Determinants of cardiac Na+/Ca2+exchanger temperature dependence: NH2-terminal transmembrane segments

2002 ◽  
Vol 283 (2) ◽  
pp. C512-C520 ◽  
Author(s):  
Christian Marshall ◽  
Chadwick Elias ◽  
Xiao-Hua Xue ◽  
Hoa Dinh Le ◽  
Alexander Omelchenko ◽  
...  
Keyword(s):  
Temperature Dependence ◽  
Temperature Sensitivity ◽  
Xenopus Oocytes ◽  
Energy Of Activation ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Inhibitory Peptide ◽  
Transmembrane Segments ◽  
Lower Temperature ◽  
Patch Technique

The cardiac Na+/Ca2+ exchanger (NCX) in trout exhibits profoundly lower temperature sensitivity in comparison to the mammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout and canine NCX cDNA and expressed in Xenopus oocytes. NCX-mediated currents were measured at 7, 14, and 30°C using the giant excised-patch technique. By using this approach, the differential temperature dependence of NCX was found to reside within the NH2-terminal region of the molecule. Specifically, we found that ∼75% of the Na+/Ca2+ exchange differential energy of activation is attributable to sequence differences in the region that include the first four transmembrane segments, and the remainder is attributable to transmembrane segment five and the exchanger inhibitory peptide site.

Temperature dependence of cloned mammalian and salmonid cardiac Na+/Ca2+ exchanger isoforms

2001 ◽  
Vol 281 (3) ◽  
pp. C993-C1000 ◽  
Author(s):  
Chadwick L. Elias ◽  
Xiao-Hua Xue ◽  
Christian R. Marshall ◽  
Alexander Omelchenko ◽  
Larry V. Hryshko ◽  
...  
Keyword(s):  
Steady State ◽  
Temperature Dependence ◽  
Effect Of Temperature ◽  
Inactivation Kinetics ◽  
Wild Type ◽  
Current Decay ◽  
Transmembrane Segments ◽  
Dependent Inactivation ◽  
Patch Technique

The cardiac Na+/Ca2+ exchanger (NCX), an important regulator of cytosolic Ca2+ concentration in contraction and relaxation, has been shown in trout heart sarcolemmal vesicles to have high activity at 7°C relative to its mammalian isoform. This unique property is likely due to differences in protein structure. In this study, outward NCX currents ( I NCX) of the wild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressed in oocytes were measured to explore the potential contributions of regulatory vs. transport mechanisms to this observation. cRNA was transcribed in vitro from both wild-type cDNA and was injected into Xenopus oocytes. I NCX of NCX-TR1.0 and NCX1.1 were measured after 3–4 days over a temperature range of 7–30°C using the giant excised patch technique. The I NCX for both isoforms exhibited Na+-dependent inactivation and Ca2+-dependent positive regulation. The I NCX of NCX1.1 exhibited typical mammalian temperature sensitivities with Q10 values of 2.4 and 2.6 for peak and steady-state currents, respectively. However, the I NCX of NCX-TR1.0 was relatively temperature insensitive with Q10values of 1.2 and 1.1 for peak and steady-state currents, respectively. I NCX current decay was fit with a single exponential, and the resultant rate constant of inactivation (λ) was determined as a function of temperature. As expected, λ decreased monotonically with temperature for both isoforms. Although λ was significantly greater in NCX1.1 compared with NCX-TR1.0 at all temperatures, the effect of temperature on λ was not different between the two isoforms. These data suggest that the disparities in I NCX temperature dependence between these two exchanger isoforms are unlikely due to differences in their inactivation kinetics. In addition, similar differences in temperature dependence were observed in both isoforms after α-chymotrypsin treatment that renders the exchanger in a deregulated state. These data suggest that the differences in I NCX temperature dependence between the two isoforms are not due to potential disparities in either the I NCX regulatory mechanisms or structural differences in the cytoplasmic loop but are likely predicated on differences within the transmembrane segments.

Both the wild type and a functional isoform of CFTR are expressed in kidney

1996 ◽  
Vol 270 (6) ◽  
pp. F1038-F1048 ◽  
Author(s):  
M. M. Morales ◽  
T. P. Carroll ◽  
T. Morita ◽  
E. M. Schwiebert ◽  
O. Devuyst ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Transmembrane Domain ◽  
Renal Medulla ◽  
Regulatory Domain ◽  
Wild Type ◽  
Transmembrane Segments ◽  
Binding Domains ◽  
Cl Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

scholarly journals Interaction of PomB with the Third Transmembrane Segment of PomA in the Na+-Driven Polar Flagellum of Vibrio alginolyticus

2004 ◽  
Vol 186 (16) ◽  
pp. 5281-5291 ◽  
Author(s):  
Toshiharu Yakushi ◽  
Shingo Maki ◽  
Michio Homma
Keyword(s):  
Marine Bacterium ◽  
Vibrio Alginolyticus ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Mutant Proteins ◽  
Transmembrane Segments ◽  
The Third ◽  
Close Proximity ◽  
Flagellar Rotation ◽  
Lines Of Evidence

