Voltage-Dependent Inactivation of MscS Occurs Independently of the Positively Charged Residues in the Transmembrane Domain

MscS (mechanosensitive channel of small conductance) is ubiquitously found among bacteria and plays a major role in avoiding cell lysis upon rapid osmotic downshock. The gating of MscS is modulated by voltage, but little is known about how MscS senses membrane potential. Three arginine residues (Arg-46, Arg-54, and Arg-74) in the transmembrane (TM) domain are possible to respond to voltage judging from the MscS structure. To examine whether these residues are involved in the voltage dependence of MscS, we neutralized the charge of each residue by substituting with asparagine (R46N, R54N, and R74N). Mechanical threshold for the opening of the expressed wild-type MscS and asparagine mutants did not change with voltage in the range from-40 to +100 mV. By contrast, inactivation process of wild-type MscS was strongly affected by voltage. The wild-type MscS inactivated at +60 to +80 mV but not at-60 to +40 mV. The voltage dependence of the inactivation rate of all mutants tested, that is, R46N, R54N, R74N, and R46N/R74N MscS, was almost indistinguishable from that of the wild-type MscS. These findings indicate that the voltage dependence of the inactivation occurs independently of the positive charges of R46, R54, and R74.

Download Full-text

Arginine mutations within a transmembrane domain of Tar, an Escherichia coli aspartate receptor, can drive homodimer dissociation and heterodimer association in vivo

Biochemical Journal ◽

10.1042/bj20041022 ◽

2004 ◽

Vol 385 (1) ◽

pp. 29-36 ◽

Cited By ~ 11

Author(s):

Neta SAL-MAN ◽

Yechiel SHAI

Keyword(s):

Amino Acids ◽

Transmembrane Domain ◽

Genetic Diseases ◽

Ion Pairs ◽

Aspartate Receptor ◽

Charged Residues ◽

Arginine Residues ◽

Tm Domain ◽

Positively Charged

The interactions between the TM (transmembrane) domains of many membrane proteins are important for their proper functioning. Mutations of residues into positively charged ones within TM domains were reported to be involved in many genetic diseases, possibly because these mutations affect the self- and/or hetero-assembly of the corresponding proteins. To our knowledge, despite significant progress in understanding the role of various amino acids in TM–TM interactions in vivo, the direct effect of positively charged residues on these interactions has not been studied. To address this issue, we employed the N-terminal TM domain of the aspartate receptor (Tar-1) as a dimerization model system. We expressed within the ToxR TM assembly system several Tar-1 constructs that dimerize via polar- or non-polar amino acid motifs, and mutated these by replacement with a single arginine residue. Our results have revealed that a mutation in each of the motifs significantly reduced the ability of the TMs to dimerize. Furthermore, a Tar-1 construct that contained two arginine residues was unable to correctly integrate itself into the membrane. Nevertheless, an exogenous synthetic Tar-1 peptide containing these two arginine residues was able to inhibit in vivo the marked dimerization of a mutant Tar-1 construct that contained two glutamate residues at similar positions. This indicates that hetero-assembly of TM domains can be mediated by the interaction of two oppositely charged residues, probably by formation of ion pairs. This study broadens our knowledge regarding the effect of positively charged residues on TM–TM interactions in vivo, and provides a potential therapeutic approach to inhibit uncontrolled dimerization of TM domains caused by mutations of polar amino acids.

Download Full-text

Effect of divalent heavy metals on epithelial Na+ channels in A6 cells

AJP Renal Physiology ◽

10.1152/ajprenal.00002.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. F236-F244 ◽

Cited By ~ 22

Author(s):

Ling Yu ◽

Douglas C. Eaton ◽

My N. Helms

Keyword(s):

