Alterations of gating parameters by neutral substitutions of transmembrane Leu52 of slow potassium channel.
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Amino Acid Sequence↗
Biomedical subjects
S Oiki
Publications and source records attributed to S Oiki.
A dipolar amino acid substitution induces voltage-dependent transitions between two stable conductance states in gramicidin channels.
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Amino Acids↗
Alteration of channel activities and gating by mutations of slow ISK potassium channel.
ISK is a small membrane protein consisting of 129-130 amino acid residues with a single putative transmembrane domain and induces a very slow voltage-dependent K+ channel activity in the Xenopus oocyte expression system. We investigated the nature and structure-function relation of ISK by examining the effects of various mutations of ISK on the K+ channel activities measured in Xenopus oocytes. Deletion and truncation of the ISK protein indicated that the 63-amino acid sequence covering a transmembrane domain is sufficient for eliciting a K+ channel activity characteristic of ISK. Amino acid substitutions at a total of 31 positions within and surrounding the transmembrane domain caused different effects on the channel activity. A channel activity was enhanced by substitution of leucine with isoleucine at position 52 within the transmembrane domain, and the kinetic analysis of this mutation indicated that the enhancement of the channel activity is due to an alteration of a gating property of the ISK protein and thus supported the view that ISK forms an integral part of the K+ channel itself. The substitutions at many positions of the membrane-following region produced drastic reduction of the channel activity, and this is in marked contrast to the lack of effects of amino acid substitutions at the membrane-preceding region. Thus, the cytoplasmic portion immediately following the transmembrane domain plays a crucial role in inducing the channel activity of ISK.
Amino Acid Sequence↗
Bundles of amphipathic transmembrane alpha-helices as a structural motif for ion-conducting channel proteins: studies on sodium channels and acetylcholine receptors.
Channel proteins are transmembrane symmetric (or pseudosymmetric) oligomers organized around a central ionic pore. We present here a molecular model of the pore forming structures of two channel proteins with different primary structures and oligomeric size: the voltage-sensitive sodium channel and the nicotinic cholinergic receptor. We report low-energy arrangements of alpha-helical bundles calculated by semiempiricial potential energy functions and optimization routines and further refined using molecular dynamics. The ion-conducting pore is considered to be a symmetric or pseudosymmetric homooligomer of 3-5 amphipathic alpha-helices arranged such that the polar residues line a central hydrophilic pathway and the apolar residues face the hydrophobic bilayer interior. The channel lining exposes either charged (Asp, Glu, Arg, Lys) or polar-neutral (Ser, Thr) residues. A bundle of four parallel helices constrained to C4 symmetry, the helix axis aligned with the symmetry axis, and the helices constrained to idealized dihedral angles, produces a structure with a pore of the size inferred for the sodium channel protein (area approximately 16 A2). Similarly, a pentameric array optimized with constraints to maintain C5 symmetry and backbone torsions characteristic of alpha-helices adopts a structure that appears well suited to form the lining of the nicotinic cholinergic receptor (pore area approximately 46 A2). Thus, bundles of amphipathic alpha-helices satisfy the structural, energetic, and dynamic requirements to be the molecular structural motif underlying the function of ionic channels.
Amino Acid Sequence↗
Intestinal secretagogues increase cytosolic free Ca2+ concentration and K+ conductance in a human intestinal epithelial cell line.
A human intestinal epithelial cell line (Intestine 407) is known to retain receptors for intestinal secretagogues such as acetylcholine (ACh), histamine, serotonin (5-HT) and vasoactive intestinal peptide (VIP). The cells were also found to possess separate receptors for secretin and ATP, the stimulation of which elicited transient hyperpolarizations coupled to decreased membrane resistances. These responses were reversed in polarity at the K+ equilibrium potential. The hyperpolarizing responses to six agonists were reversibly inhibited by quinine or quinidine. By means of Ca2(+)-selective microelectrodes, increases in the cytosolic free Ca2+ concentration were observed in response to individual secretagogues. The time course of Ca2+ responses coincided with that of hyperpolarizing responses. The responses to ACh and 5-HT were abolished by a reduction in the extracellular Ca2+ concentration down to pCa 7 or by application of Co2+. Thus, in Intestine 407 cells, not only the intestinal secretagogues, which are believed to act via increased cytosolic Ca2+ (ACh, 5-HT and histamine), but also those which elevate cyclic AMP (VIP, secretin and ATP) induce increases in cytosolic Ca2+, thereby activating the K+ conductance. It is likely that the origin of increased cytosolic Ca2+ is mainly extracellular for ACh- and 5-HT-induced responses, whereas histamine, VIP, secretin and ATP mobilize Ca2+ from the internal compartment.
