Ion Channels

Ion Channels

  • Refik Kanjhan added an answer:
    2
    Is there a list of Potassium channels blocked by intracellular Cesium? Does anybody know of potassium channels not blocked by intracellular Cesium?

    Usually people use intracellular cesium to block potassium channels, and then add for example TEA to also block them. Is there any knowledge available which potassium channels are targeted by either one ?

    Refik Kanjhan

    Dear Friedrich,

    My personal experience is that Cs blocks different potassium channels partially from inside. For example while you can block Ih current with 3 mM external Cs, internally applied much higher Cs will not block Ih completely. Similarly internal Cs will partially block Kv channels, but to have complete block people adds TEA to their internal solution. 

    Best wishes, Refik

    • Source
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      ABSTRACT: Here we describe two Neurobiotin electroporation techniques for combined analysis of neuronal structure and function in 300-m m-thick brain slices during development, using mouse hypoglossal (XII) motoneu-rons as a model. Individual neurons were studied with patch electrodes either in semi-loose (R ~50–300 M W) or in tight-seal (G W) con fi gurations. The semi-loose seal allows prolonged recording of spontaneous (if present) or evoked (by voltage steps or extracellular K +) spiking. In tight seal, the cell membrane is fi rst broken down with voltage pulses (50–100 mV). Resulting pore formation in tight-seal mode allows for the stable recording of spontaneous or evoked postsynaptic potentials or currents and the passage of Neurobiotin without spillover into the adjacent cells. With either con fi guration, Neurobiotin electropora-tion is achieved by passing 500-ms pulses at 1 Hz (5–25 mV equivalent of ~300–500 pA) for ~5 min. Labelling quality of recovered cells is similar between the two con fi gurations; however, the recovery rate of labelled neurons is higher with the semi-loose method. The ef fi ciency and reliability of the semi-loose seal makes it particularly suitable for the serial recording and labelling of multiple neurons within the same preparation. Using semi-loose-seal and tight-seal con fi gurations in combination can increase ef fi ciency and data collected per preparation. Intracellular sharp-electrode (1– 4) , whole-cell patch-clamp (5– 7) , and juxtacellular (8) recording and labelling methods using glass microelectrodes have previously been used for combined analysis of cellular structure and function. Each of these methods has limitations, as outlined below, both in obtaining good electrophys-iological recordings and in recovering the axonal, dendritic, and somatic morphology of the recorded neurons.
      Full-text · Article · Jan 2013 · Neuromethods

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  • Priyank Raj added an answer:
    9
    What is the relationship between action potential and signal transduction- two events that follow the binding of a neurotransmitter to its receptors?

    When GABA binds to its receptors, it creates an inhibitory potential, while serotonin and most other produce an action potential. But signal transduction is both their events. Also, one can keep in mind ligand-gated ion channels vis-a-vis GPCR when answering this question.

    Priyank Raj

    Dear Sir,

    Thanks for your reply.

    Your second answer certainly clarifies the difference in nature between GPCR and Iigand-gated ion channels.

    Sincerely,

    Priyank.

  • Thomas Launey added an answer:
    8
    Is there an easy method to study the intracellular calcium?

    I'm looking for an easy and cost effective method to study intracellular calcium.

    Thomas Launey

    Hi Gabriela, To measure absolute concentration, you will have to use ratiometric measurement. This can be with genetically encoded probes (see Miyawaki's lab work) or the AM versions of Fura2 (using UV) or Asante Calcium red (newer, visible light). I hope this helps, good luck, Thomas  

  • Alvin Uy added an answer:
    3
    What is a potential of mean force? What does it have to do with the flow of ions in and out of an ion channel?

    Currently we are studying the non-Ohmic behavior of alpha-hemolysin ion channels and our adviser said something about the potential of mean force of the ions going in and out (specifically Cl- and K+) of the ion channel. I haven't asked our adviser much detail about it so maybe my fellow researchers could give me some insights. Thank you so much!

    Alvin Uy

    Thank you! Your explanations would be of big help to our study.

  • Giustino Varrassi added an answer:
    2
    Please does anyone have an idea on articles or publications that link electrolytes to pain (not ion channels)?

    Please does anyone have an idea on articles or publications that link electrolytes to pain (not ion channels)?

    Giustino Varrassi

    Hi Isa. You should ask directly Prof. Vadalouca. She is very expert on this. Her email address is: athinajv@ath.forthnet.gr

  • Guillermo solis fernandez added an answer:
    3
    How can I calculate voltage for half-maximal activation of an ion channel, from the membrane potentials and amplitude current values?

