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

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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Uterine Excitability and Ion Channels and Their Changes with Gestation and Hormonal Environment

Annual Review of Physiology ◽

10.1146/annurev-physiol-032420-035509 ◽

2020 ◽

Vol 83 (1) ◽

Author(s):

Susan Wray ◽

Sarah Arrowsmith

Keyword(s):

Ion Channels ◽

Inward Rectifier ◽

Cation Channels ◽

Annual Review ◽

Publication Date ◽

K Channels ◽

Voltage Gated ◽

New Findings ◽

Voltage Dependent ◽

Pore Domain

We address advances in the understanding of myometrial physiology, focusing on excitation and the effects of gestation on ion channels and their relevance to labor. This review moves through pioneering studies to exciting new findings. We begin with the myometrium and its myocytes and describe how excitation might initiate and spread in this myogenic smooth muscle. We then review each of the ion channels in the myometrium: L- and T-type Ca2+ channels, KATP (Kir6) channels, voltage-dependent K channels (Kv4, Kv7, and Kv11), twin-pore domain K channels (TASK, TREK), inward rectifier Kir7.1, Ca2+-activated K+ channels with large (KCNMA1, Slo1), small (KCNN1–3), and intermediate (KCNN4) conductance, Na-activated K channels (Slo2), voltage-gated (SCN) Na+ and Na+ leak channels, nonselective (NALCN) channels, the Na K-ATPase, and hyperpolarization-activated cation channels. We finish by assessing how three key hormones— oxytocin, estrogen, and progesterone—modulate and integrate excitability throughout gestation. Expected final online publication date for the Annual Review of Physiology, Volume 83 is February 10, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Charybdotoxin blocks voltage-gated K+ channels in human and murine T lymphocytes.

The Journal of General Physiology ◽

10.1085/jgp.93.6.1061 ◽

1989 ◽

Vol 93 (6) ◽

pp. 1061-1074 ◽

Cited By ~ 87

Author(s):

S B Sands ◽

R S Lewis ◽

M D Cahalan

Keyword(s):

T Lymphocytes ◽

Time Course ◽

Leukemia Cell ◽

Lymphoid Cells ◽

Jurkat Cells ◽

Leukemia Cell Line ◽

K Channel ◽

K Channels ◽

Voltage Gated ◽

Scorpion Venoms

A variety of scorpion venoms and purified toxins were tested for effects on ion channels in human T lymphocytes, a human T leukemia cell line (Jurkat), and murine thymocytes, using the whole-cell patch-clamp method. Nanomolar concentrations of charbdotoxin (CTX), a purified peptide component of Leiurus quinquestriatus venom known to block Ca2+-activated K+ channels from muscle, blocked "type n" voltage-gated K+ channels in human T lymphoid cells. The Na+ channels occasionally expressed in these cells were unaffected by the toxin. From the time course of development and removal of K+ channel block we determined the rates of CTX binding and unbinding. CTX blocks K+ channels in Jurkat cells with a Kd value between 0.5 and 1.5 nM. Of the three types of voltage-gated K+ channels present in murine thymocytes, types n and n' are blocked by CTX at nanomolar concentrations. The third variety of K+ channels, "type l," is unaffected by CTX. Noxiustoxin (NTX), a purified toxin from Centruroides noxius known to block Ca2+-activated K+ channels, also blocked type n K+ channels with a high degree of potency (Kd = 0.2 nM). In addition, several types of crude scorpion venoms from the genera Androctonus, Buthus, Centruroides, and Pandinus blocked type n channels. We conclude that CTX and NTX are not specific for Ca2+ activated K+ channels and that purified scorpion toxins will provide useful probes of voltage-gated K+ channels in T lymphocytes. The existence of high-affinity sites for scorpion toxin binding may help to classify structurally related K+ channels and provide a useful tool for their biochemical purification.

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The myth of scorpion suicide: are scorpions insensitive to their own venom?