Calcium↗
C1q induces chemotaxis and K+ conductance activation coupled to increased cytosolic Ca2+ in mouse fibroblasts.
Cultured mouse fibroblasts (L cells) respond to whole C with a slow hyperpolarization. Among the C components tested, C1q was found to be most effective. In contrast, the cell did not respond to C1, in which the collagen-like region of the C1q molecule is masked. The C1q-induced hyperpolarizing response was inhibited by collagen or C1q-specific antisera. Human diploid skin fibroblasts (Flow 1,000 cells) also exhibited similar membrane potential changes in response to whole C or C1q. After repeated applications of C1q, the cell membrane became unresponsive (desensitized). The treatment of L cells with pronase E inhibited the C1q-induced response, whereas the response to ATP, which is known to interact to its own receptor, was still preserved. The reversal potential of C responses was close to the K+ equilibrium potential. The hyperpolarizing response was inhibited by a blocker of Ca2+-activated K+ channels in fibroblasts (quinine), by deprivation of extracellular Ca2+ or by a Ca2+ channel blocker (nifedipine). By means of Ca2+-selective microelectrodes, the cytosolic free Ca2+ concentration was found to increase from 126 to 206 nM upon stimulation of L cells with C1q. Using an agarose-well method, L cells were observed to migrate predominantly toward C1q or whole C. It is concluded that the fibroblasts have the C1q receptor sensitive to pronase E and that activation of C1q receptors gives rise to Ca2+ influx, triggering an increase in the cytosolic free Ca2+ ions, which in turn induces a hyperpolarizing response as a result of the stimulation of Ca2+-activated K+ channels and initiates chemotaxis to C1q.
Animals↗
Factors responsible for oscillations of membrane potential recorded with tight-seal-patch electrodes in mouse fibroblasts.
In giant fibroblastic L cells, penetration of a conventional microelectrode brought about marked decreases in the membrane potential and input resistance measured with a patch electrode under tight-seal whole-cell configuration, and repeated hyperpolarizations were often observed upon penetration. Therefore, the question arose whether such leakage artifact is a causal factor for generation of the membrane potential oscillation even in giant L cells. During whole-cell recordings, however, regular potential oscillations were observed in the cells that had not been impaled with a conventional microelectrode, as far as the Ca2+ buffer was not strong in the pipette solution. Oscillatory changes in the intracellular potential were detected by extracellular recordings with a tight-seal patch electrode in the cell-attached configuration. Thus, the potential oscillation occurs even in the absence of penetration-induced leakage or without rupture of the patch membrane. Withdrawal of a micropipette from one cell was often found to induce marked cell damage and elicit oscillatory hyperpolarizations in a neighboring cell with a certain time lag. The longer the distance between the injured and recorded cells, the greater was the time lag. Application of the cell lysate on the cell surface also gave rise to oscillatory hyperpolarizations. After repeated applications of the lysate, the membrane became unresponsive (desensitized), suggesting the involvement of receptors for the lysate factor. The lysates of different cell species (mouse lymphoma L5178Y cells or human epithelial Intestine 407 cells) produced similar effects. The effective component was heat stable and distinct from ATP. Lysate-induced hyperpolarizations were inhibited by deprivation of extracellular Ca2+ and by application of a Ca2+ channel blocker (nifedipine) or a K+ channel blocker (quinine) in the same manner as spontaneous oscillatory hyperpolarizations. It is concluded that the mouse fibroblast exhibits membrane potential oscillations, when the cell was activated, presumably via receptor systems, by some diffusible factors released from damaged cells.
Animals↗
M2 delta, a candidate for the structure lining the ionic channel of the nicotinic cholinergic receptor.