    I want to calculate voltage for half-maximal activation and the slope factor from a series of membrane potential (voltage) and amplitudes currents for a given ionic channel.

    Guillermo solis fernandez

     Thanks Norbert, I am working with voltage gated-channels so I will try to apply the procedure that you explain. 

    Also thanks to Marcus for the protocol on measuring on whole cell and the software packages to do the fitting

  • Binnur Tüzün added an answer:
    3
    Is calcium Ion channel overload responsible for the sexual side effects of Hyperparathyroidism?

    Trying to see the role calcium ion channels may play in rare causes of sexual dysfunction. When people are suffering from hyperparathyroidism their symptoms are thought to be caused by calcium channel overload. Is this also the same reason for the sexual symptoms? Developing research for the role of Calcium Channels and Post SSRI Sexual Dysfunction Syndrome (PSSD).

    Binnur Tüzün

    YES it may be this will be  a fine study

  • Saak V. Ovsepian added an answer:
    4
    Do microtubules have any effect on ion channels/release of neurotransmitters?

    Orch-OR theory for consciousness asserts that the microtubules are the neural structures that support the quantum effects. Let's assume that it is true. Therefore, if they have to play a role in the brain, they need to effect the signal transmission in the brain. Is there any indication for such an effect?

    Saak V. Ovsepian

    Dear Abolfazi,

     Please check the vast literature on the role of microtubules in axonal transport, dendritic spine formation and dynamics and synaptic plasticity. Although your question seems a simple one, its answer as I understand is very complex and multifarious and requires some substantial research. 

    Good luck,

    SV!

  • Maedeh Mozneb added an answer:
    6
    What are some common voltage sensitive proteins?

    Can someone please give me some names of voltage sensitive proteins that are present in the voltage gated ion channels? Which have been widely studied so far?

    What I have found so far was mucin, cadherin and ferritin and XIAPs.

    Do anyone know anything more than this?

    Maedeh Mozneb

    Thank you very much. Refik your answer really helped. Thank you

  • İbrahim Türkel added an answer:
    1
    Efflux pump detection by PCR targeting MexAB-oprM like genes is really useful or QPCR based expression of efflux gene is necessary?

    Dear researchers,

    I have identified the presence of efflux pump genes in only 65% of Pseudomonas and absent in 35% of Pseudomonas strains.

    Where detection of the gene by PCR targeting MexAB-oprM like genes doesn't mean that they overexpressed or not?

    I prefer qPCR-based quantification of expression of efflux pump gene from mRNA.

    My query is what about the 35% of strains that lack either MexAB-oprM, MexXY-oprm, MexEF-oprN, MexCD-oprJ, or none of these pumps, which means only few, or resistant strains only carry efflux pump genes or all strain will carry certain efflux gene, but only resistant strains will overexpress?

    Is PCR-based Efflux pump gene detection valuable or not?

    Thanks in advance.

    İbrahim Türkel

    Dear Nandogopal

    These efflux pumps encoded choromosally. So %35 of your strains must carry this pump. But some strains have lower expression then resistance form of your strains. In my opinion, real time pcr will be effective for measure the expression levels of your strains. Or you can sequence MeX genes for %35 strains by convential pcr and jel electrophoresis and blast it.

    Best wishes

  • Alvin Uy added an answer:
    7
    What are some of the possible explanations for the non-Ohmic behaviour of an ion channel?

    We are currently doing a study on the non-Ohmic behaviour of α-hemolysin ion channels excreted by the bacteria Staphylococcus Aureus. According to previous studies, these ion channels does not follow Ohm's law.

    I would like to know on what possible reasons that we might need looking in to for the duration of the study that could possibly explain this behaviour.

    Alvin Uy

    Thank you Mr. Zylbertal, Mr. Kanjhan, Mr. Rastqar, Mr. Kahanovitch, and Mr. Ilyin for your answers. They would be of big help to our study.

  • Linda M. Boland added an answer:
    3
    Ion channel mentors: will you please take a 2 min survey at this site on Survey Monkey: https://www.surveymonkey.com/r/FL2XFYX ?

    this survey takes about 2 mins and your help is appreciated.

    Linda M. Boland

    I will find a way to post the answers to the ion channel survey.

  • David C. Ellinsworth added an answer:
    2
    Are we any closer to identifying the EET binding site on TRPV4?

