Secondary structure, membrane localization, and coassembly within phospholipid membranes of synthetic segments derived from the N- and C-termini regions of the ROMK1 K+channel

Voltage-gated K+ channels are tetramers with each subunit containing six (S1–S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5–S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1–S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K+ channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of α-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting α-helical secondary structure. In addition, both the S1–S2 and S3–S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.

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Secondary structure analysis of the putative membrane-associated domains of the inward rectifier K+ channel ROMK1

Biochemical Journal ◽

10.1042/bj3350375 ◽

1998 ◽

Vol 335 (2) ◽

pp. 375-380 ◽

Cited By ~ 12

Author(s):

Stephen P. BRAZIER ◽

Bala. RAMESH ◽

Parvez I. HARIS ◽

David C. LEE ◽

Surjit K. S. SRAI

Keyword(s):

Gradient Centrifugation ◽

Helical Structure ◽

Structural Data ◽

Cd Spectroscopy ◽

Inward Rectifier ◽

K Channel ◽

Phospholipid Membranes ◽

K Channels ◽

Voltage Gated ◽

Secondary Structure Analysis

The inward rectifier K+ channels contain two putative membrane-spanning domains per subunit (M1, M2) and a ‘pore ’ (P) region, which is similar to the H5 domain of voltage-gated K+ channels. Here we have used Fourier transform infrared (FTIR) and CD spectroscopy to analyse the secondary structures of synthetic peptides corresponding to the M1, M2 and P regions of ROMK1 in aqueous solution, in organic solvents and in phospholipid membranes. A previous CD study was unable to provide any structural data on a similar P peptide [Ben-Efraim and Shai (1997) Biophys. J. 72, 85–96]. However, our FTIR and CD spectroscopic analyses indicate that this peptide adopts an α-helical structure when reconstituted into dimyristoyl phosphatidylcholine vesicles and lysophosphatidyl choline (LPC) micelles as well as in trifluoroethanol (TFE) solvent. This result is in good agreement with a previous study on a peptide corresponding to the pore domain of a voltage-gated K+ channel [Haris, Ramesh, Sansom, Kerr, Srai and Chapman (1994) Protein Eng. 7, 255–262]. FTIR spectra of the M1 peptide in LPC micelles displayed a strong absorbance characteristic of an intermolecular β-sheet structure, suggesting aggregation of the M1 peptide. Sucrose gradient centrifugation was used to separate aggregated peptide from peptide incorporated into micelles in an unaggregated manner; subsequent analysis by FTIR suggested that the M1 peptide adopted an α-helical structure when incorporated into phospholipid membranes. FTIR and CD spectra of the M2 peptide in phospholipids and high concentrations of TFE suggest that this peptide adopts an α-helical structure. The structural data obtained in these experiments have been used to propose a model for the structure of the membrane-associated core (M1-P-M2) of the inward rectifier K+ channel protein.

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Vacuolar membrane localization of the Arabidopsis ‘two‐pore’ K + channel KCO1

The Plant Journal ◽

10.1046/j.1365-313x.2002.01260.x ◽

2002 ◽

Vol 29 (6) ◽

pp. 809-820 ◽

Cited By ~ 78

Author(s):

Katrin Czempinski ◽

Jean‐Marie Frachisse ◽

Christophe Maurel ◽

Helene Barbier‐Brygoo ◽

Bernd Mueller‐Roeber

Keyword(s):

Vacuolar Membrane ◽

K Channel ◽

Membrane Localization

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Synthetic putative transmembrane region of minimal potassium channel protein (minK) adopts an α-helical conformation in phospholipid membranes

Biochemical Journal ◽

10.1042/bj3250475 ◽

1997 ◽

Vol 325 (2) ◽

pp. 475-479 ◽

Cited By ~ 12

Author(s):

Eric A. J. MERCER ◽

Geoffrey W. ABBOTT ◽

Stephen P. BRAZIER ◽

Bala RAMESH ◽

Parvez I. HARIS ◽

...

Keyword(s):

Secondary Structure ◽

Potassium Channel ◽

Transmembrane Domain ◽

Cd Spectroscopy ◽

Helical Conformation ◽

Phospholipid Membranes ◽

Transmembrane Region ◽

Aqueous Buffer ◽

Channel Protein ◽

Β Sheet

Minimal potassium channel protein (minK) is a potassium channel protein consisting of 130 amino acids, possessing just one putative transmembrane domain. In this study we have synthesized a peptide with the amino acid sequence RDDSKLEALYILMVLGFFGFFTLGIMLSYI, containing the putative transmembrane region of minK, and analysed its secondary structure by using Fourier-transform IR and CD spectroscopy. The peptide was virtually insoluble in aqueous buffer, forming intermolecular β-sheet aggregates. On attempted incorporation of the peptide into phospholipid membranes with a method involving dialysis, the peptide adopted a predominantly intermolecular β-sheet conformation identical with that of the peptide in aqueous buffer, in agreement with a previous report [Horvàth, Heimburg, Kovachev, Findlay, Hideg and Marsh, (1995) Biochemistry 34, 3893–3898]. However, by using an alternative method of incorporating the peptide into phospholipid membranes we found that the peptide adopted a predominantly α-helical conformation, a finding consistent with various proposed structural models. These observed differences in secondary structure are due to artifacts of aggregation of the peptide before incorporation into lipid.

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