Secondary Structure and Membrane Localization of Synthetic Segments and a Truncated Form of the IsK (minK) Protein

Ben-Efraim, Iris; Strahilevitz, Jacob; Bach, D.; Shai, Yechiel

doi:10.1021/bi00188a028

Cited by 21 publications

(22 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…No currents could be evoked, when internally applied C-27 was tested upon hyperpolarization or when it was perfused in the external bath solution (n ϭ 4, 3 batches). A 34-mer hydrophilic peptide (N-34), spanning an amino-terminal region of the rat IsK sequence (position 10 -43) (22), did not evoke any current when injected into the oocyte (at 200 M; n ϭ 5, 3 batches). Other non IsK peptides such as P1, P2, and P3 ( Fig.…”

Section: Resultsmentioning

confidence: 98%

“…A 27-mer hydrophilic peptide (C-27), spanning a carboxyl-terminal region of the rat IsK sequence (positions 68 -94) (22) and located immediately downstream from the transmembrane segment, was synthesized and microinjected into Xenopus oocytes (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…Peptide Synthesis and Purification-Peptides were synthesized by a solid-phase method on pyridine-2-aldoximine methyl-amino acid resin (0.15 meq) (21), as described previously (22). The peptides were subjected to amino acid analysis to confirm their composition.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Cytoplasmic and Extracellular IsK Peptides Activate Endogenous K and Cl Channels in Xenopus Oocytes

Ben-Efraim

Shai

Attali

1996

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The last few years have been an exciting time for our understanding of the molecular structure and diversity of voltagegated K ϩ channels belonging to the Shaker-like superfamily (for review, see Refs. 1-5). Contrastingly, since its original cloning by expression in Xenopus oocytes, the nature of the IsK protein (or minK) remained a mystery (for review, see Refs. 6 and 7). From a structural point of view, IsK is an exception to the family of K ϩ channels. IsK is a 14.5-kDa type III glycoprotein with one transmembrane segment which has no sequence homology with other cloned functional channels (8). It is a member of a family of small bitopic membrane proteins which induce upon expression in Xenopus oocytes slowly activating voltage-dependent currents (6,7,9). This family includes phospholemman (10), influenza virus M2 protein (11), CHIF (12), and Mat-8 (13). When expressed in Xenopus oocytes or in HEK 293 cells, the IsK protein evokes a unique slowly activating, voltage-dependent K ϩ -selective current that closely resembles the slow component I Ks of the cardiac delayed rectifier (8,14). Two main hypotheses concerning the nature of this protein were raised. The first was that IsK alone is sufficient to form a voltage-gated K ϩ channel, because mutations in the transmembrane domain altered the gating of the K ϩ current expressed in Xenopus oocytes and changed the relative permeabilities of NH 4 ϩ and Cs ϩ versus K ϩ (15, 16). However, attempts to express IsK as a K ϩ channel in a variety of host cells and after lipid bilayer reconstitution have failed (17). The second hypothesis was that IsK forms functional K ϩ channels by association with an endogenous oocyte factor or with pre-existing silent channels (18 -20). Furthermore, IsK mutagenesis suggested that the amino-terminal domain is critical for the induction of Cl Ϫ currents while the carboxyl-terminal domain is critical for the activation of K ϩ channel activity (18). These findings hinted at the possibility that IsK is a regulatory subunit of heteromultimeric channel complexes rather than a channel per se.To test this hypothesis, synthetic IsK hydrophilic peptides were applied to untreated Xenopus oocytes. In agreement with IsK membrane topology, we show here that internal or external application of IsK peptides derived from the carboxyl-and amino-terminal domains are sufficient to activate slow K ϩ or Cl Ϫ currents, respectively. The peptide-activated channels displayed characteristics similar to those exhibited by the native IsK protein, namely voltage dependence, activation kinetics, ion selectivity, and pharmacology. Our data provide clear evidence for the nature of IsK as a prototypic member of a family of short membrane transport proteins with regulatory function. EXPERIMENTAL PROCEDURESPeptide Synthesis and Purification-Peptides were synthesized by a solid-phase method on pyridine-2-aldoximine methyl-amino acid resin (0.15 meq) (21), as described previously (22). The peptides were subjected to amino acid analysis to confirm their composition.Electrop...

show abstract

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Cytoplasmic and Extracellular IsK Peptides Activate Endogenous K and Cl Channels in Xenopus Oocytes

Ben-Efraim

Shai

Attali

1996

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, our investigation showed that while the NT domain is predominantly non-ordered in aqueous solution at 20 o C, increased temperature or interaction with lipid increases the tendency to form helix, perhaps by adoption of some 3 10 helix, indicating conformational flexibility. This suggests some structural similarity with MinK NT domain, which adopts a predominantly helical conformation in methanol as determined by CD spectroscopy [35]. Furthermore, results from fluorescence emission spectroscopy suggest that MinK NT partially inserts into phosphatidylcholine vesicles, although it was not reported whether this is accompanied by any structural switch [36].…”