    Since Bernd Nilius predicted in his 2004 AJP Cell review that this could relate to an arachidonate recognition-like sequence (residues 402-408, for mTRPV4) I don't believe this has been clarified. Or am I wrong? Apparently mutant TRPV4 lacking the predicted ARS cannot be functionally expressed.

    David C. Ellinsworth

    Thank you, I am aware of this paper and am also in collaboration with Miguel Valverde on an ongoing project. This paper does not, however, further elucidate where EETs actually bind on TRPV4, even though it very elegantly describes an important facet of converging physiological signalling mechanisms of TRPV4 activation. Since posting this question I have had personal communications and come to the conclusion that the predicted ARS is the best bet, but no data exists to verify it. 

  • Kate Goasdoue added an answer:
    3
    We did a WB using ab10096 for nAChR-alpha7. It precipitated but the darkest band didn't have the predicted band size. What do you suggest?

    Blots were incubated with ab10096 overnight and for 90 min for the secondary antibody. Non-fat milk was used as blocking solution.

    Kate Goasdoue

    Sometimes proteins run differently on a gel than the predicted MW due to post-translational modifications such as glycosylation - but this shouldn't change the MW by too much. As Julia said it could be a dimer if it is double the weight. Could be also that your samples have a splice variant of that gene. Saying that, that band on the blot doesn't look too specific - if you altered your ab concentrations / block to clean up the blot you might flind that band disappears. First step is a positive control then if you want to be sure you could cut out the band from the gel and sequence it

  • Eleanor Martin added an answer:
    3
    Can anyone give me some advice on coexpression in yeast?

    Hello, 

    I would like to co-express a large (250 kDa) chloride ion channel, along with one of its interacting proteins (50kDa, soluble) in yeast. We have already optimized the expression of the membrane protein in yeast, so I would like to stick with the strain we have been using as this contains a protease deletion which has proved useful in reducing proteolysis of this large protein. The problem is that this particular stain is only ura3 deficient, meaning cloning into another plasmid and using another auxotrophic marker is not an option. The strain is also lys2 deficient so could I use a -ura media with alpha aminoadipate as a nitrogen source as the two selection markers? Or I was thinking of using a bidirectional expression vector such as this pBEVY one.

    Has anyone tried to do something similar and would be able to offer any advice?

    Many thanks!
    Ellie 

    Eleanor Martin

    Hi Rui, thanks for your response. Yes the strain is lysine deficient, which does seem to be a less common auxotrophic marker. Most of the examples of the use of this as a selective marker are based on the ability of lys2 deficient strains to utilize alpha-aminoadipate as a nitrogen source, rather than through complementation with a vector? I'm also struggling to find any vectors containing LYS2?? Am I missing something?

    Hi Reza, thanks for your response. I'm afraid expression in pichia is no good for our protein. 

  • David Baez-Nieto added an answer:
    10
    Is it possible to establish the reversal potential from Ca2 + channel macroscopic current, fitting a straight line to the data after the current peak?

    I/V from macroscopics currents. Just taking into account the data that preserve the linearity, avoiding the last points close to the reversal potential. It has been done before?

    David Baez-Nieto

    Thanks I'll taking it into account

  • Huanting Liu added an answer:
    3
    Why are my plasmids recombining when I transform them?

    I am working with plasmids containing aion channel, with the goal of eventually using them for transfection in Hek cells.  The problem I am having is preparing these plasmids at the bacterial transformation stage.  I have 2 different channels (both from he HCN family), on of them (HCN2) grew perfectly on the first try in XL1 Blue cells.  However I am now doing point mutations on the channel (using a Quikchange kit) and I cannot get a colony that has my intact channel.  Additionally I am trying to use HCN1, another member of the same family, and it is giving me similar problems to the mutation reaction.  Here is what I have tried so far:

    1.  I am using internal channel specific primers to screen picked colonies for the presence of my plasmid.  PCR of the unmated HCN2 plasmid produce a clean band of the appropriate size.  PCR of the mutation reaction prior to transformation produces a single band of the right size. but PCR's of the picked colonies for the mutants do not, they show multiple bands.

    2. Used Stbl2 competent cell to hopefully prevent recombination of the plasmid,but the pct's looked the same as the XL1-Blue.  

    3. Tried incubation at 37 and 30 degrees, and decreasing the antibiotic concentration, but still the same problem

    I have tried these things with both the HCN2 mutation reaction and the wild type HCN1 plasmid and have had no luck.

    Any advice would be much appreciated!  Also, if there are any extra details that would help please let me know 

    Thanks! 

