Evaluation of channel function after alteration of amino acid residues at the pore center of KCNQ1 channel
The effect of the electrical charge or the size of the amino acid residue at the pore center of a slowly activation component of the delayed rectifier potassium channel: KCNQ1 was studied. K+ currents were measured after transfection of one of four KCNQ1 mutants: substituting Isoleucine with Lysine, Glutamate, Valine or Glycine and then transfected in COS-7 cells. Both the negatively- and positive charged residue I313 K and I313E showed a loss of function when expressed alone and a dominant negative suppression when co-expressed with wild type KCNQ1. When the site was substituted with the smallest neutral amino acid residue: I313G, there was a small reduction of current when transfected alone and a gain of function when co-transfected with the wild type. I313V showed no difference from the wild type. Changes of amino acid residue at the pore center of KCNQ1 may alter the channel function but this depends on the electrical charge or the size of amino acid residue.
Keywords: Long QT syndrome; Missense mutation; KCNQ1; IKs; Pore center; Amino acid residue
What Is Climate Change Resistant Coffee?
http://buyorganiccoffee.org/1881/what-is-climate-change-resistant-coffee/
Year after year meteorologists report that average global temperatures have hit another high for the modern era. Considering that what is today the frozen arctic once supported palm trees we have wondered if growing coffee on the arctic tundra will one day be possible. But what would extreme climate change do to coffee production? Last year we asked if climate change could destroy coffee production.
Higher temperatures, more chaotic weather patterns, droughts and floods we become the norm as the world climate change, according to experts. The Tech Times writes about the effect of climate change on agriculture.
As average global temperatures begin to rise due to human activity, scientists say the drastic effects of climate change continue to take effect all over the world.
One of the most severely affected sectors is the field of agriculture. In the past decades, extreme weather conditions caused by climate change have disrupted global food production.
The researchers found that global cereal production was as much as 10% lower in the last twenty years. However, there appears to be a “fertilizer” effect of higher levels of CO2 in the atmosphere. The problem for coffee is that the fertilizer effect would not reduce the risk of leaf rust or help when crops are washed out by floods or die because of drought. Climate change may not destroy coffee production but it may well reduce it.
What should coffee producers do? Phy.org reports that Nicaragua focuses on climate change resistant coffee.
With climate change threatening crops in many parts of the world, Nicaragua is turning to a robust variety of coffee bean to protect one of its key exports.
The appropriately named robusta coffee comes from the Coffea canephora plant, which is being increasingly planted in the Central American country under government authorization.
What Is Climate Change Resistant Coffee?
http://buyorganiccoffee.org/1881/what-is-climate-change-resistant-coffee/
Year after year meteorologists report that average global temperatures have hit another high for the modern era. Considering that what is today the frozen arctic once supported palm trees we have wondered if growing coffee on the arctic tundra will one day be possible. But what would extreme climate change do to coffee production? Last year we asked if climate change could destroy coffee production.
Higher temperatures, more chaotic weather patterns, droughts and floods we become the norm as the world climate change, according to experts. The Tech Times writes about the effect of climate change on agriculture.
As average global temperatures begin to rise due to human activity, scientists say the drastic effects of climate change continue to take effect all over the world.
One of the most severely affected sectors is the field of agriculture. In the past decades, extreme weather conditions caused by climate change have disrupted global food production.
The researchers found that global cereal production was as much as 10% lower in the last twenty years. However, there appears to be a “fertilizer” effect of higher levels of CO2 in the atmosphere. The problem for coffee is that the fertilizer effect would not reduce the risk of leaf rust or help when crops are washed out by floods or die because of drought. Climate change may not destroy coffee production but it may well reduce it.
What should coffee producers do? Phy.org reports that Nicaragua focuses on climate change resistant coffee.
With climate change threatening crops in many parts of the world, Nicaragua is turning to a robust variety of coffee bean to protect one of its key exports.
The appropriately named robusta coffee comes from the Coffea canephora plant, which is being increasingly planted in the Central American country under government authorization.
Impact of percutaneous coronary intervention on the levels of interleukin-6 and C-reactive protein in the coronary circulation of subjects with coronary artery disease
A characteristic of the developing mammalian visual system is a brief interval of plasticity, termed the “critical period,” when the circuitry of
primary visual cortex is most sensitive to perturbation of visual experience. Depriving one eye of vision (monocular deprivation [MD]) during
the critical period alters ocular dominance (OD) by shifting the responsiveness of neurons in visual cortex to favor the nondeprived eye. A
disinhibitory microcircuit involving parvalbumin-expressing (PV) interneurons initiates this OD plasticity. The gene encoding the neuronal
nogo-66-receptor1(ngr1/rtn4r) is required to close the critical period.Herewecombinedmousegenetics, electrophysiology,andcircuitmapping
with laser-scanning photostimulation to investigate whether disinhibition is confined to the critical period by ngr1.We demonstrate that ngr1
mutant mice retain plasticity characteristic of the critical period as adults, and that ngr1 operates within PV interneurons to restrict the loss of
intracortical excitatory synaptic input following MD in adult mice, and this disinhibition induces a “lower PV network configuration” in both
critical-period wild-type miceandadult ngr1/mice.Wepropose that ngr1 limits disinhibition to close the critical period forODplasticityand
that a decrease in PV expression levels reports the diminished recent cumulative activity of these interneurons.