Heavy Metal ◽

Voltage Dependence ◽

Transmembrane Domain ◽

Single Channel ◽

Epithelial Cell Line ◽

Open Probability ◽

Renal Epithelial Cell ◽

Histidine Residues ◽

Voltage Dependent ◽

Renal Epithelial Cell Line

To better understand how renal Na+ reabsorption is altered by heavy metal poisoning, we examined the effects of several divalent heavy metal ions (Zn2+, Ni2+, Cu2+, Pb2+, Cd2+, and Hg2+) on the activity of single epithelial Na+ channels (ENaC) in a renal epithelial cell line (A6). None of the cations changed the single-channel conductance. However, ENaC activity [measured as the number of channels ( N) × open probability ( Po)] was decreased by Cd2+ and Hg2+ and increased by Cu2+, Zn2+, and Ni2+ but was not changed by Pb2+. Of the cations that induced an increase in Na+ channel function, Zn2+ increased N, Ni2+ increased Po, and Cu2+ increased both. The cysteine modification reagent [2-(trimethylammonium)ethyl]methanethiosulfonate bromide also increased N, whereas diethylpyrocarbonate, which covalently modifies histidine residues, affected neither Po nor N. Cu2+ increased N and stimulated Po by reducing Na+ self-inhibition. Furthermore, we observed that ENaC activity is slightly voltage dependent and that the voltage dependence of ENaC is insensitive to extracellular Na+ concentration; however, apical application of Ni2+ or diethylpyrocarbonate reduced the channel voltage dependence. Thus the voltage sensor of Xenopus ENaC is different from that of typical voltage-gated channels, since voltage appears to be sensed by histidine residues in the extracellular loops of ENaC, rather than by charged amino acids in a transmembrane domain.

Download Full-text

A molecular link between activation and inactivation of sodium channels.

The Journal of General Physiology ◽

10.1085/jgp.106.4.641 ◽

1995 ◽

Vol 106 (4) ◽

pp. 641-658 ◽

Cited By ~ 43

Author(s):

M E O'Leary ◽

L Q Chen ◽

R G Kallen ◽

R Horn

Keyword(s):

Sodium Channels ◽

Voltage Dependence ◽

Single Channel ◽

Critical Role ◽

Channel Measurements ◽

Wild Type ◽

Open Probability ◽

Voltage Dependent ◽

Steady State Inactivation ◽

Normal Coupling

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

Download Full-text

Inactivation of N-type calcium current in chick sensory neurons: calcium and voltage dependence.

The Journal of General Physiology ◽

10.1085/jgp.104.2.311 ◽

1994 ◽

Vol 104 (2) ◽

pp. 311-336 ◽

Cited By ~ 40

Author(s):

D H Cox ◽

K Dunlap

Keyword(s):

Voltage Dependence ◽

Kinetic Properties ◽

Dependent Manner ◽

Slow Inactivation ◽

Fast Inactivation ◽

Drg Neurons ◽

Rapid Phase ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Dependent Processes

We have studied the inactivation of high-voltage-activated (HVA), omega-conotoxin-sensitive, N-type Ca2+ current in embryonic chick dorsal root ganglion (DRG) neurons. Voltage steps from -80 to 0 mV produced inward Ca2+ currents that inactivated in a biphasic manner and were fit well with the sum of two exponentials (with time constants of approximately 100 ms and > 1 s). As reported previously, upon depolarization of the holding potential to -40 mV, N current amplitude was significantly reduced and the rapid phase of inactivation all but eliminated (Nowycky, M. C., A. P. Fox, and R. W. Tsien. 1985. Nature. 316:440-443; Fox, A. P., M. C. Nowycky, and R. W. Tsien. 1987a. Journal of Physiology. 394:149-172; Swandulla, D., and C. M. Armstrong. 1988. Journal of General Physiology. 92:197-218; Plummer, M. R., D. E. Logothetis, and P. Hess. 1989. Neuron. 2:1453-1463; Regan, L. J., D. W. Sah, and B. P. Bean. 1991. Neuron. 6:269-280; Cox, D. H., and K. Dunlap. 1992. Journal of Neuroscience. 12:906-914). Such kinetic properties might be explained by a model in which N channels inactivate by both fast and slow voltage-dependent processes. Alternatively, kinetic models of Ca-dependent inactivation suggest that the biphasic kinetics and holding-potential-dependence of N current inactivation could be due to a combination of Ca-dependent and slow voltage-dependent inactivation mechanisms. To distinguish between these possibilities we have performed several experiments to test for the presence of Ca-dependent inactivation. Three lines of evidence suggest that N channels inactivate in a Ca-dependent manner. (a) The total extent of inactivation increased 50%, and the ratio of rapid to slow inactivation increased approximately twofold when the concentration of the Ca2+ buffer, EGTA, in the patch pipette was reduced from 10 to 0.1 mM. (b) With low intracellular EGTA concentrations (0.1 mM), the ratio of rapid to slow inactivation was additionally increased when the extracellular Ca2+ concentration was raised from 0.5 to 5 mM. (c) Substituting Na+ for Ca2+ as the permeant ion eliminated the rapid phase of inactivation. Other results do not support the notion of current-dependent inactivation, however. Although high intracellular EGTA (10 mM) or BAPTA (5 mM) concentrations suppressed the rapid phase inactivation, they did not eliminate it. Increasing the extracellular Ca2+ from 0.5 to 5 mM had little effect on this residual fast inactivation, indicating that it is not appreciably sensitive to Ca2+ influx under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Download Full-text