ABSTRACT The marine bacterium Vibrio alginolyticus has four motor components, PomA, PomB, MotX, and MotY, responsible for its Na+-driven flagellar rotation. PomA and PomB are integral inner membrane proteins having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel complex. First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB, and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent. Secondly, the Cys substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34, Asp-170, and Ala-178) were combined. Cross-linked products were detected for the combination of PomB-S38C and PomA-D170C mutant proteins. The Cys substitutions in the periplasmic boundaries of PomA TM3 (from Met-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double mutants. Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it. Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it was recovered by reducing reagent. The strain with this combination showed a lower affinity for Na+ than the wild-type combination. PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of PomA TM3 and the PomB TM, also formed the cross-linked product. From these lines of evidence, we infer that TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between these two TMs is required for coupling of Na+ conduction to flagellar rotation.

scholarly journals Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1

2004 ◽  
Vol 379 (1) ◽  
pp. 31-38 ◽  
Author(s):  
Emily R. SLEPKOV ◽  
Signy CHOW ◽  
M. Joanne LEMIEUX ◽  
Larry FLIEGEL
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Integral Membrane Protein ◽  
Amide Hydrogen ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Mutant Proteins ◽  
Transmembrane Segments ◽  
Induced Changes

NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167→Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE.

scholarly journals Role of the N- and C-terminal regions of FliF, the MS ring component in Vibrio flagellar basal body

2021 ◽  
Author(s):  
Seiji Kojima ◽  
Hiroki Kajino ◽  
Keiichi Hirano ◽  
Yuna Inoue ◽  
Hiroyuki Terashima ◽  
...  
Keyword(s):  
Basal Body ◽  
Ring Formation ◽  
Terminal Region ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Bacterial Flagellum ◽  
Transmembrane Segments ◽  
Ring Assembly ◽  
C Terminus ◽  
N Terminus

AbstractThe MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues at the N-terminus (ΔN30 and ΔN50), and 83 (ΔC83) or 110 residues (ΔC110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing ΔN30FliF or ΔN50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without an additional factor or FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for ΔC83FliF as with wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly.ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

Identification of Cys140 in helix 4 as an exofacial cysteine residue within the substrate-translocation channel of rat equilibrative nitrobenzylthioinosine (NBMPR)-insensitive nucleoside transporter rENT2

2001 ◽  
Vol 353 (2) ◽  
pp. 387-393 ◽  
Author(s):  
Sylvia Y. M. YAO ◽  
Manickavasagam SUNDARAM ◽  
Eugene G. CHOMEY ◽  
Carol E. CASS ◽  
Stephen A. BALDWIN ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Cysteine Residue ◽  
Integral Membrane Proteins ◽  
Structural Domain ◽  
Nucleoside Transporter ◽  
Vasoactive Drugs ◽  
Wild Type ◽  
Functional Protein ◽  
Equilibrative Nucleoside Transporter ◽  
Transmembrane Segments

The human and rat equilibrative nucleoside transporter proteins hENT1, rENT1, hENT2 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs), and are distinguished functionally by differences in transport of nucleobases and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs. In the present study, we have produced recombinant hENT1, rENT1, hENT2 and rENT2 in Xenopus oocytes and investigated uridine transport following exposure to the impermeant thiol-reactive reagent p-chloromercuriphenyl sulphonate (PCMBS). PCMBS caused reversible inhibition of uridine influx by rENT2, but had no effect on hENT1, hENT2 or rENT1. This difference correlated with the presence in rENT2 of a unique Cys residue (Cys140) in the outer half of TM4 that was absent from the other ENTs. Mutation of Cys140 to Ser produced a functional protein (rENT2/C140S) that was insensitive to inhibition by PCMBS, identifying Cys140 as the exofacial Cys residue in rENT2 responsible for PCMBS inhibition. Uridine protected wild-type rENT2 against PCMBS inhibition, suggesting that Cys140 in TM4 lies within or is closely adjacent to the substrate-translocation channel of the transporter. TM4has been shown previously to be within a structural domain (TMs 3Ő6) responsible for interactions with NBMPR, vasoactive drugs and nucleobases.

scholarly journals Role of the N- and C-Terminal Regions of FliF, the MS Ring Component in the Vibrio Flagellar Basal Body

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Seiji Kojima ◽  
Hiroki Kajino ◽  
Keiichi Hirano ◽  
Yuna Inoue ◽  
Hiroyuki Terashima ◽  
...  
Keyword(s):  
Basal Body ◽  
Ring Formation ◽  
Terminal Region ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Transmembrane Segments ◽  
C Terminus ◽  
N Terminus

ABSTRACT The MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane in Vibrio species. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues from the N terminus (ΔN30 and ΔN50) and 83 (ΔC83) or 110 residues (ΔC110) at the C terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When coexpressing ΔN30FliF or ΔN50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF in the MS ring fraction, suggesting that the N terminus interacts with FlhF. MS ring formation is probably inefficient without FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for ΔC83FliF as for wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles, the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly. IMPORTANCE The bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N- and C-terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C terminus is essential for motor formation or function.

