channel

The hERG (human ether‐a‐go‐go related gene) potassium channel is a voltage‐gated potassium channel containing an N‐terminal domain, a voltage‐sensor domain, a pore domain and a C‐terminal domain. The transmembrane segment 4 (S4) is important for sensing changes of membrane potentials through positively charge residues. A construct containing partial S2–S3 linker, S3, S4 and the S4–S5 linker of the hERG channel was purified into detergent micelles. This construct exhibits good quality NMR spectrum when it was purified in lyso‐myristoyl phosphatidylglycerol (LMPG) micelles. Structural study showed that S3 contains two short helices with a negatively charged surface. The S4 and S4–S5 linker adopt helical structures. The six positively charged residues in S4 localize at different sides, suggesting that they may have different functions in channel gating. Relaxation studies indicated that S3 is more flexible than S4. The boundaries of S3–S4 and S4–S4–S5 linker were identified. Our results provided structural information of the S3 and S4, which will be helpful to understand their roles in channel gating. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Protein Disorder and Short Conserved Motifs in Disordered Regions Are Enriched near the Cytoplasmic Side of Single-Pass Transmembrane Proteins

Intracellular juxtamembrane regions of transmembrane proteins play pivotal roles in cell signalling, mediated by protein-protein interactions. Disordered protein regions, and short conserved motifs within them, are emerging as key determinants of many such interactions. Here, we investigated whether disorder and conserved motifs are enriched in the juxtamembrane area of human single-pass transmembrane proteins. Conserved motifs were defined as short disordered regions that were much more conserved than the adjacent disordered residues. Human single-pass proteins had higher mean disorder in their cytoplasmic segments than their extracellular parts. Some, but not all, of this effect reflected the shorter length of the cytoplasmic tail. A peak of cytoplasmic disorder was seen at around 30 residues from the membrane. We noted a significant increase in the incidence of conserved motifs within the disordered regions at the same location, even after correcting for the extent of disorder. We conclude that elevated disorder within the cytoplasmic tail of many transmembrane proteins is likely to be associated with enrichment for signalling interactions mediated by conserved short motifs.     

Catalytic and regulatory sites of yeast plasma membrane H(+)-ATPase studied by directed mutagenesis

More than 35 site-directed mutants of the plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae have been constructed and expressed to investigate the function of N- and C-termini and of conserved amino acids. Conserved motif TGES seems to form part of both the catalytic machinery for the hydrolysis of the phosphorylated intermediate and the vanadate binding site. In addition, it is involved in the coupling of ATP hydrolysis to H+ transport. The phosphorylated intermediate is also essential for this coupling, but not for ATP hydrolysis. The aspartate residues of conserved motifs DPPR, TGD and TGDGVND (the last one) seem to form part of the ATP binding site. The positive charge of the conserved motif KGAP is important for the kinase or phosphorylating activity. A conserved proline and a conserved aspartate predicted to have a transmembrane location are essential for activity. The N-terminus contains a conserved acidic region which may be involved in assembly into the plasma membrane. All the hydrophobic stretches at the C-terminus are also required for assembly. The last 11 amino acids constitute a non-essential inhibitory domain involved in regulation of the enzyme by glucose metabolism.