Intrinsic gating mechanisms of epithelial sodium channels

AJP Cell Physiology ◽

10.1152/ajpcell.00610.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. C646-C650 ◽

Cited By ~ 7

Author(s):

Hong-Long Ji ◽

Catherine M. Fuller ◽

Dale J. Benos

Keyword(s):

Sodium Channels ◽

Xenopus Oocytes ◽

Transmembrane Domain ◽

Receptor Site ◽

Epithelial Sodium Channels ◽

Gating Mechanisms ◽

Putative Receptor ◽

Arginine Residues ◽

Insight Into ◽

Positively Charged

The hypothesis that there is a highly conserved, positively charged region distal to the second transmembrane domain in α-ENaC (epithelial sodium channel) that acts as a putative receptor site for the negatively charged COOH-terminal β- and γ-ENaC tails was tested in mutagenesis experiments. After expression in Xenopus oocytes, α-ENaC constructs in which positively charged arginine residues were converted into negatively charged glutamic acids could not be inhibited by blocking peptides. These observations provide insight into the gating machinery of ENaC.

Download Full-text

The Transmembrane Domain of PDGFR-β Plays An Important Role in ETV6-PDGFR-β Activation

Blood ◽

10.1182/blood.v112.11.5320.5320 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5320-5320

Author(s):

Federica Toffalini ◽

Jean-Baptiste Demoulin

Keyword(s):

Tyrosine Kinases ◽

Transmembrane Domain ◽

Myeloproliferative Neoplasms ◽

Chromosomal Translocations ◽

Wild Type ◽

Sam Domain ◽

Helix Loop Helix ◽

Hybrid Proteins ◽

Tm Domain ◽

Myelomonocytic Leukemia

Abstract The ETV6-PDGFRβ hybrid protein (EPβ) is found in a subset of patients with chronic myelomonocytic leukemia (CMML) associated with eosinophilia. EPβ is the archetype of a larger group of hybrid receptors that are produced by chromosomal translocations of PDGFR genes and cause atypical myeloproliferative neoplasms. In EPβ, the N-terminal portion of the ETV6 transcription factor replaces the PDGFRβ ligand-binding domain and induces the activation of the chimera through its pointed domain (PNT domain, also called helix-loop-helix or SAM domain). This domain mediates ligand-independent EPβ oligomerization, resulting in constitutive tyrosine kinase activity. In addition, we showed that EPβ and other hybrid receptor tyrosine kinases are much more stable than wild-type receptors and that the deletion of the PNT domain induced EPβ protein degradation, suggesting a link between the clustering and the stabilization of the EPβ protein. The PDGFRβ transmembrane domain (TM domain) is retained in EPβ and in most PDGFRβ hybrid proteins that have been described. We observed that the deletion of the TM domain (EPβ-ΔTM mutant) strongly impaired the ability of EPβ to sustain growth factor-independent proliferation of Ba/F3 and 32D cells. The phosphorylation of the mutant protein was also markedly reduced. We confirmed that EPβ is not inserted in membranes but resides in the cytosol, indicating that the PDGFRβ TM domain does not act as a transmembrane domain in EPβ but has a completely different function. The EPβ-ΔTM mutant retained the ability to self-associate in co-immunoprecipitation experiments, but showed a decreased level of polymerization when using cross-linking agents, suggesting that this domain is required for optimal clustering of EPβ. In line with our findings on the PNT domain, the EPβ-ΔTM protein was less stable and, as a result, was expressed at a lower level. In conclusion, we demonstrate that the TM domain plays a role in EPβ activation by promoting the clustering of the protein and by preventing its degradation in cooperation with the PNT domain.

