scholarly journals A forward genetic screen identifies chaperone CNX-1 as a conserved biogenesis regulator of ERG K+ channels

2018 ◽  
Vol 150 (8) ◽  
pp. 1189-1201 ◽  
Author(s):  
Xue Bai ◽  
Kai Li ◽  
Li Yao ◽  
Xin-Lei Kang ◽  
Shi-Qing Cai
Keyword(s):  
Action Potentials ◽  
K Channel ◽  
Translation System ◽  
Loss Of Function ◽  
K Channels ◽  
Type K ◽  
Evolutionarily Conserved ◽  
Protein Biogenesis ◽  
Free Translation ◽  
Current Densities

The human ether-a-go-go–related gene (hERG) encodes a voltage-gated potassium channel that controls repolarization of cardiac action potentials. Accumulating evidence suggests that most disease-related hERG mutations reduce the function of the channel by disrupting protein biogenesis of the channel in the endoplasmic reticulum (ER). However, the molecular mechanism underlying the biogenesis of ERG K+ channels is largely unknown. By forward genetic screening, we identified an ER-located chaperone CNX-1, the worm homologue of mammalian chaperone Calnexin, as a critical regulator for the protein biogenesis of UNC-103, the ERG-type K+ channel in Caenorhabditis elegans. Loss-of-function mutations of cnx-1 decreased the protein level and current density of the UNC-103 K+ channel and suppressed the behavioral defects caused by a gain-of-function mutation in unc-103. Moreover, CNX-1 facilitated tetrameric assembly of UNC-103 channel subunits in a liposome-assisted cell-free translation system. Further studies showed that CNX-1 act in parallel to DNJ-1, another ER-located chaperone known to regulate maturation of UNC-103 channels, on controlling the protein biogenesis of UNC-103. Importantly, Calnexin interacted with hERG proteins in the ER in HEK293T cells. Deletion of calnexin reduced the expression and current densities of endogenous hERG K+ channels in SH-SY5Y cells. Collectively, we reveal an evolutionarily conserved chaperone CNX-1/Calnexin controlling the biogenesis of ERG-type K+ channels.

scholarly journals Changes in D-type K^+ channel properties influence on synaptic integration and back propagation of action potentials in dendrites

2003 ◽  
Vol 43 (supplement) ◽  
pp. S218
Author(s):  
S. Sato ◽  
Y. Shimokawa ◽  
M. Saito ◽  
H. Takagi ◽  
S. Koyama ◽  
...  
Keyword(s):  
Action Potentials ◽  
Back Propagation ◽  
Synaptic Integration ◽  
K Channel ◽  
Type K ◽  
Channel Properties

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.

scholarly journals Low molecular weight poly(A)+ mRNA species encode factors that modulate gating of a non-Shaker A-type K+ channel.

1993 ◽  
Vol 102 (4) ◽  
pp. 713-728 ◽  
Author(s):  
L D Chabala ◽  
N Bakry ◽  
M Covarrubias
Keyword(s):  
Molecular Weight ◽  
Xenopus Oocytes ◽  
Membrane Potentials ◽  
K Channel ◽  
Low Molecular Weight ◽  
K Channels ◽  
Fourfold Increase ◽  
Mrna Species ◽  
Type K

Voltage-dependent K+ channels control repolarization of action potentials and help establish firing patterns in nerve cells. To determine the nature and role of molecular components that modulate K+ channel function in vivo, we coinjected Xenopus oocytes with cRNA encoding a cloned subthreshold A-type K+ channel (mShal1, also referred to as mKv4.1) and a low molecular weight (LMW) fraction (2-4 kb) of poly(A)+ mRNA (both from rodent brain). Coinjected oocytes exhibited a significant (fourfold) increase in the surface expression of mShal1 K+ channels with no change in the open-channel conductance. Coexpression also modified the gating kinetics of mShal1 current in several respects. Macroscopic inactivation of whole oocyte currents was fitted with the sum of two exponential components. Both fast and slow time constants of inactivation were accelerated at all membrane potentials in coinjected oocytes (tau f = 47.2 ms vs 56.5 ms at 0 mV and tau s = 157 ms vs 225 ms at 0 mV), and the corresponding ratios of amplitude terms were shifted toward domination by the fast component (Af/As = 2.71 vs 1.17 at 0 mV). Macroscopic activation was characterized in terms of the time-to-peak current, and it was found to be more rapid at all membrane potentials in coinjected oocytes (9.9 ms vs 13.5 ms at 0 mV). Coexpression also leads to more rapid recovery from inactivation (approximately 2.4-fold faster at -100 mV). The coexpressed K+ currents in oocytes resemble currents expressed in mouse fibroblasts (NIH3T3) transfected only with mShal1 cDNA. These results indicate that mammalian regulatory subunits or enzymes encoded by LMW mRNA species, which are apparently missing or expressed at low levels in Xenopus oocytes, may modulate gating in some native subthreshold A-type K+ channels.

