Cloning and functional expression of a liver isoform of the small conductance Ca2+-activated K+ channel SK3

2001 ◽  
Vol 280 (4) ◽  
pp. C836-C842 ◽  
Author(s):  
Elisabeth T. Barfod ◽  
Ann L. Moore ◽  
Steven D. Lidofsky
Keyword(s):  
Rat Liver ◽  
Metabolic Stress ◽  
Functional Expression ◽  
Mammalian Brain ◽  
K Channel ◽  
Molecular Characteristics ◽  
Amino Acid Residues ◽  
Sk Channels ◽  
Hek 293 ◽  
Small Conductance

Small conductance Ca2+-activated K+(SK) channels have been cloned from mammalian brain, but little is known about the molecular characteristics of SK channels in nonexcitable tissues. Here, we report the isolation from rat liver of an isoform of SK3. The sequence of the rat liver isoform differs from rat brain SK3 in five amino acid residues in the NH3terminus, where it more closely resembles human brain SK3. SK3 immunoreactivity was detectable in hepatocytes in rat liver and in HTC rat hepatoma cells. Human embryonic kidney (HEK-293) cells transfected with liver SK3 expressed 10 pS K+ channels that were Ca2+ dependent (EC50 630 nM) and were blocked by the SK channel inhibitor apamin (IC50 0.6 nM); whole cell SK3 currents inactivated at membrane potentials more positive than −40 mV. Notably, the Ca2+ dependence, apamin sensitivity, and voltage-dependent inactivation of SK3 are strikingly similar to the properties of hepatocellular and biliary epithelial SK channels evoked by metabolic stress. These observations raise the possibility that SK3 channels influence membrane K+ permeability in hepatobiliary cells during liver injury.

scholarly journals Evidence for a Deep Pore Activation Gate in Small Conductance Ca2+-activated K+ Channels

2007 ◽  
Vol 130 (6) ◽  
pp. 601-610 ◽  
Author(s):  
Andrew Bruening-Wright ◽  
Wei-Sheng Lee ◽  
John P. Adelman ◽  
James Maylie
Keyword(s):  
Cysteine Residue ◽  
Selectivity Filter ◽  
K Channel ◽  
Homologous Region ◽  
Sk Channels ◽  
Voltage Gated ◽  
Kv Channels ◽  
Substituted Cysteine Accessibility ◽  
Activation Gate ◽  
Small Conductance

Small conductance calcium-gated potassium (SK) channels share an overall topology with voltage-gated potassium (Kv) channels, but are distinct in that they are gated solely by calcium (Ca2+), not voltage. For Kv channels there is strong evidence for an activation gate at the intracellular end of the pore, which was not revealed by substituted cysteine accessibility of the homologous region in SK2 channels. In this study, the divalent ions cadmium (Cd2+) and barium (Ba2+), and 2-aminoethyl methanethiosulfonate (MTSEA) were used to probe three sites in the SK2 channel pore, each intracellular to (on the selectivity filter side of) the region that forms the intracellular activation gate of voltage-gated ion channels. We report that Cd2+ applied to the intracellular side of the membrane can modify a cysteine introduced to a site (V391C) just intracellular to the putative activation gate whether channels are open or closed. Similarly, MTSEA applied to the intracellular side of the membrane can access a cysteine residue (A384C) that, based on homology to potassium (K) channel crystal structures (i.e., the KcsA/MthK model), resides one amino acid intracellular to the glycine gating hinge. Cd2+ and MTSEA modify with similar rates whether the channels are open or closed. In contrast, Ba2+ applied to the intracellular side of the membrane, which is believed to block at the intracellular end of the selectivity filter, blocks open but not closed channels when applied to the cytoplasmic face of rSK2 channels. Moreover, Ba2+ is trapped in SK2 channels when applied to open channels that are subsequently closed. Ba2+ pre-block slows MTSEA modification of A384C in open but not in closed (Ba2+-trapped) channels. The findings suggest that the SK channel activation gate resides deep in the vestibule of the channel, perhaps in the selectivity filter itself.