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Early sensory experience instructs the maturation of neural circuitry in the cortex1, 2. This has been studied extensively in the primary visual cortex, in which loss of vision to one eye permanently degrades cortical responsiveness to that eye3, 4, a phenomenon known as ocular dominance plasticity (ODP). Cortical inhibition mediates this process4, 5, 6, but the precise role of specific classes of inhibitory neurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediately drop by half when vision is restricted to one eye, but gradually return to normal over the following twenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation results from a rapid, although transient, reduction in the firing rates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turn can be attributed to a decrease in local excitatory circuit input onto PV interneurons. This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODP and appears to be necessary for subsequent shifts in excitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. These findings define the microcircuit changes initiating competitive plasticity during critical periods of cortical development. Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
Severe Long QT Phenotypes Associated with Novel Mutation of I313K at the Centre of KCNQ1 Potassium Channel Pore, (Authors:
Taruna Ikrar
Division of Multidisciplinary of Neuroscience, School of Medicine, Universality of California, Irvine, CA 92617, USA
Division of Cardiology, First Department of Internal Medicine, School of Medical Sciences, Niigata, Japan)
Forces associated with blood flow are major determinants of vascular morphogenesis and physiology. Blood flow is crucial for blood vessel development during embryogenesis and for regulation of vessel diameter in adult life. It is also a key factor in atherosclerosis, which, despite the systemic nature of major risk factors, occurs mainly at regions of arteries that experience disturbances in fluid flow. Recent data have highlighted the potential endothelial mechanotransducers that mediate responses to flow, the effects of atheroprotective versus atherogenic flow, and the mechanisms that contribute to progression of the disease over time and how systemic factors interact with flow patterns to cause atherosclerosis.
Frm:- Nat Rev Mol Cell Biol. 2009 Jan; 10(1): 53–62. doi: 10.1038/nrm2596
Impact of percutaneous coronary intervention on the levels of interleukin-6 and C-reactive protein in the coronary circulation of subjects with coronary artery disease
A characteristic of the developing mammalian visual system is a brief interval of plasticity, termed the “critical period,” when the circuitry of
primary visual cortex is most sensitive to perturbation of visual experience. Depriving one eye of vision (monocular deprivation [MD]) during
the critical period alters ocular dominance (OD) by shifting the responsiveness of neurons in visual cortex to favor the nondeprived eye. A
disinhibitory microcircuit involving parvalbumin-expressing (PV) interneurons initiates this OD plasticity. The gene encoding the neuronal
nogo-66-receptor1(ngr1/rtn4r) is required to close the critical period.Herewecombinedmousegenetics, electrophysiology,andcircuitmapping
with laser-scanning photostimulation to investigate whether disinhibition is confined to the critical period by ngr1.We demonstrate that ngr1
mutant mice retain plasticity characteristic of the critical period as adults, and that ngr1 operates within PV interneurons to restrict the loss of
intracortical excitatory synaptic input following MD in adult mice, and this disinhibition induces a “lower PV network configuration” in both
critical-period wild-type miceandadult ngr1/mice.Wepropose that ngr1 limits disinhibition to close the critical period forODplasticityand
that a decrease in PV expression levels reports the diminished recent cumulative activity of these interneurons.
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Early sensory experience instructs the maturation of neural circuitry in the cortex1, 2. This has been studied extensively in the primary visual cortex, in which loss of vision to one eye permanently degrades cortical responsiveness to that eye3, 4, a phenomenon known as ocular dominance plasticity (ODP). Cortical inhibition mediates this process4, 5, 6, but the precise role of specific classes of inhibitory neurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediately drop by half when vision is restricted to one eye, but gradually return to normal over the following twenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation results from a rapid, although transient, reduction in the firing rates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turn can be attributed to a decrease in local excitatory circuit input onto PV interneurons. This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODP and appears to be necessary for subsequent shifts in excitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. These findings define the microcircuit changes initiating competitive plasticity during critical periods of cortical development. Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
Severe Long QT Phenotypes Associated with Novel Mutation of I313K at the Centre of KCNQ1 Potassium Channel Pore, (Authors:
Taruna Ikrar
Division of Multidisciplinary of Neuroscience, School of Medicine, Universality of California, Irvine, CA 92617, USA
Division of Cardiology, First Department of Internal Medicine, School of Medical Sciences, Niigata, Japan)
Forces associated with blood flow are major determinants of vascular morphogenesis and physiology. Blood flow is crucial for blood vessel development during embryogenesis and for regulation of vessel diameter in adult life. It is also a key factor in atherosclerosis, which, despite the systemic nature of major risk factors, occurs mainly at regions of arteries that experience disturbances in fluid flow. Recent data have highlighted the potential endothelial mechanotransducers that mediate responses to flow, the effects of atheroprotective versus atherogenic flow, and the mechanisms that contribute to progression of the disease over time and how systemic factors interact with flow patterns to cause atherosclerosis.
