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

Abstract

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...

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

“…The demonstration of an α-helical conformation for the peptide in phospholipid membranes is in good agreement with a proposed model based on site-directed mutagenesis studies. Point mutations reveal the importance in activation gating of every third amino acid residue in the transmembrane region which, in an α-helical conformation, would align these amino acid residues along one face of the helix, forming one side of the pore as part of a multimer [6]. An α-helical conformation of the 28-residue transmembrane spanning region of minK would traverse the membrane once, placing the N-terminus on the extracellular side and the C-terminus on the intracellular side of the membrane.…”

“…Evidence suggests that the N-and Cterminal domains serve to regulate the minK channel [5]. Point mutations in the putative transmembrane domain of minK resulting in altered gating and ion selectivity strongly suggest that minK forms an integral part of the channel [6].…”

KCNE2 is colocalized with KCNQ1 and KCNE1 in cardiac myocytes and may function as a negative modulator of IKs current amplitude in the heart

“…24 Nuclear magnetic resonance spectroscopy of a peptide corresponding to KCNE1's transmembrane domain suggests that this region may adopt an ␣-helical structure when embedded in the membrane lipid bilayer. 25 Importantly, positions in the KCNE1 transmembrane domain where mutations can impact on KCNE1 modulation of KCNQ1 pore conductance and/or gating kinetics are found to cluster to one face of the putative KCNE1 ␣-helix, 24,26,27 suggesting that this may be part of the "KCNQ1-interacting" surface. 25 It has been suggested that the side chain at position 331 on KCNQ1's S6 (inner helix of the pore domain) may be in close proximity to side chains at positions 54 and 55 on KCNE1's transmembrane domain, 28 providing a further constraint on how KCNE1's transmembrane domain can interact with KCNQ1's pore domain.…”

Conformation and Ion-Channeling Activity of a 27-Residue Peptide Modeled on the Single-Transmembrane Segment of the IsK (minK) Protein,

“…The self-assembly of IsK in vivo is such that glycines 53 and 56 are in contact with the lipid chains in the cell membrane, while phenylalanines 54, 55, 57, and 58 are not (58). From the model of the helical K27 structure, it becomes clear that the corresponding glycines 12 and 15 of K27 are indeed on the same side of the helix, while the corresponding phenylalanines 13, 14, 16, and 17 are on its opposite side, in keeping with the functional results obtained with IsK.…”