Cloning and Expression of a Small-Conductance Ca2+-Activated K+ Channel From the Mouse Cochlea: Coexpression with α9/α10 Acetylcholine Receptors

2004 ◽  
Vol 91 (4) ◽  
pp. 1536-1544 ◽  
Author(s):  
Liping Nie ◽  
Haitao Song ◽  
Mei-Fang Chen ◽  
Nipavan Chiamvimonvat ◽  
Kirk W. Beisel ◽  
...  
Keyword(s):  
Acetylcholine Receptors ◽  
Outward Current ◽  
Nerve Fibers ◽  
Outer Hair Cells ◽  
Cloning And Expression ◽  
K Channel ◽  
Sk Channels ◽  
Dequalinium Chloride ◽  
Small Conductance

Functional interactions between ligand-gated, voltage-, and Ca2+-activated ion channels are essential to the properties of excitable cells and thus to the working of the nervous system. The outer hair cells in the mammalian cochlea receive efferent inputs from the brain stem through cholinergic nerve fibers that form synapses at their base. The acetylcholine released from these efferent fibers activates fast inhibitory postsynaptic currents mediated, to some extent, by small-conductance Ca2+-activated K+ channels (SK) that had not been cloned. Here we report the cloning, characterization, and expression of a complete SK2 cDNA from the mouse cochlea. The cDNAs of the mouse cochlea α9 and α10 acetylcholine receptors were also obtained, sequenced, and coexpressed with the SK2 channels. Human cultured cell lines transfected with SK2 yielded Ca2+-sensitive K+ current that was blocked by dequalinium chloride and apamin, known blockers of SK channels. Xenopus oocytes injected with SK2 in vitro transcribed RNA, under conditions where only outward K+ currents could be recorded, expressed an outward current that was sensitive to EGTA, dequalinium chloride, and apamin. In HEK-293 cells cotransfected with cochlear SK2 plus α9/α10 receptors, acetylcholine induced an inward current followed by a robust outward current. The results indicate that SK2 and the α9/α10 acetylcholine receptors are sufficient to partly recapitulate the native hair cell efferent synaptic response.

Mineralocorticoids decrease the activity of the apical small-conductance K channel in the cortical collecting duct

2005 ◽  
Vol 289 (5) ◽  
pp. F1065-F1071 ◽  
Author(s):  
Yuan Wei ◽  
Elisa Babilonia ◽  
Hyacinth Sterling ◽  
Yan Jin ◽  
Wen-Hui Wang
Keyword(s):  
Protein Tyrosine Kinase ◽  
Collecting Duct ◽  
K Channel ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Herbimycin A ◽  
Sk Channel ◽  
Small Conductance

We used the patch-clamp technique to examine the effect of DOCA treatment (2 mg/kg) on the apical small-conductance K (SK) channels, epithelial Na channels (ENaC), and the basolateral 18-pS K channels in the cortical collecting duct (CCD). Treatment of rats with DOCA for 6 days significantly decreased the plasma K from 3.8 to 3.1 meq and reduced the activity of the SK channel, defined as NPo, from 1.3 in the CCD of control rats to 0.6. In contrast, DOCA treatment significantly increased ENaC activity from 0.01 to 0.53 and the basolateral 18-pS K channel activity from 0.67 to 1.63. Moreover, Western blot analysis revealed that DOCA treatment significantly increased the expression of the nonreceptor type of protein tyrosine kinase (PTK), cSrc, and the tyrosine phosphorylation of ROMK in the renal cortex and outer medulla. The possibility that decreases in apical SK channel activity induced by DOCA treatment were the result of stimulation of PTK activity was further supported by experiments in which inhibition of PTK with herbimycin A significantly increased NPo from 0.6 to 2.1 in the CCD from rats receiving DOCA. Also, when rats were fed a high-K (10%) diet, DOCA treatment did not increase the expression of c-Src and decrease the activity of the SK channel in the CCD. We conclude that DOCA treatment decreased the apical SK channel activity in rats on a normal-K diet and that an increase in PTK expression may be responsible for decreased channel activity in the CCD from DOCA-treated rats.

scholarly journals Flow-dependent K+ secretion in the cortical collecting duct is mediated by a maxi-K channel

2001 ◽  
Vol 280 (5) ◽  
pp. F786-F793 ◽  
Author(s):  
Craig B. Woda ◽  
Alvina Bragin ◽  
Thomas R. Kleyman ◽  
Lisa M. Satlin
Keyword(s):  
Collecting Duct ◽  
K Channel ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Flow Rates ◽  
K Secretion ◽  
Tubular Flow ◽  
Flow Stimulation ◽  
Small Conductance