Frm:- Nat Rev Mol Cell Biol. 2009 Jan; 10(1): 53–62. doi: 10.1038/nrm2596
Biochemical and physiological target sites of insecticides on insectNikita Negi
Different insecticide group act on different target site and mechanism of their toxicity lies in differential actions on the target receptors/channels.
To understand the mode of action of insecticides that target the insect nervous system, it is important to have a basic understanding of how the nervous system operates. In insects, the nervous system is composed of a series of highly specialized, interconnected cells, along which travel electrical charges called impulses. A nervous system is essential for the passage of information through the body by means of electrical signals.
Impulses are driven by the movement of electrically charged sodium, potassium and chloride ions into and out of nerve cells. The uninterrupted transmission of impulses along this series of cells is required for a nervous system to function properly. In insects, prolonged or irreversible disruption of a normal-functioning nervous system will result in death.
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Earlysensoryexperienceinstructsthematurationofneuralcircuitry in the cortex1,2. This has been studied extensively in the primary visualcortex,inwhichlossofvisiontooneeyepermanentlydegrades corticalresponsivenesstothateye3,4,aphenomenonknownasocular dominance plasticity (ODP). Cortical inhibition mediates this process4–6,butthepreciseroleofspecificclassesofinhibitoryneurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediatelydropbyhalfwhenvisionisrestrictedtooneeye,butgradually return to normal over the followingtwenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation resultsfromarapid,althoughtransient,reductioninthefiringrates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turncanbeattributedtoadecreaseinlocalexcitatorycircuitinput onto PV interneurons.This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODPandappearstobenecessaryforsubsequentshiftsinexcitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. Thesefindingsdefinethemicrocircuitchangesinitiatingcompetitive
plasticityduringcriticalperiodsofcorticaldevelopment.Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
An inhibitory pull–push circuit in frontal cortexTaruna Ikrar
Push–pull is a canonical computation of excitatory cortical circuits. By contrast, we identify a pull–push inhibitory circuit in frontal cortex that originates in vasoactive intestinal polypeptide (VIP)-expressing interneurons. During arousal, VIP cells rapidly and directly inhibit pyramidal neurons; VIP cells also indirectly excite these pyramidal neurons via parallel disinhibition. Thus, arousal exerts a feedback pull–push influence on excitatory neurons—an inversion of the canonical push–pull of feedforward input.
Pten and eph b4 regulate the establishment of perisomatic inhibition in mouse...Taruna Ikrar
Perisomatic inhibition of pyramidal neurons is established by fast-spiking, parvalbuminexpressing interneurons (PV cells). Failure to assemble adequate perisomatic inhibition is thought to underlie the aetiology of neurological dysfunction in seizures, autism spectrum disorders and schizophrenia. Here we show that in mouse visual cortex, strong perisomatic inhibition does not develop if PV cells lack a single copy of Pten. PTEN signalling appears to drive the assembly of perisomatic inhibition in an experience-dependent manner by suppressing the expression of EphB4; PVcells hemizygous for Pten show an B2-fold increase in expression of EphB4, and over-expression of EphB4 in adult PV cells causes a dismantling of perisomatic inhibition. These findings implicate a molecular disinhibitory mechanism driving the establishment of perisomatic inhibition whereby visual experience enhances Pten signalling, resulting in the suppression of EphB4 expression; this relieves a native synaptic repulsion between PV cells and pyramidal neurons, thereby promoting the assembly of perisomatic inhibition.
Abstract In the mammalian neocortex, excitatory neurons provide excitation in both columnar and laminar dimensions, which is modulated further by inhibitory neurons. However, our understanding of intracortical excitatory and inhibitory synaptic inputs in relation to principal excitatory neurons remains incomplete, and it is unclear how local excitatory and inhibitory synaptic connections to excitatory neurons are spatially organized on a layer-by-layer basis. In the present study, we combined whole cell recordings with laser scanning photostimulation via glutamate uncaging to map excitatory and inhibitory synaptic inputs to single excitatory neurons throughout cortical layers 2/3–6 in the mouse primary visual cortex (V1). We find that synaptic input sources of excitatory neurons span the radial columns of laminar microcircuits, and excitatory neurons in different V1 laminae exhibit distinct patterns of layer-specific organizationofexcitatoryinputs.Remarkably,thespatialextentofinhibitoryinputsofexcitatory neurons for a given layer closely mirrors that of their excitatory input sources, indicating that excitatory and inhibitory synaptic connectivity is spatially balanced across excitatory neuronal networks. Strong interlaminar inhibitory inputs are found, particularly for excitatory neurons in layers 2/3 and 5. This differs from earlier studies reporting that inhibitory cortical connections to excitatory neurons are generally localized within the same cortical layer. On the basis of the functional mapping assays, we conducted a quantitative assessment of both excitatory and inhibitory synaptic laminar connections to excitatory cells at single cell resolution, establishing precise layer-by-layer synaptic wiring diagrams of excitatory neurons in the visual cortex.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
How to Create Map Views in the Odoo 17 ERPCeline George
The map views are useful for providing a geographical representation of data. They allow users to visualize and analyze the data in a more intuitive manner.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
This is a presentation by Dada Robert in a Your Skill Boost masterclass organised by the Excellence Foundation for South Sudan (EFSS) on Saturday, the 25th and Sunday, the 26th of May 2024.