Journal of Experimental Biology ◽

10.1242/jeb.201.18.2625 ◽

1998 ◽

Vol 201 (18) ◽

pp. 2625-2636

Author(s):

C Legros ◽

MF Martin-Eauclaire ◽

D Cattaert

Keyword(s):

Action Potential ◽

Procambarus Clarkii ◽

Scorpion Venom ◽

K Channel ◽

Muscle Fibres ◽

Channel Blockers ◽

Na Channels ◽

Nerve Fibres ◽

K Channels ◽

Voltage Gated

The resistance of the scorpion Androctonus australis to its own venom, as well as to the venom of other species, was investigated. A comparison of the electrical and pharmacological properties of muscle and nerve fibres from Androctonus australis with those from the crayfish Procambarus clarkii enabled us to understand the lack of effect of scorpion venom (110-180 microg ml-1) and purified toxins, which are active on voltage-gated Na+ and K+ channels, Ca2+-activated K+ channels, on scorpion tissues. Voltage-clamp experiments showed that peptide K+ channel blockers from scorpion and snake have no effect on currents in muscle and nerve fibres from either scorpions or crayfish. The scorpion toxin kaliotoxin (KTX), a specific blocker of Kv1.1 and Kv1.3 K+ channels, had no effect on muscle fibres of A. australis (2 micromol l-1) or P. clarkii (400 nmol l-1). Similarly, charybdotoxin (ChTX) had no effect on the muscle fibres of A. australis (10 micromol l-1) or P. clarkii (200 nmol l-1) and neither did the snake toxin dendrotoxin (DTX) at concentrations of 100 nmol l-1 in A. australis and 200 nmol l-1 in P. clarkii. These three toxins (KTX, ChTX and DTX) did not block K+ currents recorded from nerve fibres in P. clarkii. The pharmacology of the K+ channels in these two arthropods did not conform to that previously described for K+ channels in other species. Current-clamp experiments clearly indicated that the venom of A. australis (50 microg ml-1) had no effect on the shape of the action potential recorded from nerve cord axons from A. australis. At a concentration of 50 microg ml-1, A. australis venom greatly prolonged the action potential in the crayfish giant axon. The absence of any effect of the anti-mammal <IMG src="/images/symbols/&agr ;.gif" WIDTH="9" HEIGHT="12" ALIGN="BOTTOM" NATURALSIZEFLAG="3">-toxin AaH II (100 nmol l-1) and the anti-insect toxin AaH IT1 (100 nmol l-1) on scorpion nerve fibres revealed strong pharmacological differences between the voltage-gated Na+ channels of scorpion and crayfish. We conclude that the venom from A. australis is pharmacologically inactive on K+ channels and on voltage-sensitive Na+ channels from this scorpion.

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Potassium channel block does not affect metabolic responses of brown fat cells

AJP Cell Physiology ◽

10.1152/ajpcell.1992.262.3.c678 ◽

1992 ◽

Vol 262 (3) ◽

pp. C678-C681 ◽

Cited By ~ 14

Author(s):

P. A. Pappone ◽

M. T. Lucero

Keyword(s):

Potassium Channels ◽

Metabolic Response ◽

Brown Fat ◽

Neonatal Rat ◽

K Channel ◽

Fat Cells ◽

Adrenergic Stimulation ◽

K Channels ◽

Voltage Gated ◽

Calcium Activated Potassium Channels

Hormonally stimulated brown fat cells are capable of extremely high metabolic rates, making them an excellent system in which to examine the role of plasma membrane ion channels in cell metabolism. We have previously shown that brown fat cell membranes have both voltage-gated and calcium-activated potassium channels (Voltage-gated potassium channels in brown fat cells. J. Gen. Physiol. 93: 451-472, 1989; Membrane responses to norepinephrine in cultured brown fat cells. J. Gen. Physiol. 95: 523-544, 1990). Currents through both the voltage-activated potassium channels, IK,V, and the calcium-activated potassium channels, IK,Ca, can be blocked by the membrane-impermeant K channel blocker tetraethylammonium (TEA). We used microcalorimetric measurements from isolated neonatal rat brown fat cells to assess the role these potassium conductances play in the metabolic response of brown fat cells to adrenergic stimulation. Concentrations of TEA as high as 50 mM, sufficient to block approximately 95% of IK,V and 100% of IK,Ca, had no effect on norepinephrine-stimulated heat production. These results show that neither voltage-gated nor calcium-activated K channels are necessary for a maximal thermogenic response in brown fat cells and suggest that K channels are not involved in maintaining cellular homeostasis during periods of high metabolic activity.