K+ secretion by the cortical collecting duct (CCD) is stimulated at high flow rates. Patch-clamp analysis has identified a small-conductance secretory K+ (SK) and a high-conductance Ca2+-activated K+ (maxi-K) channel in the apical membrane of the CCD. The SK channel, encoded by ROMK, is believed to mediate baseline K+ secretion. The role of the stretch- and Ca2+-activated maxi-K channel is still uncertain. The purpose of this study was to identify the K+ channel mediating flow-dependent K+ secretion in the CCD. Segments isolated from New Zealand White rabbits were microperfused in the absence and presence of luminal tetraethylammonium (TEA) or charybdotoxin, both inhibitors of maxi-K but not SK channels, or apamin, an inhibitor of small-conductance maxi-K+ channels. Net K+ secretion and Na+ absorption were measured at varying flow rates. In the absence of TEA, net K+ secretion increased from 8.3 ± 1.0 to 23.4 ± 4.7 pmol · min−1 · mm−1( P < 0.03) as the tubular flow rate was increased from 0.5 to 6 nl · min−1 · mm−1. Flow stimulation of net K+ secretion was blocked by luminal TEA (8.2 ± 1.2 vs. 9.9 ± 2.7 pmol · min−1 · mm−1 at 0.6 and 6 nl · min−1 · mm−1 flow rates, respectively) or charybdotoxin (6.8 ± 1.6 vs. 8.3 ± 1.6 pmol · min−1 · mm−1 at 1 and 4 nl · min−1 · mm−1 flow rates, respectively) but not by apamin. These results suggest that flow-dependent K+ secretion is mediated by a maxi-K channel, whereas baseline K+ secretion occurs through a TEA- and charybdotoxin-insensitive SK (ROMK) channel.

scholarly journals Small-conductance, Ca2+-activated K+ channel 2 is the key functional component of SK channels in mouse urinary bladder

2008 ◽  
Vol 294 (5) ◽  
pp. R1737-R1743 ◽  
Author(s):  
K. S. Thorneloe ◽  
A. M. Knorn ◽  
P. E. Doetsch ◽  
E. S. R. Lashinger ◽  
A. X. Liu ◽  
...  
Keyword(s):  
Urinary Bladder ◽  
Action Potentials ◽  
Electrical Field Stimulation ◽  
K Channel ◽  
Sk Channels ◽  
Bladder Smooth Muscle ◽  
Sk Channel ◽  
Urinary Bladder Smooth Muscle ◽  
Insight Into ◽  
Small Conductance

Small-conductance Ca2+-activated K+ (SK) channels play an important role in regulating the frequency and in shaping urinary bladder smooth muscle (UBSM) action potentials, thereby modulating contractility. Here we investigated a role for the SK2 member of the SK family (SK1-3) utilizing: 1) mice expressing β-galactosidase (β-gal) under the direction of the SK2 promoter (SK2 β-gal mice) to localize SK2 expression and 2) mice lacking SK2 gene expression (SK2−/− mice) to assess SK2 function. In SK2 β-gal mice, UBSM staining was observed, but staining was undetected in the urothelium. Consistent with this, urothelial SK2 mRNA was determined to be 4% of that in UBSM. Spontaneous phasic contractions in wild-type (SK2+/+) UBSM strips were potentiated (259% of control) by the selective SK channel blocker apamin (EC50 = 0.16 nM), whereas phasic contractions of SK2−/− strips were unaffected. Nerve-mediated contractions of SK2+/+ UBSM strips were also increased by apamin, an effect absent in SK2−/− strips. Apamin increased the sensitivity of SK2+/+ UBSM strips to electrical field stimulation, since pretreatment with apamin decreased the frequency required to reach a 50% maximal contraction (vehicle, 21 ± 4 Hz, n = 6; apamin, 12 ± 2 Hz, n = 7; P < 0.05). In contrast, the sensitivity of SK2−/− UBSM strips was unaffected by apamin. Here we provide novel insight into the molecular basis of SK channels in the urinary bladder, demonstrating that the SK2 gene is expressed in the bladder and that it is essential for the ability of SK channels to regulate UBSM contractility.

scholarly journals Inhibition of Small-Conductance, Ca2+-Activated K+ Current by Ondansetron

2021 ◽  
Vol 12 ◽  
Author(s):  
Shuai Guo ◽  
Zhenhui Chen ◽  
Peng-Sheng Chen ◽  
Michael Rubart
Keyword(s):  
Inhibitory Effect ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Inwardly Rectifying ◽  
Small Conductance