He discussed the concept of quality improvement, emphasizing its applicability to various aspects of life, including personal, project, and program improvements. He defined quality as doing the right thing at the right time in the right way to achieve the best possible results and discussed the concept of the "gap" between what we know and what we do, and how this gap represents the areas we need to improve. He explained the scientific approach to quality improvement, which involves systematic performance analysis, testing and learning, and implementing change ideas. He also highlighted the importance of client focus and a team approach to quality improvement.
We all have good and bad thoughts from time to time and situation to situation. We are bombarded daily with spiraling thoughts(both negative and positive) creating all-consuming feel , making us difficult to manage with associated suffering. Good thoughts are like our Mob Signal (Positive thought) amidst noise(negative thought) in the atmosphere. Negative thoughts like noise outweigh positive thoughts. These thoughts often create unwanted confusion, trouble, stress and frustration in our mind as well as chaos in our physical world. Negative thoughts are also known as “distorted thinking”.
Evaluation Of Channel Function After Alteration Of Amino Acid Residues At The Pore Center Of Kcnq1 Channel
1. YBBRC 22101 No. of Pages 6, Model 5G
29 November 2008 Disk Used
ARTICLE IN PRESS
Biochemical and Biophysical Research Communications xxx (2008) xxx–xxx
1
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
2 Evaluation of channel function after alteration of amino acid residues
F
3 at the pore center of KCNQ1 channel
OO
4 Taruna Ikrar a,b, Haruo Hanawa a, Hiroshi Watanabe a, Yoshiyasu Aizawa a,*, Mahmoud M. Ramadan a,
5 Masaomi Chinushi a, Minoru Horie c, Yoshifusa Aizawa a
6 a
Division of Cardiology, First Department of Internal Medicine, Niigata University Graduate School of Medical and Dental Sciences, 1-754 Asahimachi Dori,
7 Chuo-ku, Niigata 951-8510, Japan
8 b
The National Agency for Drug and Food Control, Republic of Indonesia, Jakarta, Indonesia
PR
9 c
Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Shiga, Japan
10
a r t i c l e i n f o a b s t r a c t
1 2
2 5
13 Article history: The effect of the electrical charge or the size of the amino acid residue at the pore center of a slowly acti- 26
14 Received 17 November 2008 vation component of the delayed rectifier potassium channel: KCNQ1 was studied. K+ currents were mea-
D 27
15 Available online xxxx sured after transfection of one of four KCNQ1 mutants: substituting Isoleucine with Lysine, Glutamate, 28
16
Valine or Glycine and then transfected in COS-7 cells. Both the negatively- and positive charged residue 29
I313 K and I313E showed a loss of function when expressed alone and a dominant negative suppression 30
TE
17 Keywords: when co-expressed with wild type KCNQ1. When the site was substituted with the smallest neutral 31
18 Long QT syndrome
amino acid residue: I313G, there was a small reduction of current when transfected alone and a gain 32
19 Missense mutation
20 KCNQ1
of function when co-transfected with the wild type. I313V showed no difference from the wild type. 33
21 IKs Changes of amino acid residue at the pore center of KCNQ1 may alter the channel function but this 34
22 Pore center depends on the electrical charge or the size of amino acid residue. 35
EC
23 Amino acid residue Ó 2008 Published by Elsevier Inc. 36
24
37
38
39 The delayed rectifier K+ current (IKs) channel is formed by the trically charged ones: Lysine and Glutamate and two neutral amino 61
RR
40 co-assembly of KCNQ1 (KvLQT1) with KCNE1 (minK) and contrib- acids: Valine and Glycine, and performed an electrophysiological 62
41 utes to repolarizing cardiac myocytes [1]. A mutation in either sub- study after transfection of each mutant. 63
42 unit is well known to cause long QT syndrome (LQTS) which
43 predisposes affected individuals to cardiac arrhythmias and sud- Materials and methods 64
44 den death [2,3].