Download Full-text

Unique Light-gated Ion Channel Properties of a Novel Modular Cation Channelrhodopsin from an Evolutionary Important Terrestrial Green Alga

10.21203/rs.3.rs-68371/v1 ◽

2020 ◽

Author(s):

Rintaro Tashiro ◽

Kumari Sushmita ◽

Shoko Hososhima ◽

Sunita Sharma ◽

Suneel Kateriya ◽

...

Keyword(s):

Ion Channel ◽

Mammalian Cells ◽

Transmembrane Domain ◽

Action Spectrum ◽

Closure Rate ◽

C Terminus ◽

Ion Channel Properties ◽

Arginine Residues ◽

Tm Domain ◽

Channel Currents

Abstract Channelrhodopsins are a family of microbial rhodopsins that function as a light-gated ion channel. We report the molecular properties of a novel channelrhodopsin KnRh3 from an evolutionary important filamentous terrestrial alga Klebsormidium nitens. KnRh3 is constituted of a 7-transmembrane domain, followed by a long C-terminus moiety that encodes a peptidoglycan binding domain (FimV). When functionally expressed in mammalian cells, KnRh3 showed light-induced cation channel currents. The maximum action spectrum exhibited was at 430 nm and 460 nm, the former making KnRh3 one of the most blue-shifted channelrhodopsins characterized thus far. The channel closure rate was relatively fast (τ0ff = 10 ms). Surprisingly, photocurrent kinetics were affected by the C-terminus moiety of KnRh3. When this moiety was truncated to various lengths, this prolonged the channel open lifetime by more than 10-fold. We identified two arginine residues, R287 and R291, those are crucial for altering the kinetics. We propose that electrostatic interaction between the 7-TM domain and the C-terminus domain accelerates the photocycle. The most blue-shifted action spectrum of KnRh3 serves as a novel prototype of channelrhodopsin for studying the molecular mechanism of color tuning. In addition, KnRh3 would expand the optogenetics tool kit, especially for when short wavelength excitation is required.

Download Full-text

Slow inward Ca current in frog heart: theoretical evidence against a voltage-dependent inactivation

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y82-172 ◽

1982 ◽

Vol 60 (9) ◽

pp. 1185-1192 ◽

Cited By ~ 3

Author(s):

Rodolphe Fischmeister ◽

Magda Horackova

Keyword(s):

Time Course ◽

Voltage Dependence ◽

Frog Heart ◽

Type Model ◽

Actual Behavior ◽

Ca Current ◽

Recovery From Inactivation ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Theoretical Evidence

The validity of a Hodgkin–Huxley type voltage-dependent inactivation of slow inward Ca current (Isi) was tested in frog heart using a computer simulation. The time course of Isi, was calculated during the development of a frog atrial action potential (AP). With a time constant of inactivation (τf) of 55 ms at a membrane potential (Em) of –15 mV, the variation of Isi was biphasic; after a transient increase followed by a decrease to zero, Isi partially "reactivated" (at the beginning of the AP repolarization phase) and then fully deactivated. The "reactivation" phase of Isi developed whether τf was an increasing, decreasing, U-shaped, or bell-shaped function of Em. The addition of an independent and slower process responsible for the recovery from inactivation only partly suppressed the "reactivation" phase. However, until now there was no experimental evidence supporting such a biphasic variation of Isi during AP repolarization. Thus our results indicate that the Hodgkin–Huxley type model of the voltage-dependence of Isi-inactivation process may not correctly represent the actual behavior of frog cardiac muscle.