A synthetic 23-mer peptide that mimics the sequence of the putative transmembrane M2 segment of the Torpedo californica acetylcholine receptor (AcChoR) delta subunit--Glu-Lys-Met-Ser-Thr-Ala-Ile-Ser-Val-Leu-Leu-Ala-Gln-Ala-Val-Phe-Leu- Leu-Leu-Thr-Ser-Gln-Arg--forms discrete ionic channels in phosphatidylcholine bilayers. In contrast, a synthetic peptide that mimics the sequence of the putative M1 transmembrane segment of the Torpedo AcChoR delta subunit--Leu-Phe-Tyr-Val-Ile-Asn-Phe-Ile-Thr-Pro-Cys-Val-Leu-Ile-Ser-Phe- Leu-Ala-Ser-Leu-Ala-Phe-Tyr--does not form channels. The synthetic M2 delta channel peptide exhibits features that are characteristic of the authentic AcChoR channel, such as single channel conductances, discrimination of cations over anions, and channel lifetimes for open and closed states in the millisecond time range. Energetic considerations suggest that an aggregate of five amphipathic alpha-helices conforms the channel. Thus, the M2 segment may be a component of the AcChoR channel structure.
Amino Acid Sequence↗
Channel protein engineering: synthetic 22-mer peptide from the primary structure of the voltage-sensitive sodium channel forms ionic channels in lipid bilayers.
A synthetic 22-mer peptide that mimics the sequence of a putative pore segment of the voltage-dependent sodium channel forms transmembrane ionic channels in lipid bilayers. Several features of the authentic sodium channel are exhibited by the synthetic peptide: (i) The single channel conductance of the most frequent event is 20 pS in 0.5 M NaCl. (ii) The single channel open and closed lifetimes are in the ms time range. (iii) The synthetic channel discriminates cations over anions but is nonselective between Na+ and K+. However, the synthetic channel displays no significant voltage dependence. Energetic considerations suggest a bundle of four parallel amphipathic alpha-helices as the most plausible channel structure. The synthetic 22-mer channel-forming peptide allows study of the mechanisms of ion permeation through sodium channels by protein engineering techniques.
Amino Acid Sequence↗
Evidence for the involvement of calmodulin in the operation of Ca-activated K channels in mouse fibroblasts.
The oscillation of membrane potential in fibroblastic L cells is known to result from periodic stimulation of Ca2+-activated K+ channels due to the oscillatory increase in the intracellular Ca2+ concentration. These repeated hyperpolarizations were inhibited by putative calmodulin antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and promethazine (PMZ), and the concentrations required for half-maximal inhibition were 25, 30 and 300 microM, respectively. These doses were lower than those for reducing the membrane resistance due to nonspecific cell damages. Another calmodulin antagonist, chlorpromazine (CPZ), was also effective, but CPZ-sulfoxide was not. Intracellular pressure injections of calmodulin-interacting divalent cations, Ca2+, Sr2+, Mn2+ and Ni2+, elicited slow hyperpolarizations, whereas Mg2+ and Ba2+, which are known to be essentially inert for calmodulin, failed to evoke any responses. The injection of purified calmodulin also brought about a similar hyperpolarization. Quinine, an inhibitor of Ca2+-activated K+ channels, abolished both Ca2+- and calmodulin-induced hyperpolarizations. TFP prevented Ca2+-induced hyperpolarizations. The TFP effect was partially reversed by the calmodulin injection. It is concluded that calmodulin is involved in the operation of Ca2+-activated K+ channels in fibroblasts.
Animals↗
Synchronous oscillation of the cytoplasmic Ca2+ concentration and membrane potential in cultured epithelial cells (Intestine 407).