45 In LQTS, the phenotype was suggested to be affected by the site
CO
Construction of plasmid DNA for gene transfer. A full-length human 65
46 of the mutation and patients with trans-membrane mutations had WT-KCNQ1, KCNE1, and mutant KCNQ1 were inserted into a plas- 66
47 more frequent diagnostic criteria, LQTS-related cardiac events and mid vector pIRES2-EGFP using BamHI restriction sites making the 67
48 a prolonged QT-interval after exercise than patients with a C-ter- pIRES2-EGFP-KCNQ1, pIRES-EGFP-KCNE1, and mutant pIRES2- 68
49 minal mutation [4]. However, this finding from Japan is in contrast EGFP-I313K plasmids as described previously [6]. In the pIRES2- 69
50 to that of the International Long QT Syndrome Registry in which EGFP-I313K plasmid, we introduced three new missense mutations 70
UN
51 the phenotypes were not related to the site of the mutation [5]. which can occur in humans. One is a mutant with Glutamate which is 71
52 We have previously characterized the physiological conse- a positively charged large amino acid residue (I313E). Then we pre- 72
53 quences of the LQTS-associated mutation at the pore center: pared two mutants, Valine (I313V), a neutral amino acid of similar 73
54 I313K, in a patient with a severe LQT1 phenotype [6]. The mutant size and homology to the Isoleucine residue in WT-KCNQ1 [7–10] 74
55 channels showed almost no current when transfected alone but and Glycine as the smallest sized neutral amino acid (I313G). 75
56 when co-expressed with WT-KCNQ1, they showed a dominant The mutation of I313E we introduced with a PCR reaction using 76
57 negative suppression [6]. the mutant primer sets: 50 -GTGGTCACAGTCACCACCgaaGGCTATGG 77
58 In this report, our aim was to explore the effects of the charge GGACAAGGTG-30 and 50 -CACCTTGTCCCCATAGCCttcGGTGGTGACTG 78
59 and the size of the amino acid residue at the pore center of KCNQ1. TGACCAC-30 (lower case letters indicate mutation sites). The mutation 79
60 In order to do this, we substituted its Isoleucine residue with elec- of I313V was introduced with a PCR using the mutant primer sets 50 - 80
GTGGTCACAGTCACCACCgtcGGCTATGGGGACAAGGTG-30 and 50 -CA 81
CCTTGTCCCCATAGCCgacGGTGGTGACTGTGACCAC-3. For the muta- 82
* Corresponding author. Fax: +81 25 228 5611.
E-mail address: aizaways@med.niigata-u.ac.jp (Y. Aizawa). tion of I313G, we used the primer sets 50 -GTGGTCACAGTCACCA 83
0006-291X/$ - see front matter Ó 2008 Published by Elsevier Inc.
doi:10.1016/j.bbrc.2008.11.076
Please cite this article in press as: T. Ikrar et al., Evaluation ofchannel function after alteration ofamino acid residues ..., Biochem. Biophys.
Res. Commun. (2008), doi:10.1016/j.bbrc.2008.11.076
2. YBBRC 22101 No. of Pages 6, Model 5G
29 November 2008 Disk Used
ARTICLE IN PRESS
2 T. Ikrar et al. / Biochemical and Biophysical Research Communications xxx (2008) xxx–xxx
84 CCggcGGCTATGGGGACAAGGTG-30 and 50 -CACCTTGTCCCCATAGC the voltage dependence of channel activation and deactivation 150
85 CgccGGTGGTGACTGTGACCAC-30 . were fitted with the Boltzmann equation: I = Imax/(1 + exp[(V1/ 151
86 For these experiments, we used the QuickChange site-directed 2 À V)/k]), where A was current amplitude, s was the time constant, 152
87 mutagenesis kit (Stratagene, La Jolla, CA, USA). The resulting prod- t was time, I was current amplitude, Imax was the maximal tail cur- 153
88 ucts were again amplified by PCR using the primers 50 -CCATTTCCATC rent, V was the test pulse potential, V1/2 was the half-maximal acti- 154
89 ATCGACCTCA-30 and 50 -AAGGAGAGCGCTGGTGAAG-30 . This final vation potential, and k was the slope of the activation curve. The 155
90 PCR product was ligated to the pIRES2-EGFP WT-KCNQ1 vector relationship of current density with side-chain residue volume 156
91 using the PstI sites at nucleotide positions 697 and 1675 of KCNQ1. was measured after 2 s during depolarization. 157
92 The PCR insert was also cleaved with PstI prior to ligation. The result- Results for continuous normal data were expressed as 158
93 ing four cloned plasmids were then transformed into Escherichia coli mean ± standard error of estimation. The comparison of means of 159
94 JM109 competent cells and purified using a Quantum Prep Plasmid continuous normal variables across a grouping variable with two 160
95 Maxi prep kit (Bio-Rad Laboratories, Hercules, CA). levels was done using the student’s t-test and the comparison of 161
F
96 Culture and transfection of COS-7 cells. A COS-7 monkey kidney means of continuous normal variables across a grouping variable 162
97 cell line was obtained from the American Type Cell Collection with several levels was undertaken with one-way analysis of var- 163
OO
98 and cultured in Dulbecco’s modified Eagles medium (Invitrogen iance (ANOVA). A two-sided significance level of 0.05 was used 164
99 Corporation, Gibco-BRL, Rockville, MD) supplemented with 1% for all analyses. 165
100 penicillin–streptomycin (prepared with 10,000 U/ml penicillin G
101 sodium and 10,000 lg/ml streptomycin sulfate in 0.85% saline)
Results 166
102 and 10% fetal bovine serum in a humidified 5% CO2 incubator at
103 37 °C. The number of cells seeded per ml of medium was 2 Â 105
PR
Wild type and mutant channel currents 167
104 on average. Cultured cells were seeded in 60 mm plates 24 h before
105 transfection, then transiently transfected with various plasmids by
The cells transfected with KCNQ1 and KCNE1 exhibited a slowly 168
106 the Fugene-6 method (Roche Applied Science, Indianapolis, IN).