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Studies of Conorfamide-Sr3 on Human Voltage-Gated Kv1 Potassium Channel Subtypes

Marine Drugs ◽

10.3390/md18080425 ◽

2020 ◽

Vol 18 (8) ◽

pp. 425

Author(s):

Estuardo López-Vera ◽

Luis Martínez-Hernández ◽

Manuel B. Aguilar ◽

Elisa Carrillo ◽

Joanna Gajewiak

Keyword(s):

Action Potentials ◽

Inhibitory Concentration ◽

Molecular Probe ◽

K Channel ◽

Dependent Effect ◽

K Channels ◽

Voltage Gated ◽

Pancreatic Cells ◽

Genes Encoding ◽

Voltage Gated Ion Channels

Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K+ channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K+ channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K+ channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC50 = 2.7 and 24 μM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.

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Isolation of putative voltage-gated epithelial K-channel isoforms from rabbit kidney and LLC-PK1 cells

AJP Renal Physiology ◽

10.1152/ajprenal.1992.262.1.f151 ◽

1992 ◽

Vol 262 (1) ◽

pp. F151-F157 ◽

Cited By ~ 1

Author(s):

G. V. Desir ◽

H. A. Hamlin ◽

E. Puente ◽

R. F. Reilly ◽

F. Hildebrandt ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Rabbit Kidney ◽

K Channel ◽

Kidney Cortex ◽

Epithelial Cell Line ◽

K Channels ◽

Voltage Gated ◽

Transmembrane Segments ◽

Partial Length

Epithelial voltage-gated potassium (K) channels have been well studied using electrophysiological methods, but little is known about their structures. We tested the hypothesis that some of these channels belong to the Shaker gene family, which encodes voltage-gated K channels in excitable tissues. From published sequences of Shaker proteins in Drosophila, rat, and mouse brain, we chose regions that were conserved between species. Based on these protein sequences, degenerate oligonucleotides flanking the putative voltage sensor (S4) were synthesized and used as primers for the polymerase chain reaction. Five Shaker-like cDNAs were amplified from rabbit kidney cortex and three from LLC-PK1, an epithelial cell line derived from pig kidney. Each partial-length rabbit kidney cDNA is approximately 850 base pairs (bp) long. The deduced amino acid sequences contain five putative transmembrane segments and are 79-97% identical to two Shaker isoforms expressed in rat brain (RBK1 and RBK2). Sequence similarity is greatest in the putative transmembrane segments S1-S5. Importantly, the S4 segment, the putative voltage gate is highly conserved in all 5 cDNAs. Southern analysis of rabbit genomic DNA suggests that each isoform is encoded by a different gene. The partial length LLC-PK1 cDNAs are 450-bp long, and the deduced amino acid sequences are 77-99% identical to the rabbit cDNAs. This is, to our knowledge, the first demonstration that Shaker-like genes are expressed in renal epithelial cells. These genes most likely encode voltage-gated K channels involved in renal epithelial K transport.

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Coupling between Voltage Sensors and Activation Gate in Voltage-gated K+ Channels

The Journal of General Physiology ◽

10.1085/jgp.20028696 ◽

2002 ◽

Vol 120 (5) ◽

pp. 663-676 ◽

Cited By ~ 227

Author(s):

Zhe Lu ◽

Angela M. Klem ◽

Yajamana Ramu

Keyword(s):

Electrical Signal ◽

K Channel ◽

Coupling Mechanism ◽

Excitable Cells ◽

Voltage Sensing ◽

K Channels ◽

Voltage Sensors ◽

Voltage Gated ◽

Transmembrane Segments ◽

Activation Gate

Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1–S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5–S6, equivalent to M1–M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4–S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.

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A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes

Journal of Experimental Medicine ◽

10.1084/jem.188.9.1593 ◽

1998 ◽

Vol 188 (9) ◽

pp. 1593-1602 ◽

Cited By ~ 123

Author(s):

George R. Ehring ◽

Hubert H. Kerschbaum ◽

Claudia Eder ◽

Amber L. Neben ◽

Christopher M. Fanger ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Cell Lines ◽

K Channel ◽

Na Channel ◽

Activated T Cells ◽

K Channels ◽

Voltage Gated

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin–resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl− channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression.