    Anna

    Huanting Liu

    Your problem is not due to plasmid itself recombination, it is caused by low mutagenesis efficiency . Try to read the paper on  http://www.biomedcentral.com/1472-6750/8/91 and repeat your mutagenesis follow my lab lab protocol as attached if you want.

  • Euan Robert Brown added an answer:
    2
    Artificial bi-layer composition.

    Anyone out there making planar or tip dipping lipid bi-layers? I have been told lipid formulation is 4:1 POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) to  POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Alternatively some authors use only DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). Any thoughts on what’s best for reconstituting with ion channels please?

    Euan Robert Brown

    Thank  you Vitaly thats a great reply!

  • Sai Bharadwaj Medapuram added an answer:
    2
    How does body effect increase the threshold voltage of a NMOS transistor?

    When substrate's voltage is made negative relative to source's voltage the p-n junction between source and substrate is reverse biased.This means that the number of negatively charged ions in the channel have increased which in turn must increase drain current but I notice the opposite

    Sai Bharadwaj Medapuram

    For NMOS when we bias the substrate negative to the source the depletion region of the reverse biased pn juctions of source-body and drain-body increases expanding into the channel .So more number of negative charged ions are available while the number of electrons available have decreased.So greater threshold voltage needs to be applied to compared to the case when no bias voltage is applied to substrate

  • Pablo J Sáez added an answer:
    10
    What is the difference between a gap junction and an ion channel in cells?

    Are they different? Or just a specialized type of any of the two.

  • Qingyao Huang added an answer:
    8
    Could someone help me to clear up the question about TTX and ICAN?

    Effect of TTX on calcium-activated non-specific cation current makes me confused recently. I have no idea whether the channel activity can be blocked by TTX in direct or indirect ways.

    Thank you who will give me a hand!

    Qingyao Huang

    Thanks for your help Asaph Zylbertal.

    It is reported that ICAN is responsible for the generation of pacemaker activity related to  respiratory rhythm, on the grounds of cadmium and TTX sensitivity, as well as the riluzole insensitivity. However, I don't know why the likelihood of voltage-dependent calcium channels is excluded if the vdcc current is sensitive to cadmium and TTX, and insensive to riluzole too. On the other side, if the vdcc current is not sensitive to TTX, what mechanism mediates TTX to inhibit ICAN, also through inhibiting calcium influx ?

    Perhaps, the location of putative contributors is concerned with the effect of TTX on ICAN as well, 

    I think I should search for more basic knowledge of CAN channel.

    THX!

  • Serguei N Skatchkov added an answer:
    1
    How do I propose an analogues for a compound like imidazopyridine?

    By knowing the inhibition of carcinoma cell growth, hERG ion channel inhibition, and the structure of imidazopyridine.

    Serguei N Skatchkov

    Dear Nazanin,

    indeed pyridine, pyridinium, based compounds are widely used trying to suppress cancer growth. This is attached (see  please, Tobo et al., PLos ONE, 2015) article  on different structures of such compounds.

    When we used pyridin(ium) based compound ASP+ we visualized that this fluorescent compound is selectively taken up by astrocytes, but not neurons (except few neurons in VTA area, Inyushin et al., 2013-Fig.2). So, the compound can be very selective for astrocytes in cortex and hippocampus, or may be in other cell type in different organs that you can test.  (please see attached Inyushin et al., 2010 to get some ideas).

    However, most surprising was our discovery that the transporters which are taking up such compounds are relocated in cancer cells from plasma membrane to nucleus membrane, and thus, the "killer compound" can not reach the cancer cell cytoplasm (See please attached Kucheryavykh et al., 2014).

    Very cordially, Serguei.

    + 3 more attachments

  • Robert Lindner added an answer:
    7
    Any advice on immuno precipitation from crude/unclarified lysate?

    I am stuck in this situation at the moment, trying to immunoprecipitate (IP) and Co-immunoprecipitate (CoIP) partners of an ion channel that have totally different solubility requirements.

    N.B. - I have functional evidence (patch clamp recordings) that this channel is present fully assembled in my cells. Also, the two partners of this channel are part of other channel complexes (<10% form the channel I am interested in).

    Protein 1: Super hydrophobic (17 transmembrane segments), I can detect it by western blot when solubilizing the PELLET in LDS sample buffer after lysate clarification. I have tried Triton-X100, CHAPS, DDM, Pierce IP lysis buffer and FivePhoton Transmembrane protein lysis buffer - none of these effectively solubilize this protein (even with additives - spermidine, glycerol). Sonicating the crude lysate helps somewhat, but this appears to disrupt the delicate interaction between protein 1 and protein 2 - i.e., I cannot see a CoIP band, but I do see a faint IP band. I can see a darker IP band when resolubilizing the pellet in lysis buffer + sonication to break up the pellet. But still no CoIP band (pellet will not re-dissolve without sonication).