Download Full-text

Signal transmission within the P2X2 trimeric receptor

The Journal of General Physiology ◽

10.1085/jgp.201411166 ◽

2014 ◽

Vol 143 (6) ◽

pp. 761-782 ◽

Cited By ~ 7

Author(s):

Batu Keceli ◽

Yoshihiro Kubo

Keyword(s):

Voltage Dependence ◽

Signal Transmission ◽

Atp Binding ◽

Ion Permeation ◽

Wild Type ◽

Extracellular Adenosine ◽

Binding Domains ◽

Voltage Dependent ◽

Pore Opening ◽

Receptor Channel

P2X2 receptor channel, a homotrimer activated by the binding of extracellular adenosine triphosphate (ATP) to three intersubunit ATP-binding sites (each located ∼50 Å from the ion permeation pore), also shows voltage-dependent activation upon hyperpolarization. Here, we used tandem trimeric constructs (TTCs) harboring critical mutations at the ATP-binding, linker, and pore regions to investigate how the ATP activation signal is transmitted within the trimer and how signals generated by ATP and hyperpolarization converge. Analysis of voltage- and [ATP]-dependent gating in these TTCs showed that: (a) Voltage- and [ATP]-dependent gating of P2X2 requires binding of at least two ATP molecules. (b) D315A mutation in the β-14 strand of the linker region connecting the ATP-binding domains to the pore-forming helices induces two different gating modes; this requires the presence of the D315A mutation in at least two subunits. (c) The T339S mutation in the pore domains of all three subunits abolishes the voltage dependence of P2X2 gating in saturating [ATP], making P2X2 equally active at all membrane potentials. Increasing the number of T339S mutations in the TTC results in gradual changes in the voltage dependence of gating from that of the wild-type channel, suggesting equal and independent contributions of the subunits at the pore level. (d) Voltage- and [ATP]-dependent gating in TTCs differs depending on the location of one D315A relative to one K308A that blocks the ATP binding and downstream signal transmission. (e) Voltage- and [ATP]-dependent gating does not depend on where one T339S is located relative to K308A (or D315A). Our results suggest that each intersubunit ATP-binding signal is directly transmitted on the same subunit to the level of D315 via the domain that contributes K308 to the β-14 strand. The signal subsequently spreads equally to all three subunits at the level of the pore, resulting in symmetric and independent contributions of the three subunits to pore opening.

Download Full-text

cAMP binds to closed, inactivated, and open sea urchin HCN channels in a state-dependent manner

The Journal of General Physiology ◽

10.1085/jgp.201812019 ◽

2018 ◽

Vol 151 (2) ◽

pp. 200-213 ◽

Cited By ~ 1

Author(s):

Vinay Idikuda ◽

Weihua Gao ◽

Zhuocheng Su ◽

Qinglian Liu ◽

Lei Zhou

Keyword(s):

Sea Urchin ◽

Transmembrane Domain ◽

Single Point ◽

Single Point Mutation ◽

Fluorescence Signal ◽

Hcn Channels ◽

Dependent Manner ◽

State Dependent ◽

Dependent Inactivation ◽

Voltage Dependent

Hyperpolarization-activated cyclic-nucleotide–modulated (HCN) channels are nonselective cation channels that regulate electrical activity in the heart and brain. Previous studies of mouse HCN2 (mHCN2) channels have shown that cAMP binds preferentially to and stabilizes these channels in the open state—a simple but elegant implementation of ligand-dependent gating. Distinct from mammalian isoforms, the sea urchin (spHCN) channel exhibits strong voltage-dependent inactivation in the absence of cAMP. Here, using fluorescently labeled cAMP molecules as a marker for cAMP binding, we report that the inactivated spHCN channel displays reduced cAMP binding compared with the closed channel. The reduction in cAMP binding is a voltage-dependent process but proceeds at a much slower rate than the movement of the voltage sensor. A single point mutation in the last transmembrane domain near the channel’s gate, F459L, abolishes inactivation and concurrently reverses the response to hyperpolarizing voltage steps from a decrease to an increase in cAMP binding. ZD7288, an open channel blocker that interacts with a region close to the activation/inactivation gate, dampens the reduction of cAMP binding to inactivated spHCN channels. In addition, compared with closed and “locked” closed channels, increased cAMP binding is observed in channels purposely locked in the open state upon hyperpolarization. Thus, the order of cAMP-binding affinity, measured by the fluorescence signal from labeled cAMP, ranges from high in the open state to intermediate in the closed state to low in the inactivated state. Our work on spHCN channels demonstrates intricate state-dependent communications between the gate and ligand-binding domain and provides new mechanistic insight into channel inactivation/desensitization.

Download Full-text