Cultured epithelial Intestine 407 cells exhibit regular oscillations of the membrane potential with repeated hyperpolarizations. These hyperpolarizations were inhibited not only by K+ channel blockers (tetraethylammonium and nonyltriethylammonium) but also by inhibitors of the Ca2+-activated K+ channel (quinine and quinidine). Using Ca2+-selective microelectrodes, cyclic increases in the cytosolic free Ca2+ concentration of more than 1 X 10(-6) M were found to coincide with the cyclic membrane hyperpolarizations. Thus, it appears that the potential oscillation is brought about by the oscillation of the intracellular free Ca2+ level which induces periodic activation of the Ca2+-dependent K+ channels. Neither the deprivation of extracellular Ca2+ nor the application of Ca2+ channel blockers (Co2+ and Ni2+) abolished the potential oscillation. Mitochondrial inhibitors (KCN, NaN3, antimycin A, FCCP and dinitrophenol) inhibited the potential oscillation, whereas glycolytic inhibitors (iodoacetic acid and NaF) had no effects. Caffeine and oxalate, which affect the microsomal Ca2+ transport, failed to exert any effect upon the potential oscillation. It is concluded that the cytosolic Ca2+ oscillation results from cyclic releases of Ca2+ from the intracellular storage site, which depends upon mitochondrial activities.
Animals↗
Oscillations of cytoplasmic concentrations of Ca2+ and K+ in fused L cells.
Using Ca2+- and K+-selective microelectrodes, the cytosolic free Ca2+ and K+ concentrations were measured in mouse fibroblastic L cells. When the extracellular Ca2+ concentration exceeded several micromoles, spontaneous oscillations of the intracellular free Ca2+ concentration were observed in the submicromolar ranges. During the Ca2+ oscillations, the membrane potential was found to oscillate concomitantly. The peak of cyclic increases in the free Ca2+ level coincided in time with the peak of periodic hyperpolarizations. Both oscillations were abolished by reducing the extracellular Ca2+ concentration down to 10(-7) M or by applying a Ca2+ channel blocker, nifedipine (50 microM). In the presence of 0.5 mM quinine, an inhibitor of Ca2+-activated K+ channel, sizable Ca2+ oscillations still persisted, while the potential oscillations were markedly suppressed. Oscillations of the intracellular K+ concentration between about 145 and 140 mM were often associated with the potential oscillations. The minimum phase of the K+ concentration was always 5 to 6 sec behind the peak hyperpolarization. Thus, it is concluded that the oscillation of membrane potential results from oscillatory increases in the intracellular Ca2+ level, which, in turn, periodically stimulate Ca2+-activated K+ channels.
Animals↗
Increases in cytosolic free Ca2+ induced by ATP, complement and beta-lipoprotein in mouse L fibroblasts.
By means of Ca2+- and K+-selective microelectrodes, the changes in intracellular free Ca2+ and K+ were measured during the hyperpolarizing responses induced by ATP, complement and beta-lipoprotein in mouse fibroblastic L cells. The cytoplasmic Ca2+ concentration [( Ca]i) was about 0.4 microM in the resting state. The hyperpolarizing responses always coincided with a phasic increase in [Ca]i. ATP or beta-lipoprotein induced about a 2-fold rise in [Ca]i, and complement did up to 3-fold. Both the hyperpolarizing responses and [Ca]i increases were prevented by removal of external Ca2+ or by application of a Ca-channel blocker, nifedipine. Quinine, a Ca-activated K-channel inhibitor, suppressed the hyperpolarizing responses but not the [Ca]i increases. During the hyperpolarizing response, the intracellular free K+ concentration gradually decreased from about 120 to 110 mM. Thus, it is concluded that ATP, complement and beta-lipoprotein caused a transient elevation of cytoplasmic free Ca2+ due to Ca2+ influxes, thereby inducing electrical membrane responses through activation of Ca-dependent K-channels in the fibroblasts.
Adenosine Triphosphate↗
Exogenous ATP induces electrical membrane responses in fibroblasts.
Mouse fibroblastic L cells responded to exogenous ATP (greater than or equal to 0.2 mM) with a transient hyperpolarization due to increased membrane permeability to K+. By contrast, intracellular injection of ATP (up to about 3 mM) produced no noticeable effects on the membrane potential. The effects of a non-hydrolysable analogue of ATP (AMP-PNP) were similar to those of ATP. After successive applications of ATP, the cell membrane became virtually unresponsive (desensitized). Extracellular ADP was also effective, but AMP or adenosine was not. Antazoline suppressed the ATP response. Thus, exogenous ATP and ADP appear to stimulate P2- purinoceptors . Similar responses to ATP (or ADP) were also observed in human normal diploid fibroblasts (Flow 1000 line).
Adenosine Diphosphate↗