    Protein 2: A little more co-operative. I can detect this by western blot and IP from CLARIFIED LYSATE. I have not tried IP from the pellet for this protein. I do not see a CoIP band from clarified lysate.

    My question is this: Is it necessary to clarify the lysate? I've been trying to come up with the "perfect lysis buffer" to solubilize everything, but not disrupt the interaction - but maybe I'm wasting my time? Could I just try my IP/CoIP from crude lysate? Has anyone ever tried this?

    Happy to give more info on this. Thanks in advance for the help!

    Robert Lindner

    Hello David,

    I also would suggest to try crosslinking on cells. Perhaps a disulfide-cleavable, water-soluble x-linker might be optimal for this purpose. This would allow you to use really dissociative lysis conditions afterwards, as long as you avoid cleaving the linker by reducing agent (include iodoacetamide or NEM in the lysis buffer). After IP, you can cleave by reduction and should be able to detect your two bands of interest. Good luck!

    Regards,

    Robert

  • Hang Li added an answer:
    3
    When the cell potassium ion channels were blocked by chemical, can they still will be detected by ion channel antibody?

    I want to use 4-AP to block cell potassium ion channels, and try to confirmed with antibody to see if it is blocked or not. I was wondering even ion channel were blocked it still may can be detected  by antibody, then my method won't work, anybody has any ideas? I just want to find a way to show potassium Ion channel has been blocked by 4-AP

    Hang Li

    Thanks a lot,  Norbert Weiss and  Sandipan Chowdhury, I appreciate all the time you spend to help me. I know electrophysiology would be the best way to do it, I just want to find another way to prove it 4-AP blocked potassium ion channel besides electrophysiology. Hi  Sandipan Chowdhury, I will think about your suggestions. Thanks a lot.

  • Alessandro Bilella added an answer:
    15
    DREADD vs. optogenetics
    I am thinking of employing both these techniques in my lab and was wondering if people would like to share their thoughts/experience.

    From a behavioural point of view I suppose that a major advantage of optogenetics is that you have greater temporal control over stimulation: once a dose of CNO is administered to an animal expressing DREADD there is presumably a time-to-onset and later a decline in receptor occupation and effect, whereas with ChR2/NpHR simulation is phase-locked to light stimulation.

    By contrast, I would imagine that light scattering (optic fibre in brain)/failure of light to penetrate tissue sufficiently (stimulation of peripheral nerves in skin) is a drawback of optogenetic stimulation compared to oral administration of CNO, which has known efficacy at different DREADDs.

    Any thoughts/comments welcomed!
    Alessandro Bilella

    Yes, in both cases is Cre-recombinase.

  • Jitendra Singh added an answer:
    12
    Why are there current fluctuations in hERG patch clamp assay using port a patch (Nanion)?

    I am doing hERG voltage clamp protocol using Port a patch but I am unable to get a steady current. The current fluctuations are so high that I can't even test a single conc. A brief about Port a Patch- its a semi automated patch clamp instrument where solutions, test compounds/ vehicle/ positive control all are added manually. This patch clamp has been recently installed in our lab and has passed the initial validation tests.  

    I have checked the pulse protocol and other settings but somehow I am not able to rectify this problem. Also I am using inducible CHO-TREx hERG expressing cell line. Kindly suggest..

    Jitendra Singh

    who is the supplier of CHO-TREx hERG expressing cell line.  make sure that it is expressing the herg channel by some florescence methods.

    Proto col suggested by the Marry is ok u can try making following changes to your protocol

    Protocol Voltage (mili-volts)            Duration (mili-seconds)
    Holding potential   -80                                 50
                                 -40                                    500
    depolarization     +40                                   2000

    depolarization     +40                                     2000

    Holding potential  -80                                      200

    Measure the current in 4th segment (+40 mV to -40 mV).

    Also write in details how you are culturing the cell specifically media and antibiotic anti mycotic. 

About Ion Channels

Gated, ion-selective glycoproteins that traverse membranes. The stimulus for ION CHANNEL GATING can be due to a variety of stimuli such as LIGANDS, a TRANSMEMBRANE POTENTIAL DIFFERENCE, mechanical deformation or through INTRACELLULAR SIGNALING PEPTIDES AND PROTEINS.

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