activated outward current compatible with IKs from native cardiac 169
107 Electrophysiological experiments. The whole-cell patch-clamp
myocytes. Each mutant was then transfected with KCNE1 and the 170
108 method was applied to COS-7 cells transfected with the wild type
current–voltage relationships of the peak current during depolar- 171
109 and/or mutant plasmids as described previously [6,11,12]. Briefly,
ization and the tail current were measured as Fig. 1. 172
110 cells were allowed to settle at the bottom of a bath (0.5 ml)
D I313 G (n = 17 cells) showed an approximately 20% reduction in 173
111 mounted on an inverted microscope (Olympus Corp., Tokyo, Ja-
peak current and the densities of the peak and tail currents were 174
112 pan). Cells were superfused with the bath solution (140 mmol
less than those of the wild type (n = 10 cells) but the differences 175
TE
113 NaCl, 5.4 mmol KCl, 0.5 mmol MgCl2, 1.8 mmol CaCl2, 0.33 mmol
were not significant (P = 0.987). The activation curve shifted to- 176
114 NaH2PO4, 5.5 mmol glucose and 5 mmol HEPES) and the pH was
wards the left (Fig. 1A, B, F and Table 1). I313V (n = 15 cells) exhib- 177
115 adjusted to 7.4 by using NaOH. When inserted into the cell/bath
ited no significant change in the current compared to those of the 178
116 solution, a glass pipette with an internal diameter of 1.0–1.5 lm
wild type (P = 1.00, Fig. 1A–C and F). The shape of the membrane 179
117 had a resistance of 4–6 MX when filled with the following internal
potential vs. the current density curve was also similar among cells
EC
180
118 solution: 100 mmol/l K-aspartate, 20 mmol/l KCl, 5 mmol/l ATP-
transfected with the wild type and I313V (P = 1.00, Fig. 1A, C and 181
119 Mg, 5 mmol/l phosphocreatine-dipotassium, 5 mmol/l EGTA,
F). 182
120 5 mmol/l HEPES and 1 mmol/l CaCl2 (the pH was adjusted to 7.2
I313K (n = 14 cells) produced almost no current and I313E 183
121 with KOH). A patch-clamp amplifier Axopatch 200B (Axon Instru-
(n = 15 cells) showed a marked reduction of current compared to 184
122 ments, Foster City, CA) was used to record membrane currents.
the wild type (P < 0.01 for both, Fig. 1A, D, E and Table 1). 185
RR
123 After obtaining a whole-cell configuration, cell membrane
124 capacitance was estimated by analyzing the transient capacitance
Co-expression of WT and mutant KCNQ1 channels 186
125 elicited by 5 mV hyperpolarizing pulses. Cells were held at a start-
Cells were then co-transfected with 0.5 lg of each mutant and 187
126 ing potential of À80 mV and depolarizing pulses of various poten-
127 tials ranging from À80 to +80 mV in 20 mV increments for 2 s were 0.5 lg of the wild type together with KCNE1 (Fig. 2). Co-transfec- 188
128 applied, followed by repolarization to À40 mV for 2 s to record tail tion of I313 G with the wild type (n = 16 cells) showed a 2-fold in- 189
CO
129 currents. The pCLAMP 8.0 software (Axon Instruments, Foster City, crease of the current compared to the cells transfected with the 190
130 CA) was used to generate the pulse protocol, data acquisition, and wild type: 110.7 ± 4.7 vs. 57.1 ± 6.2 pA/pF (P < 0.001) (Fig. 2A and 191
131 analyses. B) and the tail current was also significantly augmented: 192
132 To be confident of the currents obtained, our analyses only in- 26.2 ± 1.7 vs. 16.0 ± 2.3 pA/pF (P < 0.05). The activation curve was 193
133 cluded recordings obtained by Giga-seal after applying the follow- shifted towards the left (Fig. 2E and F). I313 V co-transfected with 194
the wild type (n = 9 cells) showed similar current intensities with- 195
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134 ing quality control criteria for the patch-clamp technique [13]: (1)
135 the starting seal resistance was required to be more than 1 GX, (2) out significant differences (P = 1.00) compared to the wild type 196
136 the series resistance was required to be lower than 20 MX (n = 11 cells) (Fig. 2A, C and Table 1). Co-transfection of I313E with 197
137 throughout the recording, (3) the membrane potential was re- the wild type (n = 15 cells) exhibited a similar current to that of 198