scholarly journals Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1)

1999 ◽  
Vol 344 (3) ◽  
pp. 699-711 ◽  
Author(s):  
Jonathan D. GROVES ◽  
Michael J. A. TANNER
Keyword(s):  
Structural Model ◽  
Functional Expression ◽  
Band 3 ◽  
Current Evidence ◽  
Translation System ◽  
Cdna Clones ◽  
Red Cell ◽  
Loss Of Function ◽  
Free Translation ◽  
High Level

We have examined the functional co-assembly of non-complementary pairs of N- and C-terminal polypeptide fragments of the anion transport domain (b3mem) of human red-cell band 3. cDNA clones encoding non-contiguous pairs of fragments with one transmembrane (TM) region omitted, or overlapping pairs of fragments with between one and ten TM regions duplicated, were co-expressed in Xenopus oocytes and a cell-free translation system. Stilbene disulphonate-sensitive chloride uptake assays in oocytes revealed that the omission of any single TM region of b3mem except spans 6 and 7 caused a complete loss of functional expression. In contrast, co-expressed pairs of fragments overlapping a single TM region 5, 6, 7, 8, 9-10 or 11-12 retained a high level of functionality, whereas fragments overlapping the clusters of TM regions 2-5, 4-5, 5-8 and 8-10 also mediated some stilbene disulphonate-sensitive uptake. The co-assembly of N- or C-terminal fragments with intact band 3, b3mem or other fragments was examined by co-immunoprecipitation in non-denaturing detergent solutions by using monoclonal antibodies against the termini of b3mem. All the fragments, except for TM spans 13-14, co-immunoprecipitated with b3mem. The medium-sized N-terminal fragments comprising spans 1-6, 1-7 or 1-8 co-immunoprecipitated particularly strongly with the C-terminal fragments containing spans 8-14 or 9-14. The fragments comprising spans 1-4 or 1-12 co-immunoprecipitated less extensively than the other N-terminal fragments with either b3mem or C-terminal fragments. There is sufficient flexibility in the structure of b3mem to allow the inclusion of at least one duplicated TM span without a loss of function. We propose a working model for the organization of TM spans of dimeric band 3 based on current evidence.

scholarly journals Castration Induces Down-Regulation of A-Type K+ Channel in Rat Vas Deferens Smooth Muscle

2019 ◽  
Vol 20 (17) ◽  
pp. 4073 ◽  
Author(s):  
Susumu Ohya ◽  
Katsunori Ito ◽  
Noriyuki Hatano ◽  
Akitoshi Ohno ◽  
Katsuhiko Muraki ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vas Deferens ◽  
Oral Treatment ◽  
Functional Expression ◽  
Rat Vas Deferens ◽  
K Channel ◽  
Α Subunit ◽  
K Channels ◽  
Expression Levels ◽  
Type K

A-type K+ channels contribute to regulating the propagation and frequency of action potentials in smooth muscle cells (SMCs). The present study (i) identified the molecular components of A-type K+ channels in rat vas deferens SMs (VDSMs) and (ii) showed the long-term, genomic effects of testosterone on their expression in VDSMs. Transcripts of the A-type K+ channel α subunit, Kv4.3L and its regulatory β subunits, KChIP3, NCS1, and DPP6-S were predominantly expressed in rat VDSMs over the other related subtypes (Kv4.2, KChIP1, KChIP2, KChIP4, and DPP10). A-type K+ current (IA) density in VDSM cells (VDSMCs) was decreased by castration without changes in IA kinetics, and decreased IA density was compensated for by an oral treatment with 17α-methyltestosterone (MET). Correspondingly, in the VDSMs of castrated rats, Kv4.3L and KChIP3 were down-regulated at both the transcript and protein expression levels. Changes in Kv4.3L and KChIP3 expression levels were compensated for by the treatment with MET. These results suggest that testosterone level changes in testosterone disorders and growth processes control the functional expression of A-type K+ channels in VDSMCs.