Section: Mirp1 N-terminal (Nt) Extracellular Domainmentioning

confidence: 99%

Secondary Structure of the MiRP1 (KCNE2) Potassium Channel Ancillary Subunit

Abbott

Ramesh²,

Srai³

2008

PPL

View full text Add to dashboard Cite

MiRP1 (encoded by the KCNE2 gene) is one of a family of five single transmembrane domain voltage-gated potassium (Kv) channel ancillary subunits currently under intense scrutiny to establish their position in channel complexes and elucidate alpha subunit contact points, but its structure is unknown. MiRP1 mutations are associated with inherited and acquired cardiac arrhythmia. Here, synthetic peptides corresponding to human MiRP1 (full-length and separate domains) were structurally analyzed using FTIR and CD spectroscopy. The N-terminal (extracellular) domain was soluble and predominantly non-ordered in aqueous media, but predominantly alpha-helical in L-alpha-lysophosphatidylcholine (LPC) micelles. The MiRP1 transmembrane domain was predominantly a mixture of alpha-helix and non-ordered structure in LPC micelles, with a minor contribution from non-aggregated beta-strand. The intracellular C-terminal domain was insoluble in aqueous solution; reconstitution into non-aqueous environments resulted in solubility and adoption of increasing amounts of alpha-helix, with the solvent order sodium dodecyl sulphate < dimyristoyl L-alpha-phosphatidylcholine (DMPC) < LPC < trifluoroethanol. Correlation of secondary structure changes with lipid transition temperature during heating suggested that the MiRP1 C-terminus incorporates into DMPC bilayers. Full-length MiRP1 was soluble in SDS micelles and calculated to contain 34% alpha-helix, 23% beta-strand and 43% non-ordered structure in this environment, as determined by CD spectroscopy. Thus, MiRP1 is highly dependent upon hydrophobic interaction via lipid and/or protein contacts for adoption of ordered structure without nonspecific aggregation, consistent with a role as a membrane-spanning subunit within Kv channel complexes. These data will provide a structural framework for ongoing mutagenesis-based in situ structure-function studies of MiRP1 and its relatives.

show abstract

“…It has been suggested that several I~K subunits may aggregate together to directly form a pore-containing structure [7]. An alternative proposal is that IsK itself is not a channel forming protein but acts as a modulator of endogenous oocyte channels [8,9] In this study we have investigated the functional role of a region of the putative transmembrane domain of IsK by constructing several site-directed mutants, in which individual amino acid residues have been substituted by cysteine, followed by expression in Xenopus oocytes. …”

mentioning

confidence: 99%

Changes in activation gating of I_sK potassium currents brought about by mutations in the transmembrane sequence

et al. 1994

View full text Add to dashboard Cite

Expression of the rat kidney IsK protein in Xenopus oocytes produces slowly-activating potassium channel currents. We have investigated the relationship between structure and function of the single putative membrane-spanning domain using site-directed mutagenesis. Six mutants were constructed in which consecutive individual amino acids (53 to 58) of the transmembrane region were substituted by cysteine. Expression of four of these mutants in Xenopus oocytes resulted in currents which were similar to wild-type. However, for one mutant (position 55) activation curves were shifted in a hyperpolarising direction and for another mutant (position 58) activation curve were shifted in a depolarising direction. This suggests that the hydrophobic phenylalanine residues at positions 55 and 58 may play a critical role in I,K activation gating. This spacing of functional amino acids at every third residue may indicate an or-helical conformation for the membrane-spanning domain of I,K. Furthermore, these results also indicate that one face of the helix may represent a region of subunit association. Key words." Potassium channel; Delayed rectifier l.~u~onPotassium channels play an essential role in the generation of electrical responses of all excitable cells [1]. Different classes of potassium channel protein have been categorised by their molecular structure. One type, exemplified by the delayed rectifier potassium channel, consist of proteins which have a relatively large molecular weight (up to around 100 kDa) and possess six putative transmembrane segments with a characteristic H5 pore-forming region [2]. Recently, several inwardly rectifying potassium channels have also been sequenced which appear to be rather similar to this type in structure except that they only seem to possess two putative transmembrane segments [3]. Much attention has been focused on the above channel types which has widened our insight into their structurefunction relationships [4]. The subject of this study, however, is the further type of potassium channel known as IsK or minK which has a smaller molecular weight (around 15 kDa), possessing only around 130 amino acids [5]. Much less has been reported concerning the relationship between structure and function for this channel type.Hydropathy analysis of the amino acid sequence of Is~¢ from rat kidney suggests a single putative transmembrane sequence and current models predict an extracellular amino-terminal domain and an intracellular carboxyl-terminal domain [5]. Expression of I~K in Xenopus oocytes [5] and in HEK293 cells [6] has been shown to induce a very slowly-activating voltagedependent potassium current with no inactivation. It is unclear, however, how the protein is able to evoke potassium currents. Two mechanisms have been proposed. It has been suggested that several I~K subunits may aggregate together to directly form a pore-containing structure [7]. An alternative proposal is that IsK itself is not a channel forming protein but acts as a modulator of endogenous oocyte channels...

show abstract

Secondary Structure and Membrane Localization of Synthetic Segments and a Truncated Form of the IsK (minK) Protein

Cited by 21 publications

References 45 publications

Cytoplasmic and Extracellular IsK Peptides Activate Endogenous K and Cl Channels in Xenopus Oocytes

Cytoplasmic and Extracellular IsK Peptides Activate Endogenous K and Cl Channels in Xenopus Oocytes

Secondary Structure of the MiRP1 (KCNE2) Potassium Channel Ancillary Subunit

Changes in activation gating of I_sK potassium currents brought about by mutations in the transmembrane sequence

Contact Info

Product

Resources

About

Secondary Structure and Membrane Localization of Synthetic Segments and a Truncated Form of the IsK (minK) Protein

Cited by 21 publications

References 45 publications

Cytoplasmic and Extracellular IsK Peptides Activate Endogenous K and Cl Channels in Xenopus Oocytes

Cytoplasmic and Extracellular IsK Peptides Activate Endogenous K and Cl Channels in Xenopus Oocytes

Secondary Structure of the MiRP1 (KCNE2) Potassium Channel Ancillary Subunit

Changes in activation gating of IsK potassium currents brought about by mutations in the transmembrane sequence

Contact Info

Product

Resources

About

Changes in activation gating of I_sK potassium currents brought about by mutations in the transmembrane sequence