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Expression and function of potassium channels in the human placental vasculature

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00040.2006 ◽

2006 ◽

Vol 291 (2) ◽

pp. R437-R446 ◽

Cited By ~ 42

Author(s):

Mark Wareing ◽

Xilian Bai ◽

Fella Seghier ◽

Claire M. Turner ◽

Susan L. Greenwood ◽

...

Keyword(s):

Vascular Function ◽

Vascular Reactivity ◽

Muscle Tone ◽

K Channel ◽

K Channels ◽

Voltage Gated ◽

Chorionic Plate ◽

And Function ◽

Vascular Smooth Muscle Tone ◽

Arteries And Veins

In the placental vasculature, where oxygenation may be an important regulator of vascular reactivity, there is a paucity of data on the expression of potassium (K) channels, which are important mediators of vascular smooth muscle tone. We therefore addressed the expression and function of several K channel subtypes in human placentas. The expression of voltage-gated (Kv)2.1, KV9.3, large-conductance Ca2+-activated K channel (BKCa), inward-rectified K+ channel (KIR)6.1, and two-pore domain inwardly rectifying potassium channel-related acid-sensitive K channels (TASK)1 in chorionic plate arteries, veins, and placental homogenate was assessed by RT-PCR and Western blot analysis. Functional activity of K channels was assessed pharmacologically in small chorionic plate arteries and veins by wire myography using 4-aminopyridine, iberiotoxin, pinacidil, and anandamide. Experiments were performed at 20, 7, and 2% oxygen to assess the effect of oxygenation on the efficacy of K channel modulators. KV2.1, KV9.3, BKCa, KIR6.1, and TASK1 channels were all demonstrated to be expressed at the message level. KV2.1, BKCa, KIR6.1, and TASK1 were all demonstrated at the protein level. Pharmacological manipulation of voltage-gated and ATP-sensitive channels produced the most marked modifications in vascular tone, in both arteries and veins. We conclude that K channels play an important role in controlling placental vascular function.

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A four-disulphide-bridged toxin, with high affinity towards voltage-gated K+ channels, isolated from Heterometrus spinnifer (Scorpionidae) venom

Biochemical Journal ◽

10.1042/bj3280321 ◽

1997 ◽

Vol 328 (1) ◽

pp. 321-327 ◽

Cited By ~ 74

Author(s):

Bruno LEBRUN ◽

Régine ROMI-LEBRUN ◽

Marie-France MARTIN-EAUCLAIRE ◽

Akikazu YASUDA ◽

Masaji ISHIGURO ◽

...

Keyword(s):

Receptor Site ◽

K Channel ◽

Disulphide Bridge ◽

K Channels ◽

Voltage Gated ◽

Disulphide Bonds ◽

Immobilized Trypsin ◽

Blocking Activity ◽

Kv1.3 Channels ◽

Bridge Pattern

A new toxin, named HsTX1, has been identified in the venom of Heterometrus spinnifer (Scorpionidae), on the basis of its ability to block the rat Kv1.3 channels expressed in Xenopus oocytes. HsTX1 has been purified and characterized as a 34-residue peptide reticulated by four disulphide bridges. HsTX1 shares 53% and 59% sequence identity with Pandinus imperator toxin1 (Pi1) and maurotoxin, two recently isolated four-disulphide-bridged toxins, whereas it is only 32-47% identical with the other scorpion K+ channel toxins, reticulated by three disulphide bridges. The amidated and carboxylated forms of HsTX1 were synthesized chemically, and identity between the natural and the synthetic amidated peptides was proved by mass spectrometry, co-elution on C18 HPLC and blocking activity on the rat Kv1.3 channels. The disulphide bridge pattern was studied by (1) limited reduction-alkylation at acidic pH and (2) enzymic cleavage on an immobilized trypsin cartridge, both followed by mass and sequence analyses. Three of the disulphide bonds are connected as in the three-disulphide-bridged scorpion toxins, and the two extra half-cystine residues of HsTX1 are cross-linked, as in Pi1. These results, together with those of CD analysis, suggest that HsTX1 probably adopts the same general folding as all scorpion K+ channel toxins. HsTX1 is a potent inhibitor of the rat Kv1.3 channels (IC50 approx. 12 pM). HsTX1 does not compete with 125I-apamin for binding to its receptor site on rat brain synaptosomal membranes, but competes efficiently with 125I-kaliotoxin for binding to the voltage-gated K+ channels on the same preparation (IC50 approx. 1 pM).

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