scholarly journals Twelve-Transmembrane-Segment (TMS) Version (ΔTMS VII-VIII) of the 14-TMS Tet(L) Antibiotic Resistance Protein Retains Monovalent Cation Transport Modes but Lacks Tetracycline Efflux Capacity

2001 ◽  
Vol 183 (8) ◽  
pp. 2667-2671 ◽  
Author(s):  
Jie Jin ◽  
Arthur A. Guffanti ◽  
Catherine Beck ◽  
Terry A. Krulwich
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Monovalent Cation ◽  
Cation Transport ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Transmembrane Segments ◽  
Resistance Protein ◽  
Transport Modes ◽  
Better Than

ABSTRACT A “Tet(L)-12” version of Tet(L), a tetracycline efflux protein with 14 transmembrane segments (TMS), was constructed by deletion of two central TMS. Tet(L)-12 catalyzed Na+/H+antiport and antiport with K+ as a coupling ion as well as or better than wild-type Tet(L) but exhibited no tetracycline-Me2+/H+ antiport inEscherichia coli vesicles.

scholarly journals Cloning, expression, and characterization of the trout cardiac Na+/Ca2+exchanger

1999 ◽  
Vol 277 (4) ◽  
pp. C693-C700 ◽  
Author(s):  
Xiao-Hua Xue ◽  
Larry V. Hryshko ◽  
Debora A. Nicoll ◽  
Kenneth D. Philipson ◽  
Glen F. Tibbits
Keyword(s):  
Leader Peptide ◽  
Cytoplasmic Loop ◽  
Inhibitory Peptide ◽  
Reading Frame ◽  
Transmembrane Segments ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Hydropathy Plot ◽  
Dependent Inactivation ◽  
Patch Technique ◽  
High Level

Isoform 1 of the cardiac Na+/Ca2+exchanger (NCX1) is an important regulator of cytosolic Ca2+ concentration in contraction and relaxation. Studies with trout heart sarcolemmal vesicles have shown NCX to have a high level of activity at 7°C, and this unique property is likely due to differences in protein structure. In this study, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAP II cDNA library constructed from rainbow trout ( Oncorhynchus mykiss) heart RNA. The NCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plot indicates the protein contains 12 hydrophobic segments (of which the first is predicted to be a cleaved leader peptide) and a large cytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have nine transmembrane segments. The sequences demonstrated to be the exchanger inhibitory peptide site and the regulatory Ca2+ binding site in the cytoplasmic loop of mammalian NCX1 are almost completely conserved in NCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3–4 days currents were measured by the giant excised patch technique. NCX-TR1 currents measured at ∼23°C demonstrated Na+-dependent inactivation and Ca2+-dependent activation in a manner qualitatively similar to that for NCX1 currents.

scholarly journals A Role for the S0 Transmembrane Segment in Voltage-dependent Gating of BK Channels

2007 ◽  
Vol 129 (3) ◽  
pp. 209-220 ◽  
Author(s):  
Olga M. Koval ◽  
Yun Fan ◽  
Brad S. Rothberg
Keyword(s):  
Channel Gating ◽  
Large Change ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
K Channel ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Amino Terminal ◽  
Transmembrane Segments

BK (Maxi-K) channel activity is allosterically regulated by a Ca2+ sensor, formed primarily by the channel's large cytoplasmic carboxyl tail segment, and a voltage sensor, formed by its transmembrane helices. As with other voltage-gated K channels, voltage sensing in the BK channel is accomplished through interactions of the S1–S4 transmembrane segments with the electric field. However, the BK channel is unique in that it contains an additional amino-terminal transmembrane segment, S0, which is important in the functional interaction between BK channel α and β subunits. In this study, we used perturbation mutagenesis to analyze the role of S0 in channel gating. Single residues in the S0 region of the BK channel were substituted with tryptophan to give a large change in side chain volume; native tryptophans in S0 were substituted with alanine. The effects of the mutations on voltage- and Ca2+-dependent gating were quantified using patch-clamp electrophysiology. Three of the S0 mutants (F25W, L26W, and S29W) showed especially large shifts in their conductance–voltage (G-V) relations along the voltage axis compared to wild type. The G-V shifts for these mutants persisted at nominally 0 Ca2+, suggesting that these effects cannot arise simply from altered Ca2+ sensitivity. The basal open probabilities for these mutants at hyperpolarized voltages (where voltage sensor activation is minimal) were similar to wild type, suggesting that these mutations may primarily perturb voltage sensor function. Further analysis using the dual allosteric model for BK channel gating showed that the major effects of the F25W, L26W, and S29W mutations could be accounted for primarily by decreasing the equilibrium constant for voltage sensor movement. We conclude that S0 may make functional contact with other transmembrane regions of the BK channel to modulate the equilibrium between resting and active states of the channel's voltage sensor.

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