138 quired to be at a higher negative level than À50 mV if normal cells co-transfected with I313 K and the wild type (n = 14 cells) 199
139 high-potassium intracellular solution was used; and (4) cell capac- both showing >70% reduction of current amplitude compared to 200
140 itance and resistance were required to be stable. Furthermore, , to the wild type (P < 0.001) (Fig. 2D–E, and Table 1). An increased cur- 201
141 check the quality of our findings, COS-7 cells transfected with wild rent in I313G with the wild type suggested a gain of function and a 202
142 type KCNQ1 were compared with the results of Barhanin et al. [1] markedly reduced current in I313E and I313K when co-expressed 203
143 and Sanguinetti et al. [15] regarding the properties and biophysical with the wild type would reflect a dominant negative suppression 204
144 characteristics of the wild type KCNQ1 potassium current. (Fig. 2F–G). 205
145 Data analyses. Analyses of the data were performed with Clamp-
146 fit 9.1 (Axon Instruments, Foster City, CA) and SPSS for Windows Kinetic analysis of mutant KCNQ1 channels 206
The hetero-tetrameric of the wild type and mutant channel in 207
147 ver.15 (SPSS Inc., Chicago, IL). The time constants for activation
148 and deactivation were determined by fitting the current recordings the presence of KCNE1 showed peak and tail currents which were 208
149 with a single-exponential function [16]: f(t) = A0 + A.exp(Àt/s) and well fitted with the Boltzmann equation or a single-exponential 209
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Fig. 1. Results of the whole-cell patch-clamp experiments in COS-7 cells. COS-7 cells were transfected with plasmids of wild type KCNQ1 (Q1) and four mutants together with
(E1)KCNE1 (A–E). The activation is rapid but the peak current was about 20% smaller in I313G (B) compared with the wild type (A). I313K and I313E showed almost no
current (D,E). The current–voltage relationships of cells transfected with five plasmids are shown on the right and a shift to the left can be seen in I313G (F). The differences in
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the peak and tail currents among the wild type and four mutants were not significant. Pulse protocol and graph scale are shown at the top.
Table 1
Comparison of kinetics and peak currents in COS-7 cells transfected with wild type and/or mutant KCNQ1 plasmids.
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Plasmid DNA Peak current Activation curve (mV) Time constants (s, ms)
(pA/pF) (V1/2) Slope (k) Activation Deactivation
WT 1.0 ng (n = 10) 70.4 ± 8.7 2 17.0 ± 1.4 17.0 ± 1.4 1252.0 ± 14.2 2.0 ± 14.2220
I313G 1.0 ng (n = 17) 56.4 ± 2.7 17.6 ± 1.3à 19.9 ± 1.2 607.4 ± 5.2à 397.2 ± 14.5
I313V 1.0 ng (n = 15) 61.8 ± 2.2 20.9 ± 1.6 17.3 ± 1.1 1389.6 ± 5.7 369.3 ± 110.6
I313E 1.0 ng (n = 15) O.9 ± 1.1à – – – –
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I313K 1.0 ng (n = 14) 0.4 ± 0.2à – – – –
WT# (n = 11) 57.1 ± 6.2 23.2 ± 1.3 16.7 ± 1.1 1154.3 ± 62.8 213.6 ± 11.85
WT/I313G (n = 16) 110.7 ± 4.7* 15.6 ± 1.2* 20.2 ± 1.3* 350.8 ± 3.0* 648.4 ± 126.0*
WT/I313V (n = 9) 59.5 ± 1.5§ 22.5 ± 1.1§ 16.9 ± 1.0§ 1297.5 ± 5.7§ 327.53 ± 110.6§
WT/I313E (n = 15) 9.3 ± 0.5* 19.0 ± 1.1§ 18.1 ± 1.0§ 1242.2 ± 91.4§ 289.0 ± 74.4§
WT/I313K (n = 14) 14.6 ± 1.7* 23.9 ± 1.8§ 17.1 ± 1.6§ 1302.1 ± 88.2§ 388.4 ± 64.4§
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Data represents the mean ± SEM.
WT, wild type; KCNQ1 (a subunit of the potassium voltage-gated channel KQT-like subfamily member 1); KCNE1, b subunit of the potassium voltage-gated channel IKs-
related family member 1; I313K;I313E;I313V and I313G: mutant KCNQ1.
à
P < 0.01 vs. WT 1.0 lg/KCNE1 1.0 lg.
P > 0.05 vs. WT 1.0 lg/KCNE1 1.0 lg.
#
0.5 lg of WT was transfected and subsequent study was transfected 0.5 lg of mutant.
*
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P < 0.001 vs. WT 0.5 lg/KCNE1 1.0 lg.
§
P > 0.05 vs. WT 0.5 lg/KCNE1 1.0 lg. Each plasmid was transfected with KCNE1 (see text).
