Structural characterisation of a slowly activating potassium channel (IsK).

IsK (minK) is a small protein found in epithelial cells [I], heart[2] and uterus[3]. When expressedinXenr~~rrsoocytesII1 and HEK293 cells 141, IsK protein induces a very slowly activating, voltage-dependent, inwardly rectifying potassium current, reminiscent to that of the slow component of the cardiac delayed rectifier [S]. IsK protein consists of 130 amino acids, possessing just one putative transmembrane domain and bearing no sequence homology to the other multi-transmembrane domained potassium channels [ I ] . Current models predict the amino and carboxyl terminal domains to be extra and intracellular respectively [I] . Structure-function studies using site-directed mutagenesis (SDM) support the proposal that the IsK protein forms an integral part of the potassium channel itself [6]. In order to gain information on the functional mechanism of this channel its three dimensional structure needs to be determined. Fourier transform infrared (FT-IR) spectroscopy permits the secondary structure of proteins or peptides to be determined in an aqueous or phospholipid environment [7,8] and therefore in a physiologically relevant environment. In order to investigate the structure of IsK protein we have synthesised polypeptides corresponding to the predicted transmembrane domain and the carboxyl terminal domain. The secondary structure of these peptides was determined using FT-IR spectroscopy. The two peptides (sequences shown in Fig.lA and Fig.lB) were synthesised individually on an automated peptide synthesiser Rainin PS3 (Protein Technologies, Inc.) by a step-wise solid-phase procedure [8). The polypeptides were cleaved, and purified isocratically on a reverse-phase HPLC column [8]. The amino acid composition of the synthetic polypeptides was confirmed by amino acid analysis. Both polypeptides were insoluble in H,O hence it was not possible to obtain their spectra in aqueous solution. However we were able to record spectra of the polypeptide corresponding to the transmembrane domain in dimyristoyl L-a-phosphatidyl choline (DMPC) vesicles and the spectra of the carboxyl terminal polypeptide in SDS micelles. The buffer employed in this study was H,O phosphate-buffered saline (pH 7.4) and the polypeptide to SDS or DMPC molar ratio was 1:45. Samples were placed in lop1 calcium fluoride sample cell and infrared spectra were recorded using a 1750 Perkin-Elmer FTIR spectrometer continuously purged with dry air and maintained at 3OoC with a circulating water bath. For each sample 400 scans were averaged at a resolution of 4cm.'. The polypeptide concentration used for the FT-IR measurements was 1 5mg/ml. Absorbance spectra of the polypeptides were obtained by digital subtraction of a spectrum of H,O PBS. Detailed analysis of the amide I band was carried out using second derivative procedures [7]. Transmembrane peptide: The FT-IR second derivative spectrum of the transmembrane peptide in DMPC ('H,O) is shown in Figure 1 A. The strong bands at 1727cm.l and 1743cm-' arise from the vibration of the carbonyl ester proups of DMPC. The main amide I band is centred at 16S4cm.. This can be assigned to ahelical structure. There are some minor components at 1627cm-' and 1674cm" which occur in a region normally associated with aggregated or 0-type structures. In general, the peptide in DMPC adopts a predominantly a-helical structure. Carboxyl terminal peptide: The FT-IR second derivative spectrum of the carboxyl terminal domain in SDS ('H,O) is s h o y in Figure 1B. The main amide I component is centred at 1654cm' indicating the presence of a-helical structure. The band at 1638cm" can be attributed to absorption of @-sheet structure. Our FT-IR spectra of the transmembrane domain polypeptide, performed in phospholipid micelles, demonstrate that the putative transmembrane domain of IsK protein forms a predominantly a-helical structure in phospholipid membranes thereby agreeing with circular dichroism studies performed on synthetic peptides corresponding to the transmembrane domain in organic solvents [9]. These results also support SDM studies Flg. 1A UI 01 Iraiieiiaiiibraiic pcptlde RDDSKLEALYILMVLGFFGFF ILGIMLSY