Molecular determinants of ion conduction and inactivation in K+ channels

1995 ◽  
Vol 268 (3) ◽  
pp. C535-C556 ◽  
Author(s):  
M. Kukuljan ◽  
P. Labarca ◽  
R. Latorre
Keyword(s):  
K Channel ◽  
Amino Acid Residues ◽  
Fast Inactivation ◽  
Transmembrane Segment ◽  
Ion Conduction ◽  
K Channels ◽  
Type K ◽  
Molecular Determinants ◽  
Inward Rectifiers ◽  
Membrane Spanning

K+ channel-forming proteins can be grouped into three families that differ by the number of potential membrane-spanning segments. The largest of these families is composed of tetrameric channels with subunits containing six putative membrane-spanning segments (S1-S6). Inward rectifiers comprise a second family of K+ channels with subunits having two transmembrane domains (M1, M2). Monomers in the third family are proteins containing only one membrane-spanning segment, and they give origin to minK+ channels. Joining together segments S5 and S6 in the case of voltage-gated K+ channels and M1 and M2 in inward rectifiers, there is a highly conserved region with a hairpin shape called the H5 or P region. The P region, the loop connecting the S4 and S5 domains and the S6 transmembrane segment in Shaker-type K+ channels and the COOH-terminal in inward rectifiers, appears to play crucial roles in ion conduction. In Shaker K+ channels the NH2-terminal has been identified as responsible for fast inactivation (N-type inactivation). If the fast-inactivation gate is removed, a slower inactivation process persists, and its rate can be altered by mutations of amino acid residues forming part of the region in the neighborhood of the COOH-terminal (C-type inactivation). In this review we discuss the strategies followed to identify the different structures of K+ channels involved in ion conduction and inactivation processes and how they interplay.

scholarly journals Delayed rectifying and calcium-activated K+ channels and their significance for action potential repolarization in mouse pancreatic beta-cells.

1990 ◽  
Vol 95 (6) ◽  
pp. 1041-1059 ◽  
Author(s):  
P A Smith ◽  
K Bokvist ◽  
P Arkhammar ◽  
P O Berggren ◽  
P Rorsman
Keyword(s):  
Beta Cells ◽  
Action Potentials ◽  
Pancreatic Beta Cells ◽  
Single Channel ◽  
Outward Current ◽  
K Channel ◽  
K Channels ◽  
Dependent Component ◽  
Voltage Dependent ◽  
Action Potential Repolarization

The contribution of Ca2(+)-activated and delayed rectifying K+ channels to the voltage-dependent outward current involved in spike repolarization in mouse pancreatic beta-cells (Rorsman, P., and G. Trube. 1986. J. Physiol. 374:531-550) was assessed using patch-clamp techniques. A Ca2(+)-dependent component could be identified by its rapid inactivation and sensitivity to the Ca2+ channel blocker Cd2+. This current showed the same voltage dependence as the voltage-activated (Cd2(+)-sensitive) Ca2+ current and contributed 10-20% to the total beta-cell delayed outward current. The single-channel events underlying the Ca2(+)-activated component were investigated in cell-attached patches. Increase of [Ca2+]i invariably induced a dramatic increase in the open state probability of a Ca2(+)-activated K+ channel. This channel had a single-channel conductance of 70 pS [( K+]o = 5.6 mM). The Ca2(+)-independent outward current (constituting greater than 80% of the total) reflected the activation of an 8 pS [( K+]o = 5.6 mM; [K+]i = 155 mM) K+ channel. This channel was the only type observed to be associated with action potentials in cell-attached patches. It is suggested that in mouse beta-cells spike repolarization results mainly from the opening of the 8-pS delayed rectifying K+ channel.

scholarly journals Physiological role of dendrotoxin sensitive k channels in the rat cerebellar Purkinje neurons

2007 ◽  
pp. 807-813 ◽  
Author(s):  
H Haghdoust ◽  
M Janahmadi ◽  
G Behzadi
Keyword(s):  
Action Potentials ◽  
Physiological Role ◽  
Neuronal Firing ◽  
Purkinje Neurons ◽  
Repetitive Firing ◽  
Neuronal Membrane ◽  
K Channels ◽  
Type K ◽  
Cerebellar Purkinje Neurons

To understand the contribution of potassium (K+) channels, particularly alpha-dendrotoxin (D-type)-sensitive K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits), to the generation of neuronal spike output we must have detailed information of the functional role of these channels in the neuronal membrane. Conventional intracellular recording methods in current clamp mode were used to identify the role of alpha-dendrotoxin (alpha-DTX)-sensitive K+ channel currents in shaping the spike output and modulation of neuronal properties of cerebellar Purkinje neurons (PCs) in slices. Addition of alpha-DTX revealed that D-type K+ channels play an important role in the shaping of Purkinje neuronal firing behavior. Repetitive firing capability of PCs was increased following exposure to artificial cerebrospinal fluid (aCSF) containing alpha-DTX, so that in response to the injection of 0.6 nA depolarizing current pulse of 600 ms, the number of action potentials insignificantly increased from 15 in the presence of 4-AP to 29 action potentials per second after application of DTX following pretreatment with 4-AP. These results indicate that D-type K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits) may contribute to the spike frequency adaptation in PCs. Our findings suggest that the activation of voltage-dependent K+ channels (D and A types) markedly affect the firing pattern of PCs.

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