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

1992 ◽  
Vol 262 (1) ◽  
pp. F151-F157 ◽  
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.

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

2002 ◽  
Vol 120 (5) ◽  
pp. 663-676 ◽  
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.

Identification of a novel K-channel gene (KC22) that is highly expressed in distal tubule of rabbit kidney

1993 ◽  
Vol 264 (1) ◽  
pp. F128-F133 ◽  
Author(s):  
G. V. Desir ◽  
H. Velazquez
Keyword(s):  
Amino Acid ◽  
Gene Family ◽  
Rat Brain ◽  
Distal Tubule ◽  
Amino Acid Identity ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Degenerate Primers ◽  
Acid Identity ◽  
Partial Length

The Shaker gene family encodes voltage-gated K channels. Five partial-length Shaker-like cDNAs (KC2, 4, 10, 19, and 22) were previously isolated from rabbit kidney using polymerase chain reaction (PCR) [G. V. Desir, E. Hamlin, A.H. Puente, R.F. Reilly, F. Hiledebrandt, and P. Igarashi. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F151-F157, 1992]. We now report the cloning of another Shaker-like cDNA (KC6) from rabbit kidney and the identification of one isoform that is highly expressed in rabbit distal tubule cells grown in culture. A partial-length cDNA (859 bp) for KC6 was isolated by PCR amplification of rabbit kidney cDNA using Shaker-specific degenerate primers. KC6 was most similar to the rat brain clone RBK2 (77% amino acid identity) and to the rabbit clone KC19 (78% amino acid identity). Transcript levels for KC2, 4, 6, 10, 19, and 22 were quantified using the ribonuclease protection assay. Transcripts for all six isoforms were detected in renal tissues. KC22 was the most abundant isoform in kidney cortex and medulla (20- to 40-fold greater than the other isoforms). Furthermore, KC22 expression levels were fivefold higher in primary cultures of rabbit distal convoluted tubules and connecting tubules than in whole kidney cortex. Although the partial-length sequence for KC22 represents the most conserved regions in the Shaker gene family it only has 35-88% amino acid identity with other Shaker channels, suggesting that KC22 represents a novel isoform. In contrast, KC4 and KC19 (less abundant in kidney than KC22) are highly homologous to the rat brain clones RBK1 and RBK2, respectively (97% amino acid identity).(ABSTRACT TRUNCATED AT 250 WORDS)

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

1989 ◽  
Vol 93 (6) ◽  
pp. 1061-1074 ◽  
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.

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

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.

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

1992 ◽  
Vol 262 (3) ◽  
pp. C678-C681 ◽  
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.

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

Marine Drugs ◽  
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.

ROMK inwardly rectifying ATP-sensitive K+ channel. II. Cloning and distribution of alternative forms

1995 ◽  
Vol 268 (6) ◽  
pp. F1132-F1140 ◽  
Author(s):  
M. A. Boim ◽  
K. Ho ◽  
M. E. Shuck ◽  
M. J. Bienkowski ◽  
J. H. Block ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Functional Expression ◽  
Amino Acid Sequences ◽  
K Channel ◽  
K Channels ◽  
Noncoding Regions ◽  
Nephron Segments ◽  
Alternatively Spliced ◽  
Polymerase Chain ◽  
Inwardly Rectifying

The rat ROMK gene encodes inwardly rectifying, ATP-regulated K+ channels [K. Ho, C. G. Nichols, W. J. Lederer, J. Lytton, P. M. Vassilev, M. V. Kanazirska, and S. C. Hebert. Nature Lond. 362: 31–38, 1993; H. Zhou, S. S. Tate, and L. G. Palmer. Am. J. Physiol. 266 (Cell Physiol. 35): C809-C824, 1994], and mRNA encoding these channels is widely expressed in distal cortical and outer medullary nephron segments [see companion study; W.-S. Lee and S. C. Hebert. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F1124-F1131, 1995]. Using approaches based on homology to ROMK1, we have identified two additional ROMK isoforms, ROMK2b and ROMK3. Analysis of the nucleotide sequences of the ROMK isoforms indicates that molecular diversity of ROMK transcripts is due to alternative splicing at both the 5'-coding and 3'-noncoding regions. The splicing at the 5' end of ROMK gives rise to channel proteins with variable-length NH2 termini containing different initial amino acid sequences. Functional expression of these isoforms in Xenopus oocytes showed that they form functional Ba(2+)-sensitive K+ channels. The nephron distribution of mRNAs encoding alternatively spliced isoforms of ROMK (ROMK1-ROMK3) was investigated by reverse transcription-polymerase chain reaction (RT-PCR) of nephron segments dissected from rat kidney. Nondegenerate PCR primer pairs were designed to span at least one intron and to amplify specific alternatively spliced forms of ROMK.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and localization of a double-pore K channel, KCNK1: exclusive expression in distal nephron segments

1997 ◽  
Vol 273 (4) ◽  
pp. F663-F666 ◽  
Author(s):  
Marcelo Orias ◽  
Heino Velázquez ◽  
Freeman Tung ◽  
George Lee ◽  
Gary V. Desir
Keyword(s):  
Amino Acid ◽  
Collecting Duct ◽  
Human Kidney ◽  
Distal Tubule ◽  
Amino Acid Identity ◽  
Northern Analysis ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
K Channels

The K-selective channel, TOK1, recently identified in yeast, displays the unusual structural feature of having two putative pore regions, in contrast to all previously cloned K channels. Using the TOK1 pore regions as probes, we identified a human kidney cDNA encoding a 337-amino acid protein (hKCNK1) with four transmembrane segments and two pore regions containing the signature sequence of K channels. Amino acid identity to TOK1 is only 15% overall but 40% at the pores. Northern analysis indicates high expression of a 1.9-kb message in brain > kidney >> heart. Nephron segment localization, carried out in rabbit by reverse transcription-polymerase chain reaction, reveals that KCNK1 is expressed in cortical thick ascending limb, connecting tubule, and cortical collecting duct. It was not detected in the proximal tubule, medullary thick ascending limb, distal convoluted tubule, and glomerulus. We conclude that KCNK1 is a unique, double-pore, mammalian K channel, distantly related to the yeast channel TOK1, that is expressed in distal tubule and is a candidate to participate in renal K homeostasis.

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

1998 ◽  
Vol 188 (9) ◽  
pp. 1593-1602 ◽  
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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