210 function [16]. The activation curves for cells transfected with the The activation curves for cells transfected with I313I, I313K 222
211 wild type and that of co-transfection with I313K, I313E or I313V and I313V together with the wild type revealed statistically 223
212 were not significantly different (Table 1). non-significant differences compared to cells transfected with 224
213 Expression of I313G alone showed a more rapid initial activa- the wild type alone (P = 0.99, Fig. 2). The time constants were 225
214 tion compared to the wild type: 607.4 ± 5.2 vs. 1252.0 ± 14.2 ms similar among these three mutants but that of I313G was larger 226
215 (P < 0.01) and smaller V1/2: 17.6 ± 1.3 vs. 23.5 ± 1.4 mV (P < 0.01) at the membrane potential<À25 mV and smaller at >À25 mV 227
216 as shown in Fig. 1 and Table 1. I313 G co-transfected with the wild (Fig. 3A). 228
217 type showed a significant shift in the activation curve towards the The current density was also affected by the side-chain volume 229
218 left compared with the wild type and the time constant of activa- of amino acid residue at position 313 and a significantly larger cur- 230
219 tion was smaller: 350.8 ± 3.0 vs. 1154.3 ± 62.8 ms (P < 0.001) while rent density was found only when I313G was co-expressed with 231
220 that of deactivation of the tail current was larger: 648.4 ± 126.0 vs. the wild type compared with when it was expressed alone 232
221 213.6 ± 11.85 ms (P < 0.001) as shown in Fig. 2A and B, Table 1). (Fig. 3B). Both the homo-tetrameric and hetero-tetrameric I313G 233
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Fig. 2. Results of the whole-cell patch-clamp experiments in COS-7 cells. COS-7 cells were co-transfected with wild type KCNQ1 and four mutants together with KCNE1 (A–E).
Current–voltage relationships are shown on the right (F,G). Augmented peak (P < 0.001) and tail current (P < 0.05) and a shift to the left in activation curve was observed when
I313G was co-transfected with the wild type KCNQ1 (B and F). Dominant negative suppression was seen in D and E in which the amino acid residue was replaced by an either
positive or negative charged one (D,E). Pulse protocol and graph scale are shown at the top.
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234 with the wild type showed a shift to the left in the activation curve showed a reduction of the peak K+ current by about 20% compared 264
235 (Fig. 3C) suggesting a altered gating. with the wild type, but the initial current was larger (Fig. 1B). Fur- 265
thermore when I313G was co-expressed with the wild type 266
236 Discussion KCNQ1, the K+ current increased 2-fold which suggests a gain of 267
function (Fig. 2B). 268
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237 Both negatively charged (I313K) and positively charged (I313E) The conductive conformation of the K+ channel represents a 269
238 residues at the pore center of KCNQ1 resulted in an apparent loss match between the ion-binding sites and the size of K+ ions [26] 270
239 of channel function when they were transfected alone (with and the filter atoms and the surrounding protein atoms are impor- 271
240 KCNE1). The loss of function was not due to a trafficking defect tant for selective ion-binding and conduction [9,10]. The volume of 272
241 and when they were co-transfected with wild type KCNQ1 (with side-chain residues located in position 313 may affect conduction 273
242 KCNE1), a dominant negative suppression was observed. of K+ ions [27]. 274
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243 The IKs channel has six trans-membrane domains (S1–S6), a For the augmented K+ currents observed in the I313G mutant, 275
244 voltage sensor (S4) and a pore helix selectivity filter segment (P- we postulate as follows. The homo-tetramer by the smallest amino 276
245 loop) that connects S5 and S6 [10,17]. The selectivity filter is re- acid residue Glycine minimized the selectivity filter size (pore) and 277
246 flected in a highly conserved amino acid sequence for specific ion resulted in a reduced peak current. However, when the mutant was 278
247 conductance as elegantly defined by the crystal structure of the co-transfected with the wild type, the pore was composed of 279
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248 bacterial KcsA channel [18] and the carbonyl oxygen atoms of mixed amino acid residue, Glycine and Isoleucine rendered the 280
249 these residues which bind dehydrated K+ ions and act for selectiv- channel pore larger and augmented the K+ current (Fig. 3C). Using 281
250 ity [19,20]. An altered charge at the pore center, I313K and I313E, Brownian dynamics on a simplified model of the KcsA structure, it 282
251 is expected to result in a crucial change of the electrostatic envi- was shown that altering the pore size of the cytosolic entrance to 283
252 ronment of the selectivity filter, with serious consequences that re- the selectivity filter led to a change in conductance [28]. 284
253 duce the conduction of K+ ions [21–24]. The presence of charged As limitations, except for I313K, other mutants are virtual and 285
254 amino acid residues at the pore center may also disturb the we have no clinical counterparts so far, but if I313G is associated 286
255 closed/open equilibrium and lead to the destabilization of the with short QT syndrome or not is of interest [29]. The change in 287
256 open-state of the channel [25] but this was not confirmed in the ion selectivity in each mutant and the relation to the gating mech- 288
257 present study. anism was not fully studied in the present report [30]. 289
258 The current density was also affected by the side-chain volume As clinical implications, the functional consequences of muta- 290
259 of the amino acid residue at the pore center (Fig. 3B and Table 1). tions at the pore center of KCNQ1 varied: from dominant negative 291
260 When we substituted the neutral Isoleucine residue of KCNQ1 with suppression to a gain of function when co-transfected with wild 292
261 Valine (I313V) which has a similar size and polarity to Isoleucine, type KCNQ1. Severe reduction of IKs would be detectable as LQTS 293
262 the normalized current–voltage relationship was very similar to but a subtle change in K+ channel function of varying degrees 294
263 the wild type. The homo-tetramer of the Glycine residue (I313G) might go undetected. 295
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396
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