Background: Small-conductance Ca2+-activated K+ channels (SK channels) have been proposed as antiarrhythmic targets for the treatment of atrial fibrillation. We previously demonstrated that the 5-HT3 receptor antagonist ondansetron inhibits heterologously expressed, human SK2 (hSK2) currents as well as native cardiac SK currents in a physiological extra-/intracellular [K+] gradient at therapeutic (i.e., sub-micromolar) concentrations. A recent study, using symmetrical [K+] conditions, challenged this result. The goal of the present study was to revisit the inhibitory effect of ondansetron on hSK2-mediated currents in symmetrical [K+] conditions.Experimental Approach: The whole-cell patch clamp technique was used to investigate the effects of ondansetron and apamin on hSK2-mediated currents expressed in HEK 293 cells. Currents were measured in symmetrical [K+] conditions in the presence of 100 nM [Ca2+]o.Results: Expression of hSK2 produced inwardly rectifying whole-cell currents in the presence of 400 nM free cytosolic Ca2+. Ondansetron inhibited whole-cell hSK2 currents with IC50 values of 154 and 113 nM at −80 and 40 mV, respectively. Macroscopic current inhibited by ondansetron and current inhibited by apamin exhibited inwardly rectifying current-voltage relationships with similar reversal potentials (apamin, ∼5 mV and ondansetron, ∼2 mV). Ondansetron (1 μM) in the continuing presence of apamin (100 nM) had no effect on hSK2-mediated whole-cell currents. Wild-type HEK 293 cells did not express ondansetron- or apamin-sensitive currents.Conclusion: Ondansetron in sub-micromolar concentrations inhibits hSK2 currents even under altered ionic conditions.

scholarly journals Hydrolyzable ATP and PIP2 Modulate the Small-conductance K+ Channel in Apical Membranes of Rat Cortical-Collecting Duct (CCD)

2002 ◽  
Vol 120 (5) ◽  
pp. 603-615 ◽  
Author(s):  
Ming Lu ◽  
Steven C. Hebert ◽  
Gerhard Giebisch
Keyword(s):  
Kinase Inhibitors ◽  
Collecting Duct ◽  
K Channel ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Sk Channel ◽  
Phosphoinositide Kinase ◽  
Excised Patches ◽  
Small Conductance

The small-conductance K+ channel (SK) in the apical membrane of the cortical-collecting duct (CCD) is regulated by adenosine triphosphate (ATP) and phosphorylation-dephosphorylation processes. When expressed in Xenopus oocytes, ROMK, a cloned K+ channel similar to the native SK channel, can be stimulated by phosphatidylinositol bisphosphate (PIP2), which is produced by phosphoinositide kinases from phosphatidylinositol. However, the effects of PIP2 on SK channel activity are not known. In the present study, we investigated the mechanism by which hydrolyzable ATP prevented run-down of SK channel activity in excised apical patches of principal cells from rat CCD. Channel run-down was significantly delayed by pretreatment with hydrolyzable Mg-ATP, but ATPγS and AMP-PNP had no effect. Addition of alkaline phosphatase also resulted in loss of channel activity. After run-down, SK channel activity rapidly increased upon addition of PIP2. Exposure of inside-out patches to phosphoinositide kinase inhibitors (LY294002, quercetin or wortmannin) decreased channel activity by 74% in the presence of Mg-ATP. PIP2 added to excised patches reactivated SK channels in the presence of these phosphoinositide kinase inhibitors. The protein kinase A inhibitor, PKI, reduced channel activity by 36% in the presence of Mg-ATP. PIP2 was also shown to modulate the inhibitory effects of extracellular and cytosolic ATP. We conclude that both ATP-dependent formation of PIP2 through membrane-bound phosphoinositide kinases and phosphorylation of SK by PKA play important roles in modulating SK channel activity.

scholarly journals Molecular Characteristics of Rat Liver Arginase

1968 ◽  
Vol 243 (23) ◽  
pp. 6123-6129
Author(s):  
H Hirsch-Kolb ◽  
D M Greenberg
Keyword(s):  
Rat Liver ◽  
Molecular Characteristics

scholarly journals Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

2021 ◽  
Vol 22 (9) ◽  
pp. 4637
Author(s):  
Daniel Barth ◽  
Andreas Lückhoff ◽  
Frank J. P. Kühn
Keyword(s):  
Amino Acid Residues ◽  
Hek 293 ◽  
Complete Inactivation ◽  
293 Cells ◽  
Activation Mechanisms ◽  
Specific Regulation ◽  
Species Specific ◽  
Inside Out ◽  
Positively Charged

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), commonly referred to as PIP2, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP2. We demonstrate a crucial role of PIP2, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP2 that were dependent on the species variant became apparent. While depletion of PIP2 via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP2 differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP2 content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP2 sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP2 binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca2+ in a species-specific manner.

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